We have previously shown that monocytes derived from neonatal cord blood (CBMO) undergo less apoptosis following infection with *E. coli* or group B-streptococci (GBS), while monocytes from adult donors (PBMO) show a strong apoptotic response to infection [4,5]. Moreover, infected PBMO were able to induce apoptosis of monocytes, which were not infected (bystander-apoptosis), thereby actively terminating inflammatory immune response [6]. The mechanisms by which cell death was induced were activation of internal and external apoptosis pathways mediated through CD95L/CD95 (receptor) [7], and apoptosis induction by tumor-necrosis-factor-alpha (TNF) via tumor-necrosis-factor-receptor-1 (TNFR1) followed by caspase-cleaving and consecutive cell death [8].

Previous work revealed that CBMO show less CD95L-expression, TNFR1-internalization and TNF secretion compared to PBMO. This difference could be the result of a distinct posttranslational regulation of these pro-apoptotic factors in adults and neonates. One possible regulatory mechanism is the limited proteolysis of surface. This process, designated as shedding, can critically reduce ligands/receptors on the cell surface and thereby down-regulate the signal transmission [9,10]. The responsible enzymes are termed ectodomain sheddases and often represent zinc containing metalloproteinases.

The most relevant metalloproteinase for shedding of TNF and TNFR-1 is the tumor necrosis factor-converting enzyme (TACE/ADAM17). TACE belongs to the ADAM family proteases that share the characteristics of type-1-transmembrane proteins containing a catalytically active metalloproteinase, a disintegrin, an EGF and a transmembrane domain. Many other substrates for TACE have been identified including the IL-6 receptor [10], CD62L [11,12] and growth factors such as AREG and EGF [13].

Cleavage of CD95L can be mediated by the matrix metalloproteinase-9 (MMP-9, Gelatinase-B). This protease is well known to cleave extracellular matrix proteins such as collagen IV and V. De novo synthesized MMP-9 (pro-MMP-9) is activated by a two-step auto-proteolysis [14,15]. Moreover, MMP-9 activity can be regulated by TNF [16] and lipopolysaccharide (LPS), therefore playing an important role in endotoxin tolerance [17].

To better understand differences in PICD between neonatal and adult monocytes we here study the regulation of MMP-9 and TACE expression in CBMO and PBMO after *E. coli* infection. By blocking experiments with distinct metalloproteinase inhibitors we then investigate the role of MMP-9 and TACE for surface-expression and secretion of death ligands in monocytes from adults and neonates. Next, we perform blocking experiments to obtain information on the involvement of metalloproteinase activities for PICD of CBMO and PBMO. Finally, we compare the relevance of cell contact formation for bystander apoptosis induced in CBMO and PBMO target cells. Our results suggest that MMP9 and TACE are differentially regulated and have opposite functions in PICD. MMP9 expression is comparatively high in CBMO and seems in part responsible for shedding of CD95L and the low rate of PICD in these cells.

#### **2. Results**

#### *2.1. Plasma-Membrane Expression of Metalloproteinases is Induced by E. coli Infection*

In previous studies we observed that phagocytosing PBMO and CBMO express similar levels cell associated TNF whereas CBMO secrete much lower levels of soluble TNF than PBMO 24 h and 48 h post-infection (p.i.), (Supplemental Figure S1). These findings led us to the question whether ectodomain sheddases responsible for release of TNF and other death ligands (e.g., CD95L) could be differentially regulated in PBMO and CBMO. We here focus on the two metalloproteinase TACE and MMP-9 known as major shedding enzymes for membrane expressed TNF (memTNF) and memCD95L, respectively. First, we studied the surface expression level of TACE and MMP9 by flow cytometry before and after infection with *E. coli.* Infection caused a significant up-regulation of TACE on PBMO, whereas the expression level remained low in CBMO (Figure 1A). In contrast to TACE, the expression level of MMP-9 was significantly higher in CBMO compared to PBMO (Figure 1B).

**Figure 1.** TACE and MMP-9 expression is induced four hours p.i. by infection with *E. coli*. TACE expressing monocytes were assessed before and after infection with *E. coli*. (**A**) Histogram plots to the right compare isotype controls (filled) and anti-TACE stained PBMO which were non-infected or *E. coli* infected. Density plots (below) detail the distribution of *E. coli* infected PBMO regarding TACE surface expression (compare to the non-infected PBMO to the left). *E. coli* MMP-9 expressing monocytes were assessed before and after infection with *E. coli*. (**B**) (*n* = 5, \*\*\* *p* < 0.001, forked bars represent Student's *t*-test, blunt-ended bars represent ANOVA). Blocking of TACE activity increases its substrate CD62L. (**C**) Monocytes were infected with GFP-*E. coli* for 4 h with or without indicated inhibitors and surface expression of CD62L was determined. (**A**–**C**); (*n* = 5, \*\* *p* < 0.01, \*\*\* *p* < 0.001, forked bars represent Student's *t*-test, blunt-ended bars represent ANOVA).

To check whether expression profiles were correlated to enzyme activities and to exclude the possibility that infection caused alterations in the presence of MMP-9 and TACE only coincidentally, we performed a shedding assay for CD62L as an indicator for TACE-activity (Figure 1C). CD62L plasma membrane expression was induced after *E. coli* infection in PBMO but less in CBMO. Administration of the TACE inhibitor TAPI-0 considerably increased CD62L expression on infected PBMO and less on CBMO, indicating that the expression level of TACE is reflected by the shedding of one of the major substrates. By contrast, CHX treatment, which is known to inhibit MMP-9 activity [18] had no effect on CD62L expression (Figure 1C). These data show that under our experimental conditions PBMO exhibit higher TACE expression and activity while CBMO exhibit higher expression of MMP9. Furthermore, the results show effectiveness of the used inhibitors.

#### *2.2. Metalloproteinase-Inhibition Down-Regulates the Expression of CD95L and TNF*

Next we checked whether TACE and MMP-9 activities on *E. coli* infected monocytes could affect the expression of the two reported substrates CD95L and TNF, respectively. To this end, we added the metalloproteinase inhibitors TAPI-0 and CHX to infected monocytes (Figure 2). Addition of these inhibitors had no effect on the phagocytosis index under the chosen conditions (Supplemental Figure S2).

PBMO showed reduced levels of memCD95L after treatment with both inhibitors and an increase of memCD95L after *E. coli* infection. CBMO showed no difference regarding memCD95L levels. The memTNF levels increased significantly after *E. coli* infection in PBMO and CBMO (Supplemental Figure S3). Inhibition of MMP-9 by CHX was associated with a two-fold increase of memCD95L on PBMO and CBMO, whereas TACE inhibitor TAPI-0 had no effect (Figure 2A, compare columns 1, 2 and 3). The results are in line with the CD62L shedding assay, which shows a clear specificity of CHX for MMP-9 (Figure 1B). Infection with *E. coli* increased the number of memCD95L-positive PBMO eight-fold. This effect was much lower (four-fold) in CBMO (Figure 2A, compare columns 1 and 4). CHX but not TAPI-0 increased the number of memCD95L expressing infected PBMO. CHX also increased the number of memCD95L expressing CBMO to levels that were observed for infected but not CHX treated PBMO (Figure 2A, compare columns 4 and 11).

CHX had no effect on the expression of memTNF. However, treatment with TAPI-0 clearly increased the number of memTNF expressing PBMO and CBMO (Figure 2B, compare columns 1 and 3). Infection with *E. coli* amplified the expression level of memTNF three-fold on PBMO and two-fold on CBMO (Figure 2B, *p* < 0.05 vs. non-treated, non-infected monocytes). After blockage of TACE, nearly all PBMO expressed memTNF, while only 60% of CBMO did (Figure 2B, compare columns 4 and 6).

The results mirrored the effects of CHX and TAPI-0 on memCD95L and memTNF: While increasing memCD95L on infected monocytes (Figure 2A), CHX significantly diminished the concentration of secreted CD95L (Figure 2C). Similar results were obtained for TAPI-0 with respect to secreted TNF (Figure 2D).

**Figure 2.** Blockage of metalloproteinases modulated expression of memCD95L and memTNF. PBMO and CBMO were infected and/or treated with the indicated metalloproteinase inhibitors. Expression of memCD95L (**A**) and memTNF (**B**) were assessed on CD14-positive gated monocytes. The same groups were analyzed for secreted (s) CD95 (**C**) and TNF (**D**) 24 h p.i. via ELISA (*n* = 5, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, forked bars represent Student's *t*-test, blunt-ended bars represent ANOVA).

#### *2.3. Inhibition of TACE Reduced PICD, but Inhibition of MMP-9 Restored PICD in CBMO*

Since our experiments indicated the involvement of metalloproteinases in the regulation of death receptors and ligands we addressed the question as to whether inhibition of metalloproteinases affects the induction of apoptosis in uninfected and infected monocytes (Figure 3). Treatment with CHX doubled apoptosis rates in uninfected PBMO and CBMO (compare columns 1 and 2). TAPI-0 did not show this effect (compare columns 1 and 3). In concordance with our previous results, PICD of CBMO was diminished compared to PBMO (column 4). Treatment with CHX increased the PICD in infected PBMO significantly by about 10%. In infected CBMO, the effect of CHX on PICD was much higher and doubled PICD rates on *E. coli*. infected monocytes. PICD rates of CHX treated infected CBMO were two times higher than infected PBMO (Figure 3, right panel) In contrast, TAPI-0 addition reduced PICD in PBMO by about 50% (*p* < 0.05 vs. infected PBMO). In CBMO PICD was found to be unaltered (colums 6).

**Figure 3.** Blockage of MMP-9 increased PICD of CBMO. PBMO and CBMO were infected and/or treated with the indicated metalloproteinase inhibitors. Apoptosis was determined by Nicoletti assay 24 h p.i. The right panel gives the ratio of the values highlighted in dark-grey and grey, respectively (*n* = 5, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, forked bars represent Student's *t*-test, blunt-ended bars represent ANOVA).

#### *2.4. TNF and E. coli Infection Triggers Expression of memCD95L*

We had observed that infected PBMO and CBMO release considerable amounts of soluble TNF in a manner that most likely involves TACE (Figure 2D). Moreover, this inhibition was associated with a reduction of CD95L expression (Figure 2A). This may suggest that a cross-talk between TNF production and memCD95L expression exists. To address this possibility, monocytes were either incubated with soluble recombinant TNF and/or infected with *E. coli* and memCD95L expression was analyzed. Both addition of TNF and *E. coli* infection strongly up-regulated memCD95L on PBMO and to a lesser degree in CBMO (Figure 4, compare groups 2 and 3). As an inhibitor of TNF, we applied the TNF inhibitor etanercept to one group (Figure 4A, columns 2 and 4) resulting in considerable reduction of CD95L expression in PBMO, while the effect was not significant in CBMO. Furthermore, pre-treatment with etanercept reduced apoptosis rates significantly in TNF-incubated and *E. coli-*infected PBMO, but had no effect on CBMO (Figure 4A). Comparing the mean intensity values (MFI) of memCD95L (Figure 4B) reflected the specific effect of TNF, since addition of etanercept reduced the concentration of memCD95L on both PBMO and CBMO after TNF treatment and infection, respectively.

**Figure 4.** TNF triggers expression of CD95L. PBMO and CBMO were stimulated with TNF and/or infected with *E. coli* for four hours and analyzed for CD95L expression. Some groups were pre-treated with etanercept. The number of PBMO and CBMO expressing memCD95L were determined as the percentage (**A**) and the mean concentration given as the mean intensity (MFI), (**B**); for A and B, *n* = 5, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, forked bars represent Student's *t*-test, blunt-ended bars represent ANOVA.

#### *2.5. Apoptosis of Bystander Monocytes is Reduced Without Cellular Contact*

The surface expression levels of TNF and CD95L are separately regulated by TACE and MMP-9 leading to the release of soluble mediators. This process may affect bystander apoptosis in which cells could be directed to undergo apoptosis by either cell-to-cell contact or soluble mediators. In fact, bystander-apoptosis in PBMO required intercellular contact with phagocytosing monocytes, as we have previously shown in a transwell chamber setup [8]. To compare this aspect in CBMO and PBMO, we infected monocytes with *E. coli*-GFP in one (upper) transwell chamber and co-cultivated non-infected monocytes of the same donor in the other (lower) chamber (Figure 5A, sketch). In the upper chamber apoptosis occurred in PBMO in a significantly higher percentage compared to CBMO, as was expected. In the lower chamber, apoptosis of PBMO was reduced by 70% compared to PBMO in the upper chamber (Figure 5A). In CBMO, contact-independent ("trans") apoptosis was nearly absent.

We then questioned whether the expression of death receptors or shedding enzymes on cells that had either bound or ingested *E. coli* would be different from that on those cells, that had no contact to bacteria. As previously reported [6], we distinguished monocytes that had either bound or ingested *E. coli* (GFP-positive, GFP+) from those that had no contact with bacteria (GFP-negative, GFP−). CD95L was significantly higher up-regulated on the surface of GFP+ PBMO as compared to GFP− PBMO (Figure 5B). For CBMO, the upregulation of CD95L on GFP+ cells was less compared to on GFP+ PBMO (*p* < 0.005). Moreover, for on GFP- cells much less upregulation of CD95L was seen on CBMO than on PBMO.

Since CD95L expression can be regulated via TACE through TNF signalling and via MMP9 by cleavage of CD95L we analyzed TACE and MMP-9 expression on GFP+ and GFP− PBMO and CBMO, respectively. GFP+ PBMO expressed more TACE and MMP-9 compared to GFP− PBMO (Figure 5C, left panel, *p* < 0.001). MMP-9 was also upregulated on GFP+ CBMO compared to GFP− CBMO (Figure 5C, right panel). By contrast TACE expression was very low and comparable on GFP+ and GFP− CBMO (Figure 5C). These results suggest that CBMO are less effective in inducing bystander apoptosis than PBMO. This correlates with less upregulation of CD95L on CBMO compared to PBMO target cells, which is again associated with a low expression of TACE but still high expression of MMP-9 on CBMO compared to PBMO target cells. This correlation further supports our notion that activity of MMP-9 rather than of TACE contributes to reduced levels of PICD that are typically observed in neonate monocytes.

Additionally, the percentage of memCD95L-, memTNF-positive PBMO as well as the density of memTNF on PBMO was higher than on CBMO. In contrast, with less memCD95L-positive CBMO, their mean density was higher than on PBMO (Figure 5D).

**Figure 5.** Monocyte PICD is enhanced by cell–cell contact. Monocytes were infected with GFP-*E. coli* for one hour, washed and re-incubated for another 24 h in transwell chambers (experimental setup sketch); non-infected cells served as controls. Monocytes from the same donor were co-cultivated in compartments, separated by teflon membranes. Apoptosis was detected by hypodiploid DNA-content (**A**, *n* = 6 for PBMO and *n* = 4 for CBMO; \* *p* < 0.05). PBMO and CBMO were sub-gated (**B**) in phagocyting/binding (GFP+) and non-phagocyting (GFP−) monocytes and analyzed for memCD95L expression. Subgated PBMO and CBMO were further analyzed for TACE (**C**, left panel) and MMP9 (**C**, right panel) expression. (**D**) The expression of memCD95L and memTNF of monocytes in the lower ("trans") chamber was assessed. The percentage of surface expressing monocytes (panels to the left) and the mean values (MFI, panels to the right) were determined (*n* = 5, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, forked bars represent Student's *t*-test, blunt-ended bars represent ANOVA).

#### **3. Discussion**

The present study compared the induction of PICD in infected and non-infected monocytes (bystander cell death) from cord blood and adult blood, utilizing an in vitro *E. coli* infection model with respect to a distinct regulation pattern of the metalloproteinases TACE and MMP-9 (Figure 1). MMP expression was distinctly regulated by infection in PBMO and CBMO. Whereas infected CBMO exhibited less TACE expression and activity, they reacted with stronger expression of MMP-9 (Figure 1) as compared to PBMO. Depending on the expression pattern of MMP-9 and TACE, death ligands CD95L and TNF were either presented on the surface or shedded by distinct monocyte populations and thereby functionally controlled in regard to PICD (Figure 2, Supplemental Figure S3). We speculate that this regulation may be phagocytosis dependent (see Figure 5B), since the percentage of memCD95L appears higher on GFP+ phagocytosing monocytes (Figure 5B) than on non-phagocytosing (GFP-) monocytes. This observation was one of the reasons for monitoring the shedding of memCD95L and TNF via ELISA (Figure 2C,D). We could show, that PICD in CBMO was diminished (Figure 3), confirming earlier results [4]. TNF secretion triggered expression of memCD95L on PBMO but critically less on CBMO (Figure 4) which may explain that induction of PICD is predominantly contact-dependent (Figure 5). The data suggest that phagocytosis regulates expression of MMP-9 but not TACE in *E. coli* infected CBMO (Figure 5). To our knowledge, data on the expression and activity of metallomatrixproteinases in CBMO have been scarcely published.

The results of the actual study indicate an interlocked action of TNF and memCD95L. They suggest a functional model, where infection with *E. coli* activates TNF and consecutive TACE secretion in PBMO. TACE cleaves memTNF allowing predominantly ligation to TNFR1, since soluble TNF preferentially binds to TNFR1 [19,20]. The TNFR1/TNF ligand receptor complex is internalized to induce apoptosis [8,20]. This is in line with the findings that TAPI-0 decreases concentrations of soluble TNF resulting in reduced apoptosis (Figure 2B,D and Figure 3) However, our experiments provide evidence that soluble TNF also induces memCD95L (Figure 4). Pro-apoptotic signaling of CD95L/CD95R (receptor) can be distinguished from death receptor signaling by acting in a cell–cell contact dependent manner [21]. Increasing the amount of memCD95L by the MMP-9 inhibitor CHX is accompanied by an increase in apoptosis rates (Figure 2A,C and Figure 3).

This model is further supported by the low abundance of secreted TNF, TNFR1 [6] and CD95L in CBMO resulting in reduced PICD [4]. It explains also the significantly reduced trans-activation of PICD in PBMO and CBMO (Figure 5A). Phagocytosing monocytes express more TNF (Supplemental Figure S1A) which may cause a higher level of memCD95L in GFP+ monocytes (Figure 5B). Previous data have shown that blockage of TNF almost abolished PICD in PBMO [6]. The fact that CBMO secrete significantly less TNF than PBMO (Supplemental Figure S1B), but TNF stimulation increases the memCD95L-expression (Figure 4) may explain the observation of a reduced PICD initiation in cord blood (Figure 3). However, PBMO and CBMO showed a different reaction to factors which were secreted by phagocytosing mates in the transwell experiments (Figure 5D), pointing to a fundamental different meaning of cell-contact independent signaling in neonatal monocytes. Future experiments will elucidate these differences.

Various microbial, fungal and viral agents such as *Streptococcus pneumoniae* [22], zymosan [23] and HIV [24] were reported to initialize bystander apoptosis in monocytes. For resolving inflammation, bystander apoptosis may in the same way be essential [25]. Disbalancing this system may result in overwhelming infection or prolonged inflammation, thus the reduced bystander apoptosis of CBMO may be critical for the resulting immune reaction.

The mechanism of infection induced MMP-9/TACE expression should be clarified by future experiments. It was recently published, that soluble CD95L as well as memTNF activate the NFκ-B pathway which in turn targets metalloproteinases such as MMP-9 [26]. TACE, which also was examined in this study, is activated by infection-induced p38 MAP kinase and reactive oxygen species (ROS) [27].

MMP-9 and TACE are integrative members of a feedback control system which can skew the signal transduction of TNF-α and CD95L from pro-apoptotic to pro-inflammatory by balancing soluble and plasma-membrane bound forms and vice versa [28,29]. Reduced TACE activity was shown to promote endotoxin tolerance [30] without engagement of TLR4 [31], which circumvents an overwhelming inflammation upon infection.

The damage of organ tissues can be curtailed by controlling the expression of MMP-9. Enhanced expression of MMP-9 was correlated to COPD [32,33]. MMP-9 activation plays also a role in amniotic membrane rupture during labor [34] and enhanced MMP levels were associated with sepsis [35].

In this study we attributed a co-expression of MMP-9 and TACE with memCD95L and memTNF to the induction of apoptosis and proved this by simultaneous exposure to inhibitors. As a limitation it has to be mentioned that substrate specificity of the two enzymes is still controversial. The cleavage of memTNF by TACE is well documented [36,37] but the cleavage of the receptor TNFR1 in vivo has been discussed [38]. Furthermore it was reported that pathogenic *E. coli* synthesize proteins with metalloproteinase like properties [39,40] which could contribute to the shedding of TNF and CD95L. The results of our study are based on relatively small sample sizes and did not follow up on kinetics of cord and peripheral blood of newborns longitudinally. The neonatal situation several days postnatal is therefore not properly reflected by this study.

PICD and bystander apoptosis represent important tools to shape the immune response upon infection. Our data suggest phagocytosis-related cell death of non-phagocytosing monocytes, designated as monocytic bystander kill after infection with *E. coli*. It provides evidence that if monocytes are "at the wrong place at the wrong time", i.e., near or in contact to phagocytosing monocytes, they may be killed by fratricide. To what extent this may influence the course of neonatal sepsis, remains to be elucidated in vivo.

#### **4. Materials and Methods**

#### *4.1. Patients*

The study protocol was approved by the Ethics Committees of Aachen University Hospital (Permission No: EK150/09, 6 October 2009, signed by Profs G. Schmalzing and U. Buell, respectively). All adult participants involved gave written consent to use their blood samples. All term neonates were delivered spontaneously and did not exhibit signs of infection, as defined by clinical status, white blood cell count and C-reactive protein. Mothers with amnion infection and prolonged labour (>12 h) were excluded. Umbilical cord blood was placed in heparin-coated tubes (4 IE/mL blood), immediately following cord ligation as described before [7].

#### *4.2. Bacteria*

#### *E. coli*-GFP

*E. coli* DH5α, an encapsulated K12 laboratory strain, carrying the green fluorescent protein (*gfp*)-mut2 gene (*E. coli*-GFP) was a generous gift from Prof. Dr. Dehio (University of Basel, Switzerland) and was used for phagocytosis as previously described. Bacteria were freshly grown in Lennox-L-Broth-medium (Invitrogen) until early logarithmic growth, resuspended in phosphate-buffered-saline (PBS) and used immediately. Infection was performed at a multiplicity of infection (MOI) of 25 which was achieved by dilution with PBS. The phagocytosis assays were performed as described [7]. The phagocytosis index (CD14+GFP+ monocytes: CD14+ monocytes) was analyzed by flow cytometry. In some indicated experiments, *E. coli*-GFP was replaced by *E. coli*-EOS-FP (Supplemental Figure S1).

#### *4.3. Reagents*

Antibodies to CD14 (clone MEM18), MMP-9 (clone 56129), TACE (clone FAB9301P), CD62L (L-selectin; clone LT-TD180) and Ig-matched controls (IgG1, IgG2b) were purchased from R&D Systems and Immunotools (Abingdon, UK and Friesoythe, Germany), respectively. FITC-labeled CD95L antibody (clone SB93a) was purchased from SouthernBiotech (Birmingham, USA) and corresponding mouse IgG2b kappa isotype control (clone eBMG2b) was obtained from eBioscience (Waltham, USA). The secondary anti-mouse-PB antibody (F'ab fragment) was purchased from Invitrogen (Darmstadt, Germany). Propidium iodide (PI), isopropyl-β-D-thiogalactopyranoside (IPTG) and antibiotics were purchased from Sigma (Munich, Germany). For the blocking of TNF an anti-TNF antibody (a chimeric molecule combining the ligand-binding domain of the TNF-receptor 2 and the Fc-domain of human IgG1 (Etanercept, Pfizer–Wyeth, Hamburg, Germany), final concentration 1 μg/mL) was administered 1 h prior to infection. TACE inhibitor N-(R)-(2-(Hydroxyaminocarbonyl)Methyl)-4-Methylpentanoyl-L-Naphthylalanyl-L-Alanine amide (TAPI-0; Merck Millipore, Darmstadt, Germany) was added to a final concentration of 100 nm/L 30 min prior to infection. PBMO and CBMO were pre-incubated with MMP-9 inhibitor CHX (chlorhexidine; Santa Cruz Biotechnologies, Heidelberg, Germany) to a final concentration of 20 μg/mL 1 h prior to the infection. This concentration was chosen since it had been previously shown to be effective in MMP-9 inhibition [18].

TNF was purchased from eBiosciences (eBiosciences-Natutec, Frankfurt, Germany), aliquoted freshly after dilution in PBS and used in apoptosis induction assays in final concentrations of 5 ng/mL.

#### *4.4. Mononuclear Cell Cultures*

Peripheral blood cells from adults and cord blood mononuclear cells (PBMC and CBMC) were isolated by density gradient centrifugation on Ficoll cushions (Amersham, Freiburg, Germany) as described previously [7]. Washed cells were resuspended in VLE RPMI-1640 (Biochrom, Berlin, Germany). For analysis of post-phagocytic reactions, cells were counted in an ultraplane Neubauer hemocytometer, placed at 2 × 106 cells/ml in flat bottom 24 well cell culture plates (Costar, Bodenheim, Germany), containing 10% heat-inactivated fetal calf serum (FCS, Biochrom) and were incubated at 37 ◦C.

#### *4.5. Flow Cytometry*

A daily calibrated FACS-Canto flow cytometer (Becton Dickinson, Mountain View, CA, USA) was used to perform phenotypic analysis. To prevent nonspecific binding, cells were incubated with 10% fetal calf serum on ice for 10 min before staining with appropriate fluorophore coupled secondary antibodies, or isotype-specific immunoglobulin-labelled monoclonal antibodies for 20 min over ice in the dark. Monocytes were gated by forward (FSC), side scatter (SSC), and CD14 expression. For intracellular cytokine staining, monocytes were fixed in 2% paraformlaldehyde/PBS for 30 min at room temperature (RT) and washed three times with PBS. Afterwards monocytes were permeabilized utilizing a permeabilization buffer (purchased from Thermo Fisher Scientific, Hennef, Germany) according to the manufacturers' recommendations. Data was analyzed using the FCS Express V4.0 research Edition software (DeNovo Software, Glendale, California, USA).

#### *4.6. Detection of Hypodiploid Nuclei*

DNA fragmentation was assessed according to Nicoletti and previously described [4]. In brief, washed cells were slowly resuspended in 2 mL of −20 ◦C ethanol 70% with continuous vortexing and stored for four hours at −20 ◦C. Cells were washed twice, resuspended in 50 μL PBS containing 13 units RNAse (DNAse free; Sigma, Taufkirchen, Germany) and incubated for 15 min at 37 ◦C. 180 μL of PI (70 μg/mL) was added, incubated for 20 min and analysis was performed immediately. Alternatively, mononuclear cells were stained with CD14 antibody for 15 min at RT to identify monocytes. A fixation with paraformaldehyde (2% *v/v* in PBS) for 2 h at RT replaced the ethanol fixation. Afterwards, cells were permeabilized by incubation in PBS-T (PBS, Triton X-100 0.1% *v/v*) for 20 min at RT, washed twice in PBS, resuspended in PBS-PI (PBS, 70 μg/mL PI and 13 units RNAse) and incubated for 10 min at RT before analysis by flow cytometry. Cell-doublets were discriminated by assessment of PI-width/PI-area.

#### *4.7. Transwell Experiments*

Transwell plates (pore diameter 0.4 μm, purchased from Corning, NY, USA) were used. Cells in the upper chamber were untreated or infected as described above. Cells separated by the Teflon membrane had no contact to bacteria as assessed by plating on appropriate growth-medium and FACS analysis.

#### *4.8. ELISA*

The TNF enzyme-linked immunosorbent assay (ELISA) was purchased from eBiosciences (eBiosciences-Natutec, Frankfurt, Germany) and the CD95L ELISA from Hölzel Diagnostika (Hölzel Diagnostika Handels GmbH, Cologne, Germany). Both were used according to the manufacturer's recommendations. The read-out was executed in a spectra max 340PC ELISA reader (Molecular Devices, Sunnyvale, CA, USA) with a sensitivity from 4–500 pg/mL.

#### *4.9. Statistical Analysis*

Results are expressed as mean +/− standard deviation. Error bars represent standard deviations. Values of *p* < 0.05 were considered as significant. Analyses were done with statistical software (performing two-way ANOVA adjusted according to Bonferroni-Holm for multiple group comparisons as provided by GraphPad Software Statistical Package, La Jolla, CA 92037 USA).

#### **5. Conclusions**

Matrix metalloproteases regulate the expression of death ligands and their receptors. On neonatal monocytes CD95L density is reduced upon infection due to lower activity of MMP-9. Enhancing MMP-9 expression could be a target to restore PICD in neonatal monocytes.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/6/ 1399/s1.

**Author Contributions:** S.D., conception, acquisition of data, analysis, interpretation of data; C.P., conception, acquisition of data, analysis, interpretation of data; A.L., drafting the article, critical revise; N.K., interpretation of data, drafting the article, critical revise; C.G., drafting the article, critical revise; T.O., conception, acquisition of data, analysis, interpretation of data.

**Funding:** This research received no external funding.

**Acknowledgments:** Thanks to the team of midwives (Roja Fakhrabadi) and the Department of Obstetrics and Gynaecology at the University Hospital of Aachen (E.Stickeler), for coordinating the cord blood sampling.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Isolation of a Novel Metalloproteinase from Agkistrodon Venom and Its Antithrombotic Activity Analysis**

## **Jin Huang 1,**†**, Hui Fan 1,**†**, Xiaojian Yin 1,2,\* and Fang Huang 1,2,\***

Received: 1 July 2019; Accepted: 18 August 2019; Published: 21 August 2019

**Abstract:** Snake venom contains large amounts of active proteins and peptides. In this study, a novel snake protein, metalloproteinase SP, was successfully isolated from the venom of *Agkistrodon acutus* by multi-gel chromatography. The isolated protein exhibits anti-platelet aggregation activity. Animal experiments showed that it exhibited defibration, anticoagulation, and antithrombotic effects and contributes to improved blood rheology and antiplatelet aggregation. In vivo experiments demonstrated that it prolonged clotting time, partial thromboplastin time, prothrombin time, thrombin time, fibrinogen time and reduced fibrinogen content of mice. Also, metalloproteinase SP inhibited carrageenan-induced tail thrombosis, ADP-induced acute pulmonary embolism, and ADP, Arachidonic acid (AA), or collagen-induced platelet aggregation. In vitro experiments showed that the protein cleaved the α, β, and γ chains of fibrinogen. Metabolomic analysis upon metalloproteinase SP treatment revealed that 14 metabolites, which are mainly involved in phenylalanine, tyrosine, and tryptophan biosynthesis, responded to metalloproteinase SP treatment. In summary, the isolated snake venom protein inhibits formation of acute pulmonary embolism probably through regulating and restoring perturbed energy, lipid, and amino acid metabolism.

**Keywords:** Agkistrodon venom; metalloproteinase; fibrinogen; antithrombotic; metabolomics

#### **1. Introduction**

Thrombotic disease is one with a high morbidity and mortality rate in the world, accounting for approximately 40% of deaths yearly [1]. Thrombotic diseases include pulmonary embolism (PE), deep vein thrombosis (DVT), myocardial infarction, coronary atherosclerosis, ischemic stroke, and so on [2,3]. The formation of thrombus is closely related to platelet aggregation, blood coagulation, and fibrin network formation. At present, antithrombotic drugs are categorized into three groups: anti-platelet aggregation drugs, anticoagulants, and thrombolytic drugs.

Evidence from recent research indicates that snake venom contains many active protein or peptides with defibration, anticoagulation, antiplatelet aggregation, and antithrombotic functions [4]. The anti-thrombotic action of snake venom protein is due to its ability to cleave the fibrinogen, reduce the content of fibrinogen, activate fibrinolytic enzyme, inhibit the activation of FXa, FIIa, thrombin, and other coagulation factors [5–7]. A large number of snake venom protease components have been isolated and some snake venom preparations are widely used in clinical practice.

Deinagkistrodon is one of the most virulent snake species in China that mostly lives in China's coastal and southwestern regions [8,9]. Snake venom contains a variety of active ingredients related to blood coagulation, such as phospholipase A2, serine protease, thrombin, metalloproteinase, and thrombin-like protein. Several new proteases and peptides have been isolated from the venom of Agkistrodon, such as Agacutas, Pt-A (Glu-Asn-Trp), Pt-B (Glu-Gln-Trp), ACH-11 [10–12]. These proteases affect many coagulation factors and cascades in the hemostatic system, such as cleavage of the alpha or beta chain of fibrinogen, inhibition of FXa, and activation of plasmin [13]. Most of the studied venomous snake venom polypeptides belong to serine proteases rather than metalloproteinases [14]. Metalloproteinases constitute an important components of the Agkistrodon genus. Metalloproteinase is an essential anticoagulant factor and exhibits fibrinogenolytic activity. However, there are few studies on the isolation and characterization of metalloproteinases from the venom of snakes belonging to the Agkistrodon genus. There is also paucity of information on the pharmacological effects of the isolated metalloproteinases. Metabolomics is an emerging discipline that compares the changes in body metabolites under different physiological and pathological conditions. In recent years, metabolomics has been widely used in drug research. It can be applied to study the mechanism of drug action by examining the changes of metabolic components in the body using serum, urine, and tissue fluid. It has been widely used in the study of monomeric compounds and traditional Chinese medicinal compounds. This study, to our best knowledge, is the first to report the anti-thrombotic effect mechanism of snake venom monomer using metabolomics

This work aimed to isolate and purify a novel metalloproteinase SP, determine its chemical properties and amino acid sequence, and investigate its pharmacological effects via in vivo studies. In order to study the mechanism of action of the novel snake venom protein, we investigated its antithrombotic effect using fibrinogen solubilization assay combined with UPLC-Q/TOF-MS-based non-targeted plasma metabolomics.

#### **2. Results**

#### *2.1. Isolation of Metalloproteinase SP from Agkistrodon Venom*

Based on the molecular weight differences of the snake venom proteins, an efficient separation and purification was achieved by the functional group ion exchange. The metalloproteinase SP snake venom monomer was then isolated and purified by sequential preparation, including ultracentrifugation, molecular exclusion chromatography on Sephadex TM G-75 (Figure 1A), anion exchange chromatography on DEAE-SphadexA-50 (Figure 1B), Sephadex TM G-75 (Figure 1C), and ultrafiltration.

#### *2.2. Protein Identification*

Based on LC-MS/MS analysis and database search analysis, it was found that three specific amino acid sequence fragments including SFGEWR, STEFQR, ENPPCILNKP were identified to belong to metalloproteinase SP (Figure 1D and Figure S1). Metalloproteinase SP consists of 202 amino acids with a molecular weight of 22.945 KDa and an isoelectric point of 5.78.

#### *2.3. Anti-Thrombotic Activity*

#### 2.3.1. Anti-Coagulant Activity

Compared to the control group, the coagulation time after metalloproteinase SP treatment was significantly prolonged (\*\*\* *p* < 0.001, Figure 2A,B). Anticoagulant activity of metalloproteinase SP was evaluated by the classical coagulation assays. The parameters including activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and Fibrinogen time (FT) were checked (Figure 2C–F). Metalloproteinase SP protein also significantly prolonged the clotting time, which inevitably influenced the changes in blood components and affect blood rheology (Tables 1 and 2).

**Figure 1.** Isolation of metalloproteinase SP from Agkistrodon venom. (**A**) Snake venom (1000 mg) was subjected to Sephadex TM G-75 chromatography (1 × 100 cm) by eluting with 5 mM Tris-HCl (pH 7.4). The fraction III contains the target molecular weight protein. (**B**) Fraction III was further subjected to separation on DEAE-SphadexA-50 column (2 × 150 cm), eluted with 5 mM Tris-HCl (pH 7.4) and a segmented concentration gradient of 0.2 M NaCl. The fraction I contain the target molecular weight protein. (**C**) Fraction I was then separated on Sephadex TM G-75 column (1 × 50 cm), with 5 mM Tris-HCl (pH 7.4). Fraction II of this separation contains the target molecular weight protein. M, marker of protein molecular weight. (**D**) The peptide sequence of targeted protein. Red colors indicated unique peptides identified by LC-MS/MS, matching to targeted protein.

**Table 1.** Hemodynamic examination of arterial blood in clopidogrel and metalloproteinase SP treated mice.

In the clopidogrel sulfate treated group, the platelet-larger cell ratio was decreased compared with the blank group. In the metalloproteinase SP group, the platelet count and plateletcrit were decreased compared to the blank group. All data were expressed as mean ± SD. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 compared with the blank control.

**Table 2.** Hemodynamic examination of venous blood in clopidogrel and metalloproteinase SP treated mice.

In the clopidogrel sulfate treated group, red blood cell count, hematocrit, and platelet-larger ratio cell were decreased compared with the blank group. In the metalloproteinase SP treated group, the red blood cell count, the number of platelets and plateletcrit were decreased. All data were expressed as mean ± SD. \* *p* < 0.05 and \*\* *p* < 0.01 compared with the blank group.

**Figure 2.** Anticoagulant activity of metalloproteinase SP was evaluated by coagulation time. (**A**) Blood coagulation time was determined by using the capillary technique. (**B**) Blood coagulation time was determined by the slide method. (**C**) Activated partial thromboplastin time (APTT). (**D**) Thrombin time (TT). (**E**) Prothrombin time (PT). (**F**) Fibrinogen time (FT). All data were expressed as mean ± SD, *n* = 10, \* *p* < 0.05 and \*\*\* *p* < 0.01, compared with the blank group.

#### 2.3.2. Anti-Thrombotic Activity

Compared with the blank group, the red blood cell and platelet content in the model group were significantly changed (\*\* *p* < 0.01), indicating the successful establishment of model (Table 3). Metalloproteinase SP significantly inhibited acute pulmonary embolism formation induced by ADP (Figure 3A,B). It also inhibited Carrageenan-induced tail thrombus (Figure 3C and Figure S2). CCK-8 assay indicated that metalloproteinase SP did not have cytotoxicity within concentration of 0.5 ug/mL (Figure S3). Then, metalloproteinase SP activity was evaluated by the classical coagulation assays, thus, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen time (FT) (Figure 4A–D).

**Table 3.** Hemodynamic examination of venous blood to check clopidogrel and metalloproteinase SP effects on acute pulmonary embolism mice.

In the clopidogrel sulfate treated group, red blood cell count and hematocrit were decreased compared with the blank group. In the metalloproteinase SP treated group, the red blood cell level, hematocrit, platelet count, and plateletcrit were decreased compared with blank. All data were expressed as mean ± SD. \* *p* < 0.05 and \*\* *p* < 0.01 compared with the blank control group.

**Figure 3.** Effects of metalloproteinase SP on thrombosis. (**A**) Metalloproteinase SP inhibited the tail thrombus induced by the Carrageenan. (**B**,**C**) Metalloproteinase SP inhibited ADP-induced acute pulmonary thrombosis in mice. ADP (250 mg/kg) was intravenously injected to induce acute pulmonary thrombosis. The time that the mice righting reflex recovery was recorded as the recovery time. The hematoxylin and eosin stained section of the lung tissue shows the one for the control mouse to be dominated by thrombi. Effect of metalloproteinase SP on the ADP-induced formation of acute pulmonary thromboembolism in mice. Compared with the control group, the thrombus of the metalloproteinase SP and the clopidogrel group exhibited significant difference. All data were expressed as mean ± SD, compared with the control group, \*\* *p* < 0.01, \*\*\* *p* < 0.001 and compared with the blank group ### *p* < 0.001.

#### 2.3.3. Antiplatelet Aggregation Activity

In vitro, metalloproteinase SP showed powerful antiplatelet activity by inhibiting the stimulatory effect of ADP, AA, and collagen (Figure 5A–C). In vivo, it exhibited a dose-dependent inhibition of the ADP-induced aggregation of PRP (Figure 5D–F). However, the inhibitory anti-platelet aggregation activity in vitro was more pronounced than in vivo.

#### *2.4. Fibrinogenolytic Activity*

In vivo, metalloproteinase SP protein cleaved α, β, and γ chains of fibrinogen, and decomposed the fibrinogen chain sequence in the order, α > β > γ. In addition to degrading the α, β, and γ chains of fibrinogen, metalloproteinase SP also degraded the primary and secondary decomposition products to produce stable small molecular peptides (Figure 6A). Fibrinogen was also used to evaluate the effects of different inhibitors on the activity of metalloproteinase SP. The specific serine protease inhibitors such as benzamidine, butyl boronic acid, trasylol, trypsin, and PMSF were not able to significantly inhibit its enzymatic activity. However, EDTA significantly inhibited its enzymatic activity to a statistically significant level (Figure 6B). Regarding ideal temperature conditions, the enzyme showed high activity at 30–60 ◦C (Figure 6C). Metalloproteinase SP showed higher enzymatic activity at neutral pH values, with optimum catalysis at pH 6–7 (Figure 6D).

**Figure 4.** Effects of metalloproteinase SP on acute pulmonary embolism coagulation. Compared with blank group, the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen time (FT) of the control group exhibited significant difference (\*\*\* *p* < 0.001). Compared with control group, the metalloproteinase SP group could prolong the APTT, PT, TT, FT. (**A**) Activated partial thromboplastin time (APTT). (**B**) Thrombin time (TT). (**C**) Prothrombin time (PT). (**D**) Fibrinogen time (FT). All data were expressed as mean ± SD, compared with the control group, \*\* *p* < 0.01 \*\*\*, *p* < 0.001, and compared with the blank group ### *p* < 0.01.

**Figure 5.** The in vitro and in vivo anti-platelet aggregation activity of metalloproteinase SP. (**A**) Compared with the blank group, the clopidogrel and the metalloproteinase SP groups specifically inhibited ADP-induced platelet aggregation. Metalloproteinase SP inhibited ADP-induced platelet aggregation with an inhibition rate of 44.28%. (**B**) Metalloproteinase SP inhibited AA-induced platelet aggregation with an inhibition rate of 13.19%. (**C**) Metalloproteinase SP inhibited collagen-induced platelet aggregation with an inhibition rate of 71.09%. The anti-platelet aggregation activity of metalloproteinase SP in vitro. (**D**,**E**) Metalloproteinase SP exhibited a dose-dependent inhibition of the ADP-induced aggregation of PRP. Compared with blank group, 0.5 μg of metalloproteinase SP significantly inhibited ADP-induced platelet aggregation with an inhibition rate of 18.27%. (**F**) Metalloproteinase SP not exhibited the promoted platelet aggregation activity. All data were expressed as mean ± SD, compared with the blank control group, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.01.

**Figure 6.** (**A**) Time-dependent effect of metalloproteinase SP on fibrinogen. Fibrinogenolytic activity was evaluated on 12% SDS-PAGE after incubation of metalloproteinase SP (5 μg) with human fibrinogen at 37 ◦C for different time periods. (**B**) Effects of inhibitors on metalloproteinase SP. Metalloproteinase SP was pre-incubated at 37 ◦C for 30 min at the presence of benzamidine (5 mM), ethylenediaminetetraacetic acid (EDTA, 5 mM), butyl boronic acid (20 mM), Trasylol (400 IU), trypsin inhibitor (20 mM), and PMSF (5 mM), respectively, prior to the addition of fibrinogen. (**C**) Temperature dependent effect of metalloproteinase SP on fibrinogen. Effect of temperature on fibrinogenolytic activity was evaluated on 12% SDS-PAGE after incubation of metalloproteinase SP (5 μg) with human fibrinogen at different temperature for 30 min. (**D**) The pH-dependent effect of metalloproteinase SP on fibrinogen. Effect of pH on fibrinogenolytic activity was evaluated on 12% SDS-PAGE after incubation of metalloproteinase SP (5 μg) with human fibrinogen at different pH for 30 min. All data were expressed as mean ± SD.

#### *2.5. E*ff*ects of Snake Venom Monomer on the Chemical Constituents of Acute Pulmonary Embolism*

In this study, the effect of metalloproteinase SP in an acute pulmonary embolism animal model was explored by analyzing metabolomic changes among metalloproteinase SP, model, and blank groups. As shown in the OPLS-DA score plot in both positive and negative ion modes, the three groups could be clearly distinguished (Figure 7A,C). This suggests that metalloproteinase SP exhibited a significant effect on acute pulmonary embolism by changing the anomalous metabolic states. Therefore, the metalloproteinase SP may be able to improve the pathological process of acute pulmonary embolism. The metabolites identified herein are listed in Table 4.

**Figure 7.** Score plot of plasma metabolomic profiling of three groups mice in positive ion mode (**A**) and negative ion mode (**C**). A total of 119 differential ions were screened by "adjusted *p*-value < 0.05, Fold change > 2" in positive ion mode (**B**). A total of 42 differential ions were screened by "adjusted *p*-value < 0.05, FC > 2" in negative ion mode (**D**).

The potential metabolic pathways and networks were analyzed by Metabo Analyst 4.0. Based on the Met PA analysis of Metabo Analyst 4.0, the related biomarkers primarily participated in the following pathways: tyrosine and tryptophan biosynthesis, phenylalanine metabolism, catecholamine biosynthesis, beta-alanine metabolism, aspartate metabolism, histidine metabolism, propanoate metabolism, pyrimidine metabolism, valine, leucine and isoleucine degradation, and tyrosine metabolism (Figure 8, Table 5).

**Table 5.** Metabolic pathways and differential metabolites.

**Figure 8.** (**A**) Relative signal intensities for metabolic biomarkers in the blood. (**B**) Summary of pathway analysis with Metabo Analyst 4.0. Each ordinate in the plot represents a metabolic pathway. The ordinate is the original *p*-value obtained from the pathway analysis, and the abscissa is the influence value of pathways obtained from the topological analysis. The ordinate color and size of each circle was based on *p*-value and pathway impact value, respectively.

#### **3. Discussion**

The snake venom contains a large number of active proteins, peptides and small molecular weight compounds, which show anticoagulant, procoagulant, antithrombotic, antiplatelet aggregation, Fibrinogenolytic activity, analgesic, and other activities [15–18]. In the past few decades, various snake venom proteins have been extensively studied, and many snake venom proteins have been purified and characterized. Some snake venom preparations have been widely used in clinical settings as hemostatics, anticoagulants, antithrombotics, and thrombolytics. Examples include Ancrod, Batroxobin, and Acutase [18,19].

In the present study, metalloproteinase SP was purified form *Agkistrodon acutus* venom through ultracentrifugation, molecular exclusion chromatography on Sephadex TM G-75, anion exchange chromatography on DEAE-SphadexA-50, Sephadex TM G-75, and ultrafiltration. The molecular weight of metalloproteinase SP was determined by LC-MS/MS to be 22.9 kDa [20]. Metalloproteinase SP was proven to show anticoagulant, antithrombotic, antiplatelet aggregation, and fibrinogenolytic activities, and improve blood rheology. In vivo and in vitro experiments showed that metalloproteinase SP could cleave α, β, and γ chains of fibrinogen and further reduce fibrinogen content.

Fibrinogen plays a vital role blood coagulation, blood rheology, platelet aggregation, and thrombosis [21,22]. The coagulation process is traditionally classified into extrinsic, intrinsic, and common coagulation. In the common pathway of coagulation, fibrinogen forms the insoluble fibrin network under the stimulation of coagulation factors such as thrombin and FXa, which ultimately constitutes a fibrin clot that stops bleeding [23,24]. Fibrinogen mainly affects platelet aggregation, blood rheology, fibrinogen clot structure, atherosclerosis, plasmin activation, and fibrinogen decomposition (FgDP) to adjust the formation of thrombus [24–28]. Hence, the centrality of fibrinogen in platelet aggregation cannot be overemphasized. Fibrinogen has multiple GPIIb/IIa receptor binding sequences, such as γ400~411, α95~97, and α572~574 [29]. Fibrinogen functions as a bridge of platelet aggregation [29–31]. Moreover, fibrinogen is the most important factor affecting plasma viscosity. Increased plasma fibrinogen levels inevitably result in elevated plasma viscosity and shear stress, as well as endothelial cells and platelets activation. Activation of endothelial cells further promotes the expression or activation of various adhesion molecules and integrins, which leads

to red blood cells adhesion and platelets aggregation and thrombosis [32–34]. Fibrin also possesses multiple plasminogen binding sites. Fibrinogen was cleaved by metalloproteinase SP to form fibrin, which may be through decomposing the α, β, and γ chain of fibrinogen, to reduce the content of the fibrinogen in the blood. Furthermore, it affected the blood coagulation pathway, prolonged the clotting time, directly inhibited the aggregation of platelets, indirectly regulated blood rheology, and indirectly regulated the formation of thrombus [35–38].

Based on the in vivo and in vitro experiments, the underlying mechanism of metalloproteinase SP on ADP-induced acute pulmonary embolism in mice was further investigated by untargeted mass spectrometry-based metabolomics profiling [39]. Metalloproteinase SP inhibited the formation of acute pulmonary embolism induced by ADP, which may be related to the amino acid metabolism and synthesis, including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, catecholamine biosynthesis, and eight other metabolic pathways [40,41].

Phenylalanine is one of the essential amino acids in humans that forms tyrosine under the action of phenylalanine hydroxylase in the liver [42,43]. Tyrosine synthesizes certain hormones and neurotransmitters such as dopamine (DA), norepinephrine (NE), epinephrine (E), and melanin in the nervous system and adrenal medulla. Thus, tyrosine plays an important role in regulating energy metabolism and scavenging free radicals [40]. Metalloproteinase SP regulated the biosynthesis of tyrosine and tryptophan, thereby alleviating lipid metabolism disorder and exert indirectly inhibited the formation of thrombus.

The content of catecholamines is closely related to blood coagulation and thrombosis [44]. Excessive levels of catecholamines could result in hypertension and acute myocardial infarction. Catecholamines act on platelet alpha (2)-adrenergic receptors to regulate platelet activation and aggregation, blood coagulation, and thrombosis.

The anticoagulant and antithrombotic activity of metalloproteinase SP may be ascribed to the regulation of histidine metabolism. Histidine is a semi-essential amino acid that is decarboxylated to form histamine under the action of histidine decarboxylase. Histamine exhibits diastolic vasoactive, antihypertensive and anti-inflammatory activities, and regulates a variety of allergic reactions. Histamine is clinically used for angina pectoris, cardiac insufficiency and other diseases [45]. Histamine has a strong vasodilator effect and is associated with a variety of allergic reactions and inflammation. Histidine may indirectly regulate inflammatory factors and inflammatory reaction by regulating the synthesis of histamine, thus inhibiting the formation of thrombus.

Metalloproteinase SP has many pharmacological activities, such as prolonging coagulation time, anti-platelet aggregation, inhibiting thrombosis, and decomposing fibrinogen. Metalloproteinase SP has high affinity with fibrinogen, and its anti-platelet aggregation and inhibition of thrombosis are closely related to its ability to decompose fibrinogen. Studies have shown that fibrinogen decomposition has an important relationship with the activation of fibrinolytic enzymes, and at the same time affects them. The experimental results showed that metalloproteinase SP can rapidly decompose fibrinogen and plays an anti-thrombotic role. However, fibrinogen content that is too low can easily lead to hemorrhage and coagulation disorders. At the same time, metalloproteinase has certain blood toxicity, which may lead to the reduction of platelet count and erythrocyte count. Thus, toxicity and safety of this venome component are needed to be further studied before its application in clinical.

#### **4. Materials and Methods**

#### *4.1. Materials*

SPF grade ICR mice (18–22 g) were purchased from the Comparative Medical Center of Yangzhou University (Jiangsu province, China, License No. SYXK (Su) 2018-0019). Sprague-Dawley rats (180–220 g) were purchased from Nanjing Qing long Shan Animal Center (Jiangsu province, China, License No. SYXK (su) 2018-0022). Animals were housed in a room of temperature of 22 ± 2 ◦C, relative humidity of 50 ± 5%, and exposed to 12 h dark/light cycle. The animals had free access to

food and drinking water, and were allowed to acclimatize to their new environment for 1 week. The experiments were carried out in accordance with the guidelines and the regulations of the Ethical Committee of the China Pharmaceutical University. The protocols were approved by the Institutional Animal Care and Use Committee of the China Pharmaceutical University. The ethics approval number was AUC-37 (20180329) and the approval date was 29 March 2018.

#### *4.2. Separation and Purification of Metalloproteinase SP*

A total of 1000 mg venom powder was dissolved in 5 mM Tris-HCl solution (pH 7.4), and then centrifuged at 12,000 r/min for 15 min at 4 ◦C. After centrifugation, the supernatant was collected and loaded on a gel filtration column of Sephadex TM G-75. The transparent supernatant was further analyzed by molecular exclusion chromatography with Sephadex TM G-75 column (1 × 100 cm, GE Healthcare, Frankfurt, Germany), which was pre-conditioned with 5 mM Tris-HCl (pH 7.4). Aliquots separated samples (4 mL/tube) were collected at a flow rate of 0.5 mL/min at 15 ◦C. The target component was chosen according to its profile on sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and was pooled, lyophilized, and then stored at −20 ◦C for subsequent chromatographic analysis. The target component from Sephadex TM G-75 was solubilized in 4 mL of 5 mM Tris-HCl (pH 7.4), and centrifuged at 12,000 r/min for 15 min at 4 ◦C. The supernatant was loaded on the anion exchange chromatography column (DEAE-SphadexA-50, 2 × 150 cm GE Healthcare, Germany) previously equilibrated and eluted with 5 mM Tris-HCl (pH 7.4). The target proteins were sequentially washed out with 5 mM Tris-HCl (pH 7.4), 0−0.2 M NaCl in 5 mM Tris-HCl (pH 7.4), and 0.2 M NaCl in 5 mM Tris-HCl (pH 7.4). The 4 mL/tube samples were collected at a flow rate of 0.5 mL/min at 15 ◦C. The target component was chosen based on its SDS-PAGE profile and was re-chromatographed after lyophilization. The clear supernatant was subjected to further purification via molecular exclusion chromatography using Sephadex TM G-75 (1 × 50 cm, GE Healthcare, Germany), previously equilibrated and eluted with 5 mM Tris-HCl (pH 7.4). Samples of 3 mL/tube were collected at a flow rate of 0.5 mL/min at 15 ◦C. The target component was chosen according to its profile on sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and was pooled, lyophilized, and stored at −20 ◦C for further chromatographic analysis.

#### *4.3. Protein Quantification and Identification*

The protein concentration was determined by the Bradford Protein Concentration Assay Kit (Thermo Fisher Scientific Incorporation, MA, USA), according to the manufacturer's instructions [46]. The purified venome protein was separated by SDS-PAGE and identified by LC-MS/MS and a Uniprot database search (https://www.uniprot.org/uniprot/?query=agkistrodon+acutus&sort=score).

#### *4.4. Pharmacological Evaluation of Metalloproteinase SP*

#### 4.4.1. Anti-Coagulant Activity

#### Blood Clotting Assays

Half an hour after metalloproteinase SP intravenous administration via the tail vein, blood samples were collected from the orbital plexus of mice. Afterward, the time for the blood coagulate was recorded. The clotting time was observed when microfibril was visible. The average time of each observation was 20 s. The collected blood samples were also spotted on glass slides and clotting times determined. Clotting was achieved when the fibrin mesh appeared and could be picked by glass dissecting needle [47,48].

#### Blood Parameters Test

Half an hour after clopidogrel sulfate and metalloproteinase SP administration via the tail vein, blood samples were collected from the orbital plexus of mice or from abdominal aorta of mice. Both

venous and arterial blood were anticoagulated by heparin sodium. The anticoagulant whole blood was placed in the automatic blood cell analyzer (RT-7600Vet, Rayto, Shenzhen, China) for detection and analysis within 2 h.

#### Coagulation Function Test

One hour after the intravenous administration of test drugs or vehicles, blood samples were harvested from the orbital plexus. An aliquot of 600 μL blood was gently mixed with 66 μL citrate sodium anticoagulant. The changes of blood corpuscles in whole blood (anticoagulated by citrate sodium) were analyzed using blood cell analyzer RT-7600Vet (Rayto, Shenzhen China). After centrifugation at 3000 rpm/min for 15 min, the plasma and blood cells were collected and analyzed for four coagulation parameters using the semiautomatic blood coagulation analyzer LG-PABER-I (STEELLEX, Beijing, China) [49–51]. These parameters were: activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and Fibrinogen time (FT). Standard analytical protocols were closely followed for all determinations.

#### 4.4.2. Anti-Thrombotic Activity

The anti-thrombotic effect of metalloproteinase SP was evaluated using two thrombosis pathological models [52,53].

#### ADP-Induced Acute Pulmonary Thrombosis in Mice

After metalloproteinase SP and clopidogrel treatment for 1 h, the ADP solution (250 mg/kg) was intravenously administered to induce acute pulmonary embolism, which could probably cause the paralysis or unpredicted death of mice. The recovery time of the mice was then recorded. After establishment of the model, the whole blood cell changes were assayed and coagulation parameters determined as earlier mentioned.

Half an hour after metalloproteinase SP intravenous administration via the tail vein, blood samples were collected from the orbital plexus of mice of mice. Both venous and arterial blood were anticoagulated by heparin sodium. The anticoagulant whole blood was placed in the automatic blood cell analyzer (RT-7600Vet, Rayto, Shenzhen, China) for detection and analysis within 2 h.

#### Carrageenan-Induced Tail Thrombosis in Mice

One hour after intravenous administration of metalloproteinase SP and aspirin in each group, Carrageenan solution (20 mg/kg) was intraperitoneally injected to induce tail thrombosis. The mice were then placed in environmentally-friendly cages for 12 h and their tail lengths recorded.

#### 4.4.3. Antiplatelet Aggregation Activity

#### In Vivo Antiplatelet Aggregation Assay

After 60 min of administration in each group, the rats were anaesthetized with 50 mg/kg chloral hydrate via abdominal injection. Blood samples were collected from abdominal aorta via cannulation. An aliquot of 8 mL blood was gently mixed with 880 μL citrate sodium anticoagulant (3.8%, *v*/*v*). Afterward, platelet-rich plasma (PRP) was obtained after centrifugation at 1000 rpm/min for 10 min. The residue was further centrifuged at 3000 rpm/min for 10 min to get platelet poor plasma (PPP). Prior to the antiplatelet aggregation assay, both PRP and PPP were incubated at 37 ◦C. An aliquot of 250 μL PPP was added into the measuring cup to calibrate the baseline. Thereafter, 225 μL of PRP was added and platelet aggregation stimulated with 25 μL different stimulants; thus, ADP (150 μM), AA (1 mg/mL), Collagen (50 μg/mL). Platelet aggregation was measured by aggregometer (AggRAM, Helena, USA), and the maximal aggregation rate was recorded within 5 min [54].

#### In Vitro Antiplatelet Aggregation Activity

The blank PRP plasma was harvested from control rats. Metalloproteinase SP (1 μg, 5 μg) was pre-incubated with 225 μL PRP at 37 ◦C for 5 min, and then stimulated with 25 μL different aggregating agents at the following final concentrations (ADP 15 μM). Platelet aggregation was assayed by a platelet aggregation instrument (AggRAM, Helena, TX, USA), and the maximum aggregation rate recorded in 5 min. 225 uL aliquot of PRP was measured into each cuvette and stimulated with metalloproteinase SP 25 uL (1 μg, 5 μg). Platelet aggregation was assayed by a platelet aggregation instrument, and the maximum aggregation rate recorded in 5 min [53].

#### 4.4.4. Fibrinogenolytic Activity

Human fibrinogen (8 mg/mL) was incubated with 5 μg metalloproteinase SP at 37 ◦C for 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h, 24 h, 48h, 72 h, and 96 h [53]. Afterward, the samples mixed with loading buffer solution were heated at 100 ◦C for 10 min. All samples were transferred to SDS-PAGE system for fibrinogen degradation analysis.

#### Effect of Temperature and pH on Metalloproteinase SP Activity

Human fibrinogen (8 mg/mL) was incubated with 5 μg metalloproteinase SP at 0 ◦C, 10 ◦C, 20 ◦C, 30 ◦C, 40 ◦C, 50 ◦C, and 60 ◦C for 30 min [55]. Additionally, 5 μg metalloproteinase SP was incubated with buffer solutions of different pH (pH 3, 4, 5, 6, 7, 8, 9, 10, 11) at 37 ◦C for 30 min, and subsequently incubated with human fibrinogen (8 mg/mL) at 37 ◦C for another 30 min. All samples were subjected to SDS-PAGE to investigate the effect of temperature and pH on metalloproteinase SP activity.

#### Effects of Serine Protease and Metalloproteinase Inhibitors on Metalloproteinase SP Activity

Different inhibitors, benzamidine (5 mM), ethylenediaminetetraacetic acid (EDTA, 5 mM), butyl boronic acid (20 mM), Trasylol (400 IU), trypsin inhibitor (20 mM), and PMSF (5 mM) were incubated with 5 μg metalloproteinase SP at 37 ◦C for 30 min [54]. The samples were further incubated with human fibrinogen at 37 ◦C for 30 min. All samples were analyzed by SDS-PAGE to evaluate the effect of inhibitors on metalloproteinase SP activity.

#### *4.5. E*ff*ects of Snake Venom Monomer on the Chemical Constituents of Acute Pulmonary Embolism Based on UHPLC-Q*/*TOF-MS Non-Targeted Metabolomics*

An aliquot of 150 μL of methanol/acetonitrile (3:1) solution (containing 0.4 μg/mL l-2-chloropheylaanine and 10 μg/mL ketoprofen as the internal standard for the ESI+ and ESI− modes respectively) was added to 50 μL plasma followed by vigorous vortex-mixing for 30 s. The mixture was centrifuged at 13,000 r/min at 4 ◦C for 10 min to precipitate protein. The supernatant (150 μL) was divided into two portions and dried under a gentle stream of nitrogen gas at room temperature. One of the samples was reconstituted with 75 μL of 50% aqueous acetonitrile for ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) analysis in positive mode while the other was for negative mode. The quality control (QC) samples were prepared to improve the data quality for metabolic profiling.

#### 4.5.1. UHPLC/Q-TOF MS Analysis

Chromatographic evaluations were performed with an Agilent 1290 series (Agilent Corp, Santa Clara, CA, USA) HPLC system equipped with a binary pump, degasser, an autosampler, and a temperature-controlled column compartment. Chromatographic separations were achieved on an UPLC BEH C18 column (1.8 μm, 2.1 mm × 100 mm, Waters, Ireland) [25]. Mobile phase A was 0.1% formic acid aqueous solution and B was 0.1% formic acid-acetonitrile solution for the positive ion mode. Mobile phase A was 10 mM ammonium acetate solution and B was 10 mM ammonium acetate acetonitrile solution for the negative ion mode. This mobile phase system was run in a gradient elution

program as follows: 90–99% A, at 0–1 min; 30–90% A, at 1–3 min; 15–30% A, at 3–8 min; 0–15% A, at 8–9 min; 0% A, at 9–10 min. The post-run time was 5 min. The oven temperature was set at 30 ◦C, the injection volume was 10 μL and the flow rate was 0.4 mL/min.

Separated components were detected by Agilent 6545A Q/TOF mass spectrometer (Agilent Corp, Santa Clara, CA, USA) equipped with an ESI interface. The operating parameters were as follows: drying N2 gas flow rate, 8 L/min; temperature, 320 ◦C; nebulizer, 35 psig; capillary, 3000 V; skimmer, 65 V; OCT RFV, 750 V; fragmentor 100 V. The reference masses 254.2806 (ketoprofen) and 299.75 (L-Phenylalanine) were used for internal mass calibration during the runs in the positive and negative ion modes. At the scan rate of 1.50 spectra/s using fixed collision energies (0.00, 10.00, 20.00, 40.00 V) MS/MS data were acquired with isolation width MS/MS medium (~4 amu).

#### 4.5.2. Data Processing and Identification of Differential Metabolites

LC-MS raw data files were converted to m/z Data format using DA reprocessor (Agilent Corp., Santa Clara, CA, USA). Peak finding, filtering, and alignment were subsequently carried out using open-source R-Package XCMS [25]. Metabo Analyst 4.0 (https://www.metaboanalyst.ca/) was then employed for the analysis of the data. The use of Metabo Analyst 4.0 can be broadly categorized into three major activities: (1) missing value processing, (2) data filtering, and (3) data normalization (i.e., sample normalization, data transformation, and data scaling) [55].

Using Metabo Analyst 4.0 unsupervised principal component analysis (PCA), a general picture of the relationship that exists among the data matrix was obtained. Thereafter, the supervised orthogonal partial least-squares discrimination analysis (OPLS-DA) was carried out to examine the metabolite differences between the treatment, blank, and model groups. The inclusion criterion of the metabolites was that, the fold change between the groups compared should be greater than 1.2 and *p*-value < 0.05 (one-way ANOVA).

Metabolites that met this criterion were considered as the differential metabolites. In the identification of the biomarkers, the following databases were used: METLIN (http://metlin.scripps. edu/), HMDB database (http://www.hmdb.ca/), Metabo Analyst4.0 (https://www.metaboanalyst.ca/), SMPDB (http://www.smpdb.ca/) and KEGG (http://www.genome.jp/kegg/) [55,56]

#### *4.6. Statistical Analysis*

Functional and enzymatic activities were assessed by two individual experiments in triplicate. All data were expressed as mean ± SD. The significance of differences was analyzed by one-way ANOVA followed by the Bonferroni correction. A value of *p* < 0.05 was considered statistically significant.

#### **5. Conclusions**

In this study, a novel metalloproteinase SP was isolated from Agkistrodon venom. In vivo, the metalloproteinase SP protein showed anticoagulant, antithrombotic, and antiplatelet aggregation activities and changed blood rheology. In vitro, metalloproteinase SP strongly inhibited platelet aggregation and cleaved the α, β, and γ chains of fibrinogen, but its activity was obviously affected by temperature or pH and could be reversed by EDTA. Finally, this study successfully built a mice model with acute pulmonary embolism to explore the antithrombotic mechanism of metalloproteinase SP. Assisted by LC-MS-Q-TOF-MS analysis, 14 representative biomarkers were identified, involving in phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, catecholamine biosynthesis, and eight other metabolic pathways. All these findings indicated that metalloproteinase SP may affect blood coagulation and thrombus formation by decomposing fibrinogen and anti-platelet aggregation

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/20/17/ 4088/s1.

**Author Contributions:** J.H., F.H., and X.Y. made the major contributions to this study in the conception, design, drafting part of manuscript, and final revision. H.F. performed statistical analysis of the data and purified protein. All authors read and approved the final manuscript.

**Funding:** The work was supported by the National Natural Science Foundation of China (No.<PHONE_NUMBER>A).

**Acknowledgments:** We thank Mr Yingchun Tan providing us snake venom. Thanks to the help and support provided by the National Platform for Natural Medicine Active Components and the National Key Laboratory of Pharmacodynamics and the Platform of Chinese Medicine College.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression**

## **René Huber 1,†, Rozan Attili/Abedalkhader 1,†,‡, Daniela Küper 1,2, Lara Hauke 1, Bernadette Lüns 1, Korbinian Brand 1, Karin Weissenborn 3 and Ralf Lichtinghagen 1,†,\***

Received: 7 March 2019; Accepted: 28 March 2019; Published: 30 March 2019

**Abstract:** Blood sampling with different anticoagulants alters matrix metalloproteinase (MMP-) 9 expression, thus influencing its concentration and diagnostic validity. Here, we aimed to evaluate the effects of different anticoagulants on MMP-9 regulation. MMP-9 expression was assessed in response to ethylenediaminetetraacetic acid, citrate, and high-/low-molecular-weight heparin (HMWH, LMWH) in co-culture experiments using THP-1, Jurkat, and HT cells (representing monocytes, T, and B cells). Triple and double cell line co-culture experiments revealed that HMWH treatment of THP-1 and Jurkat led to a significant MMP-9 induction, whereas other anticoagulants and cell type combinations had no effect. Supernatant of HMWH-treated Jurkat cells also induced MMP-9 in THP-1 suggesting monocytes as MMP-9 producers. HMWH-induced cytokine/chemokine secretion was assessed in co-culture supernatant, and the influence of cytokines/chemokines on MMP-9 production was analyzed. These experiments revealed that Jurkat-derived IL-16 and soluble intercellular adhesion molecule (sICAM-) 1 are able to induce MMP-9 and IL-8 production by THP-1. As a consequence, the increased MMP-9 expression found in HMWH blood samples may be influenced by HMWH-dependent secretion of IL-16 and sICAM-1 by T cells resulting in an increased production of MMP-9 and IL-8 by monocytes. IL-8, in turn, may support MMP-9 and its own expression in a positive autocrine feedback loop.

**Keywords:** blood sampling; anticoagulants; MMP-9; high-molecular-weight heparin; IL-16; sICAM-1; IL-8; monocytes; T cells

#### **1. Introduction**

Matrix metalloproteinases (MMPs) are a major group of endopeptidases degrading extracellular matrix (ECM) and basement membrane components [1] involved in the regulation of cellular events such as proliferation, differentiation, and migration [2]. In humans, 23 zinc-dependent MMPs have been identified [3]. Emerging evidence indicate an important role of these enzymes in normal and pathological processes including tissue remodeling, wound healing, inflammation, arthritis, cardiovascular diseases, cancer, and neurological diseases [3–5]. Of particular importance is MMP-9, also known as gelatinase B or type IV collagenase, since it has recently become a subject of growing interest in diverse human pathologies such as stroke. Stroke is regarded as the one of the leading causes of death in the world [6] and MMP-9 levels in the blood have been suggested to represent a suitable biomarker supporting its prognosis [7]. It was previously shown by our group and others that the way of blood sampling may affect subsequent analytical tests and that the kind of anticoagulant used, especially heparin, may alter the amount of MMP-9 (and other proteases or protease inhibitors) measured [8–11]. The molecular mechanism underlying this phenomenon, however, is still not well elucidated. Nevertheless, currently, MMP-9 assessment in blood is performed using heparin, ethylenediaminetetraacetic acid (EDTA), citrate, or serum samples [12]. The clinical chemist who has to evaluate a patient's blood MMP-9 concentration usually does not know about the patient's medical treatment, and an impact of concurrent treatment upon measured MMP-9 levels has not been considered in practice, so far.

High-molecular-weight heparin (HMWH) is a highly sulfated glycosaminoglycan. Due to its high negative charge density, HMWH prevents clotting and is widely used as an anticoagulant in heparin blood samples [13]. Low-molecular-weight heparin (LMWH), another class of anticoagulant derived from unfractionated (i.e., high-molecular-weight) heparin, has a number of advantages over HMWH, which has led to its increasing use for a number of thromboembolic indications [13,14]. The interaction of HMWH, LMWH, and other commonly used anticoagulants such as EDTA [15] and citrate [16] with human blood cells and their potential modulation of cellular processes are not well defined, neither in general nor in respect of MMP-9 expression [17].

The aim of this study was to characterize the influence of direct and indirect effects of different anticoagulants on the regulation of MMP-9 and to identify the molecular mechanism mediating these effects so as to assess the impact of the respective substances on the suitability of MMP-9 as a biomarker.

#### **2. Results**

#### *2.1. Direct Stimulation of Different Cell Types with Anticoagulants Has No Influence on MMP-9 Expression*

Stimulation of individual cell line cultures (THP-1, Jurkat, HT cells) with 3.2 mg/well EDTA (dissolved in medium), 220 μL/well citrate, or 50 international units (IU)/well HMWH revealed that MMP-9 expression and secretion are not influenced directly by any of these anticoagulants in monocytic cells (Figure 1a), T cells (Figure 1b), or B cells (Figure 1c) since no significant induction of MMP-9 mRNA could be shown by qPCR. This indicates that the reported MMP-9 induction in heparin blood sampling [8] is mediated via an indirect mechanism.

#### *2.2. Significant Induction of MMP-9 Expression by HMWH in A Co-Culture Including THP-1, Jurkat, and HT Cells*

The analysis of MMP-9 mRNA expression in a co-culture of THP-1, Jurkat, and HT cells demonstrated that MMP-9 expression increased significantly over time after addition of HMWH (approximately 7-fold after 24 h; Figure 2a). Equivalent results were obtained when the amounts of secreted MMP-9 protein were measured in the culture supernatant (approximately 3.5-fold induction after 24 h; Figure 2b). In contrast, stimulation with other anticoagulants such as EDTA or citrate (Figure 2a) had no MMP-9-inducing effect in this co-culture model. These results suggest that both MMP-9 mRNA and protein expression in one of the cell types used depends on an interaction with another cell type present in the mixture in response to HMWH, potentially by cell-to-cell contacts or indirectly via the stimulation with a secreted mediator.

**Figure 1.** No significant induction of MMP-9 expression by HMWH, EDTA, or citrate in individual cell line cultures of THP-1, Jurkat, and HT cells. 2 × 105 THP-1 (**a**), Jurkat (**b**), or HT cells (**c**) per well were starved overnight and then stimulated with 50 IU/well HMWH, 3.2 mg/well EDTA, or 220 μL/well citrate up to 24 h. MMP-9 mRNA expression was determined using qPCR (QuantiTect Custom Assay; housekeeping gene: GAPDH); mean ± SD, *n* = 3.

**Figure 2.** Induction of MMP-9 expression by HMWH in a THP-1, Jurkat, and HT cells containing co-culture. 7 × 105 THP-1, Jurkat, and HT cells per well each (i.e., a total of 2.1 × 106 cells/well) were starved overnight and then stimulated with 50 IU/well HMWH, 3.2 mg/well EDTA, or 220 μL/well citrate up to 24 h. MMP-9 mRNA (**a**) and protein (**b**) expression were determined using qPCR (QuantiTect Custom Assay; housekeeping gene: GAPDH) and ELISA (MMP-9 Quantikine Kit); mean ± SD, *n* = 3 (measured in duplicates). Kruskal–Wallis test, \* *p* ≤ 0.05; \*\* *p* ≤ 0.01.

#### *2.3. Significant Induction of MMP-9 Expression by HMWH in the THP-1 and Jurkat Co-Culture*

To determine whether the impact of HMWH on MMP-9 expression depends on an interplay of the three cell types used or a cooperation of two cell lines, MMP-9 expression was further assessed in co-culture approaches consisting of two cell types each. Thus, cell line mixtures including HMWH-stimulated monocytes and T cells, monocytes and B cells, as well as T and B cells were performed. In control approaches, the mixtures were alternatively treated with EDTA or citrate. As expected, no increase in MMP-9 mRNA expression was observed in the cultures of THP-1 and Jurkat, THP-1 and HT, as well as Jurkat and HT cells following control stimulation with EDTA or

citrate (data not shown). Moreover, there was also no significant induction of MMP-9 levels following HMWH stimulation in a mixture of THP-1 and HT cells or HT and Jurkat cells (Figure 3a). In contrast, HMWH stimulation of a combination of THP-1 and Jurkat cells led to a significantly increased MMP-9 mRNA expression over time (approximately 8-fold after 24 h; Figure 3a). These results could also be confirmed on the protein level (approximately 3-fold induction after 24 h; Figure 3b).

**Figure 3.** Induction of MMP-9 expression by HMWH in a THP-1 and Jurkat cells containing co-culture. 1 × 106 THP-1 and HT, HT and Jurkat, or THP-1 and Jurkat cells per well (i.e., a total of 2 × 106 cells/well) were starved overnight and then stimulated with 50 IU/well HMWH up to 24 h. MMP-9 mRNA (**a**) and protein (**b**) expression were determined using qPCR (QuantiTect Custom Assay; housekeeping gene: GAPDH) and ELISA (MMP-9 Quantikine Kit); mean ± SD, *n* = 3 (measured in duplicates). Kruskal–Wallis test, \* *p* ≤ 0.05.

#### *2.4. Significant Induction of MMP-9 Expression in THP-1 Cells in Response to Culture Supernatant Derived from HMWH-Treated Jurkat Cells*

In the next step, it was analyzed whether the increased MMP-9 production depends on cell-to-cell-contacts or is rather based on an indirect effect, e.g., via soluble mediator(s). Since monocytes/macrophages are able to produce large amounts of MMP-9 [18], we hypothesized that in this context, the T cells are responsible for the secretion of soluble factor(s) activating the MMP-9 production in monocytes. Therefore, the MMP-9 expression in THP-1 cells following incubation with culture supernatant derived from HMWH-stimulated Jurkat cells was analyzed. In the respective experiments, a significant induction of MMP-9 mRNA up to 24 h could be shown (approximately 8-fold after 24 h) whereas the supernatant of HMWH-treated HT cells had only a slight (though significant) effect on MMP-9 (up to 3-fold; Figure 4a). Stimulation of THP-1 cells with the supernatant of EDTA- or citrate treated Jurkat and HT cells did not increase MMP-9 mRNA expression considerably (data not shown). THP-1 cells were also incubated with the supernatant from Jurkat cells which have been incubated with human plasma derived from heparin-containing monovettes and MMP-9 was also significantly increased in this experimental setting, albeit to a lesser extent and characterized by a divergent temporal development (peaking at 4 h with approximately 5.5-fold induction; Figure 4b). These data support the suggestion that the monocytes are the main producers of MMP-9 and that factor(s) present in the supernatant of HMWH-stimulated T cells are able to significantly induce MMP-9 expression in monocytes.

**Figure 4.** Induction of MMP-9 expression in THP-1 cells by incubation with supernatant from HMWHand HMWH plasma-stimulated Jurkat cells. 2 × 106 THP-1 cells/well were starved overnight and subsequently stimulated up to 24 h with the supernatant of Jurkat or HT cells which have been treated for 24 h with 50 IU/well HMWH (**a**) or HMWH plasma (derived from a heparin monovette) (**b**). MMP-9 mRNA expression was determined using qPCR (QuantiTect Custom Assay; housekeeping gene: GAPDH); mean ± SD, *n* = 3 (measured in duplicates). Kruskal–Wallis test, \* *p* ≤ 0.05.

#### *2.5. High-Molecular-Weight Heparin Versus Low-Molecular-Weight Heparin*

To further investigate the stimulatory effect of other types of heparin on MMP-9 expression, the effect of two different types of LMWH (i.e., Clexane and Fragmin) was analyzed. As demonstrated in Figure 5, the stimulation of THP-1 and Jurkat with LMWH had no increasing effect on MMP-9 mRNA amounts, neither using Clexane nor Fragmin, in contrast to the significant approximately 10-fold effect shown by HMWH after 24 h. These results indicate that induction of MMP-9 expression also depends on the type of heparin used.

#### *2.6. Identification of Soluble Mediators Secreted in Response to HMWH*

The basic idea of the following experiment was to identify the (combination of) T cell-derived mediator(s) which is able to increase MMP-9 synthesis in monocytes. Therefore, all three individual cell lines as well as the relevant double (THP-1 and Jurkat) and triple (THP-1, Jurkat, and HT) cell line mixtures were cultivated and stimulated with HMWH. Cell culture supernatants were then profiled for the expression of multiple cytokines and chemokines. As shown in Table 1, HMWH-stimulated THP-1 released 3 types of mediators, macrophage migration inhibitory factor (MIF), interleukin 1 receptor antagonist (IL-1RA), and CC-chemokine ligand 5 (CCL5). Jurkat secreted IL-13, IL-16, MIF, sICAM-1, and Serpin E1, whereas HT cells produced IL-13, MIF, tumor necrosis factor (TNF), SICAM-1, and Serpin E1. In the co-cultures of HMWH-stimulated THP-1 and Jurkat or THP-1, Jurkat and HT cells, virtually all identified factors could be detected, additionally supplemented with IL-8 as a factor specifically occurring in cell line mixtures. This suggested that IL-16—as the only T cell-specific soluble

mediator present under all MMP-9-inducing conditions—might be involved (either individually or in a combination with other mediators) in the induction of MMP-9 secretion from monocytes.

**Figure 5.** No Induction of MMP-9 expression by LMWH in a THP-1 and Jurkat cells containing co-culture. 1 × 106 THP-1 and Jurkat cells (i.e., a total of 2 × 106 cells/well) were starved overnight. Afterwards, the co-culture was stimulated with HMWH (as a positive control), Clexane, or Fragmin (50 IU/well each) for 24 h. Then, MMP-9 mRNA expression was determined using qPCR (QuantiTect Custom Assay; housekeeping gene: GAPDH); mean ± SD, *n* = 3 (measured in duplicates). T test, \* *p* ≤ 0.05.

Interestingly, following stimulation with LMWH, no secreted cytokines/chemokines could be detected in co-cultures of THP-1 and Jurkat cells (data not shown) supporting the consideration that only HMWH (but not LMWH) is able to activate the production of MMP-9-inducing cytokine(s) from T cells.

#### *2.7. Regulation of MMP-9 Expression in THP-1 by Jurkat-Derived IL-16 and sICAM-1*

In the next step, THP-1 cells were stimulated with IL-16 to assess its influence on MMP-9 mRNA synthesis. Unexpectedly, this approach did not result in a significant upregulation of MMP-9, and equivalent negative results were obtained when THP-1 were stimulated with IL-13, MIF, or Serpin E1, i.e., the other factors found in the supernatant of HMWH-treated T cells. Only in the case of sICAM-1 a significant (but only approximately 3-fold) MMP-9 induction was observed (data not shown). This indicates that IL-16 alone is not the driver of MMP-9 production in monocytes and suggests that sICAM-1—potentially in combination with other secreted proteins—might contribute to the stimulatory effect on MMP-9 expression.

To reveal whether an interaction between two or more factors is responsible for this effect, (T cell-specific) IL-16, sICAM-1 (due to its mild significant effect on MMP-9) and (cell mixture-specific) IL-8 were applied in different combinations. A slight but significant effect on MMP-9 was observed following stimulation of THP-1 cells with IL-16 and sICAM-1 or IL-16 and IL-8 with about 3– to 4-fold induction levels after 24 h (Figure 6). Our results suggest that the T cell-derived mediators IL-16 and sICAM-1 are acting in combination with the presumably monocyte-derived chemokine IL-8 thus mediating an induction of MMP-9. To further confirm these results, monocytes were incubated with the triple combination of these mediators. In fact, a synergistic influence of IL-16, sICAM-1, and IL-8 was confirmed (7-fold, i.e., as strong as the positive control TNF; Figure 6).

**Figure 6.** Induction of MMP-9 expression by IL-16, sICAM-1, and IL-8 in THP-1 cells. 2 × 106 THP-1 cells/well were starved overnight. Afterwards, cells were stimulated with the mediators identified via Proteome Profiler Array: IL-16 and sICAM-1, IL-16 and IL-8, or IL-16, sICAM-1, and IL-8 for 24 h (5 ng/mL each). The dashed line indicates MMP-9 mRNA expression following TNF stimulation for 2 h as a positive control. MMP-9 mRNA expression was determined using qPCR (QuantiTect Custom Assay; housekeeping gene: GAPDH); mean ± SD, *n* = 3 (measured in duplicates). T test, \* *p* ≤ 0.05.

In contrast, the analysis of further cytokine combinations including MIF and IL-13 did not yield a significant increase in MMP-9 mRNA (data not shown) indicating that these proteins do not play an influential role in the regulation of MMP-9.

#### *2.8. Regulation of IL-8 Expression in THP-1 by IL-16 and Autocrine Activation*

To further assess whether the supporting chemokine IL-8 is induced by the same mediator cocktail as MMP-9, or whether other factors are involved, monocytic cells were stimulated individually or with combinations of IL-16, sICAM-1, and IL-8 and the resulting IL-8 mRNA expression was measured. Initially, stimulation of THP-1 with individual factors as demonstrated for IL-16 and IL-8 itself resulted in a significant increase in the IL-8 measurement (3- to 4-fold induction; Figure 7). Moreover, further trials using IL-16 and sICAM-1 or IL-16, sICAM-1, and IL-8 also enhanced IL-8 expression with about 4- to 6-fold induction levels (thus roughly reaching a dimension also observed using the positive control TNF; Figure 7). With respect to the effect of IL-16, sICAM-1, and IL-8 on MMP-9 expression shown above, the results presented here substantiate the assumption that the T cell-derived factors IL-16 and sICAM-1 provoke the secretion of monocyte-derived IL-8. This combination, in turn, is able to induce both a continuing IL-8 production by monocytes and an increased MMP-9 expression.

**Figure 7.** Induction of IL-8 expression by IL-16, sICAM-1, and IL-8 in THP-1 cells. 2 × 106 THP-1 cells/well were starved overnight. Afterwards, cells were stimulated with IL-16, IL-8, IL-16 and sICAM-1, or IL-16, sICAM-1, and IL-8 for 24 h (5 ng/mL each). The dashed line indicates IL-8 mRNA expression following TNF stimulation for 2 h as a positive control. IL-8 mRNA expression was determined using qPCR (housekeeping gene: GAPDH); mean ± SD, *n* = 3 (measured in duplicates). T test, \* *p* ≤ 0.05.

#### **3. Discussion**

Like other MMPs, MMP-9 is a member of the broad ECM remodeling network [19,20] and plays a crucial role in physiological processes such as proliferation, differentiation, and wound healing, but also in tumor progression, metastasis, and angiogenesis [2,21] due to its ability to efficiently degrade (amongst others) gelatin, collagens (IV, V, VII, X, XIV), elastin, aggrecan, and fibronectin [22]. Its structure comprises an N-terminal pro-domain, the catalytically active metalloproteinase domain (including three fibronectin type II repeats and a zinc binding motif), and a hemopexin domain [20]. MMP-9 is initially synthesized as a latent pro-enzyme (the zymogen) which is activated via the so-called cysteine switch, i.e., the proteolytic cleavage of the pro-peptide [23]. To avoid deleterious effects in normal tissue, the enzymatic activity of MMP-9 is negatively regulated by tissue inhibitor of metalloproteinases (TIMP-) 3 and (to a lesser extent) 1, either by association with pro-MMP-9 or the inhibition of mature MMP-9 [24]. It has been reported that MMP-9 production is induced by TNF, interferon (IFN-) α, IL-1β, and several growth factors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF)) [21]. Its mRNA expression is positively regulated via IκB kinase (IKK-), protein kinase C (PKC-), Ras-, or mitogen-activated protein kinases (MAPK; especially p38-, extracellular signal-regulated kinase (ERK-), and Jun N-terminal kinase (JNK-)) dependent signaling pathways, resulting in a nuclear factor κB (NF-κB-), specificity protein (Sp-) 1-, or activator protein (AP-) 1-driven gene expression. The repression of MMP-9 transcription is mainly Janus kinase (Jak)/signal transducer and activator of transcription (STAT-) dependent in response to IFN-β, IL-4, and IL-10 [21]. Consequently, MMP-9 expression is pharmacologically controlled using agents negatively affecting the MMP-9-regulating pathways or transcriptions factors, or both, thus especially reducing the activity of NF-κB or AP-1. For instance, MMP-9 expression can be significantly reduced via the suppression of NF-κB as demonstrated in breast cancer cells following the application of the isothiocyanate Sulforaphane [25] and in monocytic THP-1 cells treated with the labdane diterpenoid Andrographolide [26]. Consistently, MMP-9 can be induced in THP-1 cells using NF-κB-activating agents such as the synthetic triacylated lipopeptide Pam3CSK4 [27]. In a variety of other studies, the application of small molecule inhibitors for several kinases provoked a significant reduction of MMP-9 expression in different cell types. This has been demonstrated for inhibitors of PKC (e.g., RO318220, Calphostin C, Safingol), p38-MAPK (SB203580), MEK (PD098059), ERK (Rosiglitazone), and other signaling molecules (reviewed in [28]). Alternatively, signaling pathway activation can be prevented

via neutralizing antibodies either against activating mediators (such as TNF), the respective receptors such as EGFR, or involved signaling molecules (e.g., MEK, Ras, PKC) [28].

In addition to the approaches to address MMP-9 as a target in neoplastic, vascular, and other diseases [5,29], it is also regarded as a promising diagnostic or prognostic factor, e.g., in inflammatory bowel disease [30] or stroke [7], rendering MMP-9 an interesting potential biomarker [30]. In the framework of a recently published study [8], we found a considerable relation between induction of MMP-9 expression and different anticoagulants used. Moreover, this relation affected both MMP-9 protein baseline values and kinetics, which was clearly shown in heparin samples with or without proteinase inhibitors. Consequently, the way of blood sampling influences the measurement of MMP-9. This is also reflected by further studies also showing that blood sampling influences the concentration and the diagnostic validity of MMP-9 [31] and other circulating MMPs (1, 2, 3, 7, and 8) [32]. Therefore, we aimed to characterize the effects of common anticoagulants on MMP-9 expression and to identify the cell types contributing to this effect to assess the impact of these anticoagulants in clinical applications and the suitability of MMP-9 as a biomarker. Our initial analyses showed that direct stimulation of THP-1, Jurkat, and HT cells (representing the major leukocyte cell types in the blood, i.e., monocytes, T cells, or B cells) with the common anticoagulants EDTA, citrate, or HMWH had no effect on the amounts of MMP-9 mRNA or protein. This indicates that its expression in blood cells is not induced directly by anticoagulants, but rather influenced via an indirect mechanism, i.e., an interaction of two or more cell types in the blood.

Based on this consideration, we performed an experiment with a mixture of the three cell lines and incubated them with EDTA, citrate, or HMWH. A strong (approximately 7-fold and 3.5-fold, respectively) and significant induction of MMP-9 mRNA and secreted protein in the mixture was only observed in response to HMWH, whereas EDTA and citrate had no effect on MMP-9 levels. These results are in good agreement to the literature since it has been reported that stimulation with heparin increases MMP-9 and TIMP levels in leukocytes and platelets [10], whereas citrate and EDTA appeared to be less effective [8]. We concluded that the MMP-9 expression in at least one of the included cell types depends on an activating interaction with another involved cell type in response to HMWH, either directly by cell-to-cell contacts or indirectly via the stimulation with a secreted mediator. To address this question, MMP-9 levels were analyzed in co-culture approaches including two different cell types each, i.e., THP-1/Jurkat, THP-1/HT, and Jurkat/HT. In contrast to all other combinations, stimulation of THP-1 and Jurkat cells with HMWH led to a significant increase in MMP-9 mRNA expression indicating that the interplay between monocytes and T cells is involved in the upregulation of MMP-9 in heparinized blood samples. The results may also reflect the tendency of leukocytes to generate the members of the MMP family not ubiquitously, but in distinct gene expression patterns [33] which has also been predicted bioinformatically for other cell types, e.g., fibroblasts [19].

In the next step, we analyzed whether the increased MMP-9 production depends on direct or indirect effects. With respect to the known ability of monocytes and macrophages to produce large amounts of MMP-9, e.g., in the course of tissue invasion [18], while T cells are important producers of cytokines [34], it was reasonable to speculate that during the interaction of monocytes and T cells, the latter are responsible for the secretion of one or more soluble mediator(s) to which monocytes react with increased MMP-9 production. Therefore, we measured the MMP-9 expression in THP-1 cells in response to stimulation with culture supernatant derived from HMWH-stimulated Jurkat cells. Indeed, a significant MMP-9 induction was observed under these conditions. In order to elucidate whether it is further possible to increase MMP-9 production under clinical-like circumstances, THP-1 were also incubated with the supernatant from Jurkat cells which have been activated by human plasma derived from heparin-containing monovettes. Again, even under these conditions, the MMP-9 mRNA amounts were enhanced in THP-1 cells stimulated with the transferred supernatant. Together, these results support the suggestion that in response to HMWH, T cells release monocyte-activating factor(s) while monocytes stimulated in this manner are the relevant producers of MMP-9. This assumption

also fits to earlier reports demonstrating that monocytes are the main producers of MMP-9 among leukocytes [33].

To identify the T cell-derived mediator(s) involved in this process, cytokines and chemokines secreted by T cells in response to HMWH were monitored. These analyses revealed that HMWH-challenged Jurkat cells are characterized by the production of IL-13, IL-16, MIF, sICAM-1, and Serpin E1. Since most of these factors were also produced by THP-1 (MIF) or HT cells (IL-13, MIF, sICAM-1, and Serpin E1) in response to HMWH, IL-16 proved to be the only T cell-specific factor in this setting. Thus, IL-16, which is constitutively expressed but not secreted in T cells on the mRNA and pro-protein level [35], appeared to be a promising candidate cytokine, especially in consideration of other studies that have already shown that IL-16 is able to stimulate human monocytes, e.g., to increase the expression of pro-inflammatory cytokines [36]. A subsequent stimulation of THP-1 cells with IL-16, however, did not result in an enhanced MMP-9 expression, although its MMP-9 inducing capability has been demonstrated in vascular smooth muscle cells [37]. This suggested that further factors are required to achieve that effect. Among the mixtures of cytokines/chemokines then tested, only the combination consisting of IL-16, sICAM-1 (which showed a slight but significant effect on MMP-9 when individually applied), and IL-8 (which was the only mediator specifically expressed in co-culture experiments) exhibited a monocytic MMP-9 expression equivalent to the positive control (2 h TNF) and, more important, to that observed in the co-culture of THP-1 and Jurkat cells following HMWH stimulation. These circumstances indicated that T cell-derived IL-16 and sICAM-1, which had together a significant but relatively low effect on MMP-9, require the supporting presence of IL-8 to exploit their full activating potential on MMP-9. In this context, monocytes were the presumed source of IL-8, since they are known to produce large amounts of IL-8 in response to certain stimuli such as TNF [38]. Other factors, however, such as CCL5 or MIF appear not to be involved, presumably due to the absence of further stimulating agents, e.g., TNF or IL-1β, which have been reported to be necessary for their MMP-9-inducing activity [39,40]. To monitor whether IL-8 can be induced in monocytic cells by the same cocktail as MMP-9 (or at least one mediator included), THP-1 cells were stimulated individually or with combinations of IL-16, sICAM-1, and IL-8. Indeed, THP-1 cells expressed significantly increased amounts of IL-8 chemokine following a stimulation with IL-16 or IL-8 individually—thus confirming the previously reported autocrine regulation of IL-8 [41]—as well as combinations of IL-16 and sICAM-1 or IL-16, sICAM-1, and IL-8. In the latter cases, the IL-8 levels observed were roughly comparable to those found in the presence of the positive control TNF. Considered together with the result that IL-16, sICAM-1, and IL-8 are potent drivers of MMP-9 expression, our data suggest a model in which the presence of HMWH may lead to the secretion of T cell-derived IL-16 and sICAM-1 which induces monocytic IL-8 production. The combination of these factors appears to yield both a continuing IL-8 secretion as well as an enhanced MMP-9 production by monocytes (Figure 8). However, with respect to our experimental conditions, the participation of other cell types and further mediators could not be excluded and has to be addressed by future studies. It should also be mentioned that the mechanism presented here may also have an impact on the expression of further proteases thus potentially affecting their validity as biomarkers (such as MMP-2, -11, or -13 [42]).

**Figure 8.** Proposed model of MMP-9 induction in monocytes by HMWH-treated T cells. Our data suggest that in response to HMWH, T cells secrete the mediators IL-16 and sICAM-1, which induce monocytic IL-8 production. Together, these factors yield a continuing IL-8 secretion as well as an enhanced MMP-9 production by monocytes.

In some of our experiments, TNF was used as a positive control in the cytokine stimulation experiments since it remarkably increases MMP-9 [43] and IL-8 expression [38] by monocytes. Thus, it appears surprising that the presence of HT cells had no influence on MMP-9 levels in THP-1- and HT-containing co-cultures although they secrete TNF in response to HMWH (Table 1). This might be ascribed to the absence of TNF in the case of the triple cell line culture. Whether this absence is due to an altered expression time course, ineffectively low TNF levels (i.e., under the detection limit of the proteome profiler membrane), or a suppression of HT-derived TNF expression in this cell mixture, remains to be established. In the pure THP-1/HT culture, in which TNF expression could be detected, the relatively low TNF amounts produced by HMWH-challenged HT cells (as represented by only slightly stained THF-representing dots on the detection membrane; data not shown) might be insufficient to evoke MMP-9 expression in THP-1 cells over time.

In contrast to HMWH, we did not detect a significant cytokine/chemokine secretion or MMP-9 expression in co-cultured THP-1 and Jurkat cells in response to LMWH. Although the effects of HMWH and LMWH on cytokine/chemokine or growth factor expression appear to differ among different conditions and cell lines [44], thus providing (at least in part) contradictory results, it has been observed that HMWH enhances the expression of CCL5 in endometrial carcinoma cells [44]. In heparinized plasma samples, elevated levels of methyl-accepting chemotaxis protein (MCP), stem cell factor (SCF), PDGFβ, vascular endothelial growth factor (VEGF), CXC chemokine ligand (CXCL) 11, and Serpin E1 [45] or IL-6, -8, -17, granulocyte/macrophage-colony stimulating factor (GM-CSF), and macrophage inflammatory protein (MIP-) 1 in comparison to citrate or EDTA plasma, or both, were detected [46]. In contrast, LMWH has been reported to significantly reduce the expression of pro-inflammatory cytokines and chemokines in several cell types. For instance, LMWH application inhibited the secretion of IL-4 and TNF by mast cells [47] and the expression of IL-6 and -8 in epithelial cells [48] as well as IL-4, -5, -13, and TNF in T cells, effects that fit well to the anti-inflammatory features of LMWH [49]. The remarkable difference in cytokine induction between HMWH and LMWH described in the literature and observed in our study may be attributed to the molecular structure of the respective heparins. Natural heparin is exclusively produced by mast cells and predominantly

isolated on an industrial scale from porcine intestinal mucosa [50]. Both the unfractionated heparin (i.e., HMWH) and its fractionated derivative LMWH (produced by chemical or enzymatic processing of HMWH [50]) are glycosaminoglycans exhibiting a common molecular structure, i.e., repeating disaccharide units consisting of variably sulfated glucosamine, glucuronic acid, or iduronic acid [51]. Due to the general structural similarity, the anticoagulant mechanism is comparable: both heparins interact with antithrombin and suppress the activity of factor Xa and—to a lesser extent—thrombin [50]. However, in contrast to the long-chained HMWH with an average molecular weight of 16 kDa, LMWH is characterized by considerably shorter disaccharide chains resulting in an average molecular weight of 3.5–6 kDa [51], a reduced charge per chain, and less unspecific interactions with proteins [52]. In consequence, HMWH's length, charge, and tendency towards unspecific interactions may play a crucial role in the activation of T cells. For instance, an interaction with specific surface receptors is to be assumed, since it has been shown that unfractionated heparin is able to interact with the high mobility group box 1 protein receptor on macrophages [53] as well as G-protein-coupled receptor GPR56 on a variety of human cell lines [54]. Moreover, in the case of EDTA and citrate, the—in comparison to HMWH—negligible length and charge may be crucial for the absence of an MMP-9-inducing effect of these anticoagulants under the conditions tested in our study. Taking these points into account, one can speculate that receptor crosslinking may play a role during HMWH-induced activation of T cells, an effect which could not be provided by LMWH, EDTA, or citrate.

Our results also possess some clinical implications. For instance, HMWH-containing monovettes should be avoided when MMP-9 levels have to be assessed as a biomarker to circumvent its overvaluation. More important, heparin is also administered systemically during therapeutic approaches, e.g., for the treatment of venous thromboembolism (VTE) [13] or the prevention of myocardial infraction during the acute phase of unstable angina [55]. Known tissue-damage-associated adverse effects of heparin such as increased tendency towards bleeding [56] may be influenced or intensified by its MMP-9 promoting capacity. LMWH which according to our data does not appear to possess an influence on the expression of MMP-9 nor any cytokine/chemokine monitored may represent a preferable "gentle" alternative to HMWH. In another study, even a reduction of MMP-9 concentration and activity has been reported in the plasma of LMWH-treated patients with abdominal aortic aneurysm [57], an effect which might be connected to the LMWH-dependent downregulation of IL-8 as shown in enoxaparin-treated epithelial cells [48]. Accordingly, LMWH is increasingly used as an anticoagulant, e.g., for VTE prevention [58,59].

In summary, our study suggests that the increased MMP-9 expression previously found in HMWH blood samples may be ascribed to an HMWH-induced secretion of IL-16 and sICAM-1 by T cells resulting in an increased production of MMP-9 by monocytes, a process potentially supported by IL-8 in an autocrine feedback loop (Figure 8). This implies that HMWH shall be used cautiously in clinical applications.

#### **4. Materials and Methods**

#### *4.1. Collection and Storage of Human Heparin Plasma*

A total of three venous blood samples from healthy individuals were collected at the Department of Clinical Chemistry at Hannover Medical School. Informed consent was obtained from all donors before blood sampling. The experiments were carried out in accordance with the relevant guidelines and regulations and approved by the Hannover Medical School ethics committee in accordance with the Declaration of Helsinki (No. 388-2008, 02.12.2008). Blood sampling was performed using commercially available heparin- (i.e., HMWH) containing monovette tubes (Sarstedt, Nümbrecht, Germany). Blood samples were immediately centrifuged at 1900× *g* for 10 min and after removal of the supernatant, plasma samples were aliquoted and stored at −80 ◦C until assay.

#### *4.2. Cell Lines and Cell Culture Conditions*

For the analyses, THP-1 (human acute monocytic leukemia cell line; ACC-16), Jurkat (human T cell leukemia cell line, ACC-282), and HT cells (human B cell lymphoma cell line, ACC-567) were used (DSMZ, Braunschweig, Germany), representing monocytes, T cells, and B cells, respectively. All cell lines were cultured in RPMI 1640 medium containing 300 mg/L L-glutamine (PAA, Linz, Austria), supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Biochrom, Berlin, Germany) at 37 ◦C in a humidified atmosphere containing 5% CO2 [60].

Cell culture experiments were performed in 6-well plates (Sarstedt) with 2 mL medium/well. For stimulation experiments, cells were incubated without antibiotics. For starvation, the FCS concentration in the culture medium was reduced to 1% and cells were incubated overnight. Starved cells without further treatment/stimulation served as negative controls to avoid FCS-induced disturbances of the respective measurements.

#### *4.3. Cell Culture Experiments*

#### 4.3.1. Individual and Co-Culture Experiments

In the initial experiments, 2 × 106 THP-1, Jurkat, or HT cells/well were starved in individual cultures overnight. Afterwards, cells were stimulated with 3.2 mg EDTA (Sigma Aldrich, Darmstadt, Germany), 10 μL HMWH (=50 IU; Ratiopharm, Ulm, Germany) or LMWH (Clexane; Sanofi-Aventis, Frankfurt, Germany or Fragmin; Pfizer, Berlin, Germany), or 220 μL citrate (Sarstedt) per well and harvested after 0, 4, 6, and 24 h for the analysis of MMP-9 mRNA expression.

In double co-culture experiments, THP-1 and Jurkat, THP-1 and HT, or Jurkat and HT cells were cultivated together (in total 2 × 106 cells/well, i.e., 1 × 106 cells per cell line). Following starvation overnight, cells were stimulated with the respective anticoagulants and harvested at the indicated time points. In addition, supernatants of the respective double co-cultures were collected for the analysis of secreted proteins.

Furthermore, in a triple co-culture experiment a mixture of the three cell lines used was seeded with 2.1 × 106 cells/well (i.e., 0.7 × 106 cells per cell line) and starved overnight. Then, the respective anticoagulant was added and the cells were harvested and the supernatant collected at the indicated time points.

#### 4.3.2. Cell Supernatant Transfer Experiments

In a further series of experiments, starved monocytic THP-1 cells were stimulated with the supernatant of anticoagulant-treated Jurkat or HT cells and the MMP-9 expression was analysed as indicated. Alternatively, starved THP-1 cells were incubated with supernatant from starved Jurkat or HT cells which have been stimulated for 24 h with human plasma derived from blood samples collected in HMWH-containing monovettes.

#### 4.3.3. Cytokine/Chemokine Stimulation Experiments

Following a starvation phase overnight, 2 × 106 THP-1 cells/well were stimulated with 5 ng/mL MIF, 5 ng/mL IL-13, 5 ng/mL IL-16, 5 ng/mL sICAM-1, 25 ng/mL Serpin E1, or 5 ng/mL IL-8 (Peprotech, Hamburg, Germany), either individually or in combination, for 24 h. The respective concentrations were derived from the respective product sheet by selecting a low effective concentration and then validated by dose response experiments. As a positive control, starved THP-1 cells were stimulated for 2 h with 25 ng/mL human TNF (Sigma Aldrich). Following stimulation, cells were harvested and the MMP-9 or IL-8 mRNA expression was measured.

#### *4.4. RNA Extraction, cDNA Synthesis, and qPCR*

Cells were lysed and RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions [61]. RNA was eluted in 50 μL RNase-free water and concentrations were determined using the NanoDrop ND-1000 photometer at 260/280 nm (Peqlab, Erlangen, Germany). For first-strand complementary DNA (cDNA) synthesis, 1 μg total RNA per sample were reverse transcribed using the SuperScript™ II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA) and 250 ng random primers in a total volume of 100 μL as previously described [62]. Following reverse transcription, cDNA samples were immediately frozen and stored at −80 ◦C until further use. Quantitative polymerase chain reaction (qPCR) was performed using the LightCycler 480 II (Roche Diagnostics, Mannheim, Germany) as described in [8]. In short, MMP-9 analyses were based on the QuantiTect® Custom Assay (Qiagen; forward primer: 5 -TCCAGTACCGAGAGAAAG-3 , reverse primer: 5 -CAGGATGTCATAGGTCACGTAG-3 , hybridization probe: 5 -non-fluorescent quencher-GGAGTGAGTTGAACCAG-6-carboxyfluoroscein-3 ). Suitability of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, and β2-microglobulin as housekeeping genes was assessed in 5 randomly selected cDNA samples. In all cases, expression levels were roughly comparable among tested samples. GAPDH was finally selected as housekeeping gene since it exhibited the highest expression levels. GAPDH mRNA determination was performed using sequence-specific fluorescent resonance energy transfer probes (forward primer: 5 -TGCTGAGTATGTCGTGGAGTC-3 , reverse primer: 5 -GGATGCAGGGATGATGTTCT-3 ; donor hybridization probe: 5 -GACAACTTTGGTATCGTGGAAGGACTCATGACCACA-fluorescein thiocyanate-3 , acceptor hybridization probe: 5 -Cy5.5-CTGAGCGTGGCTATTCCTTCGTGACTACTG-phosphate-3 ). 5 μL cDNA were mixed with 4 pmol hybridization probes, 10 pmol forward and reverse primers, and 10 μL LightCycler 480 Probes Master (Roche). H2O was added to a final volume of 20 μL. PCR conditions were selected as follows: initial hot start incubation (95 ◦C, 5 min), denaturation (95 ◦C, 10 s), annealing (56 ◦C, 30 s for MMP-9; 59 ◦C, 30 s for GAPDH), and extension (72 ◦C, 30 s).

IL-8 mRNA expression was determined using the SYBR Select Master Mix (Invitrogen) and the LightCycler 480 (forward primer: 5 -TCCTGTTTCTGCAGCTCTGG-3 , reverse primer: 5 -GGCCACTCTCAATCACTCTC-3 ). An amount of 5 μL cDNA was mixed with 10 pmol forward and reverse primers, and 10 μL SYBR Select Master Mix. H2O was added to a final volume of 20 μL. PCR conditions were selected as follows: enzymatic degradation of contaminating uracil-containing DNA (50 ◦C, 2 min), initial hot start incubation (95 ◦C, 2 min), denaturation (95 ◦C, 10 s), annealing (59 ◦C, 15 s) extension (72 ◦C, 20 s) [38].

For the calculation of cDNA concentration, plasmid standards were generated and used in serial dilutions ranging from 1 mg/μL to 100 fg/μL as previously described [63].

#### *4.5. Enzyme-Linked Immunosorbent Assay*

Measurement of total secreted MMP-9 protein in the cell culture supernatant was performed using the Quantikine Kit (R&D Systems, Wiesbaden, Germany). Amounts of secreted total MMP-9 protein were assessed in thawed cell culture supernatant (stored at −150 ◦C until use) according to the manufacturer's instructions as described before [8]. The mean intra- and inter-assay coefficients of variation were given as 2.3% and 7.5%, respectively. The minimum detectable concentration was given as 0.156 μg/L.

#### *4.6. Proteome Profiler Array*

To identify secreted mediators, the Proteome Profiler Human XL Cytokine Array (based on the sandwich ELISA principle; R&D Systems) was carried out according to the manufacturer's instructions [64]. In short, an array membrane (consisting of capture antibodies spotted in duplicate on nitrocellulose) was incubated with 1.5 mL diluted supernatant (derived from HMWH-stimulated individual cell lines or co-cultures) and a cocktail of biotin-coupled capture antibodies (15 μL) overnight at 4 ◦C. Afterwards, the membrane was washed and incubated with 1.5 mL Streptavidin-HRP dilution for 30 min. Subsequently, membrane-bound cytokines/chemokines were detected using the chemiluminescent detection reagent and Amersham Hyperfilm ECL films (GE Healthcare, Freiburg, Germany).

#### *4.7. Statistical Analysis*

Data were described as means ± standard deviation (SD). The Kruskal–Wallis test was applied to analyze differences in mRNA or protein levels among several time points. The unpaired T test was used to analyze differences among mRNA levels following LMWH or cytokine stimulation. Analyses were performed using IBM SPSS Statistics, version 24 (IBM Deutschland GmbH, Ehningen, Germany).

**Author Contributions:** R.A., D.K., L.H., and B.L. performed the experiments. R.H., R.A., and R.L. wrote the paper. R.H. and R.L. conceived the study and designed the experiments. K.B. and K.W. contributed to the design of the study. R.H., R.A., D.K., L.H., B.L., K.B., K.W., and R.L. analyzed the results and approved the final version of the manuscript.

**Funding:** This research was funded by the *Deutsche Vereinte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin* (DGKL; Stiftung für Pathobiochemie und Molekulare Diagnostik; grant number 2015/16 to RA and RL) and the *Doktor Robert Pfleger Stiftung Bamberg* (grant number RDR/chu to RH and RL).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Biomarker Analysis of Orally Dosed, Dual Active, Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitor, AQU-118, in the Spinal Nerve Ligation (SNL) Rat Model of Neuropathic Pain**

**Mei Yee Kwan 1, Anthony Choo 1, Taleen Hanania 1, Afshin Ghavami 1, Jose Beltran 1, John Shea 1, Amidi Barboza 1, Andrew Hu 1, Marcie Fowler 2, Venugopal Rao Neelagiri 3 and Irving Sucholeiki 3,\***

Received: 23 December 2018; Accepted: 11 February 2019; Published: 14 February 2019

**Abstract:** There is an unmet medical need for the development of non-addicting pain therapeutics with enhanced efficacy and tolerability. The current study examined the effects of AQU-118, an orally active inhibitor of metalloproteinase-2 (MMP-2) and MMP-9, in the spinal nerve ligation (SNL) rat model of neuropathic pain. Mechanical allodynia and the levels of various biomarkers were examined within the dorsal root ganglion (DRG) before and after oral dosing with AQU-118. The rats that received the SNL surgery exhibited significant mechanical allodynia as compared to sham controls. Animals received either vehicle, positive control (gabapentin), or AQU-118. After SNL surgery, the dorsal root ganglion (DRG) of those rats dosed with vehicle had elevated messenger RNA (mRNA) expression levels for MMP-2, IL1-β & IL-6 and elevated protein levels for caspase-3 while exhibiting decreased protein levels for myelin basic protein (MBP) & active IL-β as compared to sham controls. Rats orally dosed with AQU-118 exhibited significantly reduced mechanical allodynia and decreased levels of caspase-3 in the DRG as compared to vehicle controls. Results demonstrate that oral dosing with the dual active, MMP-2/-9 inhibitor, AQU-118, attenuated mechanical allodynia while at the same time significantly reduced the levels of caspase-3 in the DRG.

**Keywords:** matrix metalloproteinase; MMP-2; MMP-9; inhibitor; allodynia; caspase-3; neuropathic; pain; dorsal root ganglion; spinal nerve ligation

#### **1. Introduction**

The population suffering from neuropathic pain is large; neuropathic pain is a significant problem for patients and the healthcare system. This type of pain is difficult to treat effectively with currently available pharmacological agents, in part due to the fact that the aetiologies that drive the neuropathic pain states are varied, complex, and not well-understood. Indeed, many of the currently approved neuropathic pain medications, and in particular anticonvulsants such as gabapentin and pregabalin, do not have a direct effect on the pathophysiology of the disorder but rather modulate neurotransmission [1]. Since only one in four patients with neuropathic pain experiences greater than

a 50% reduction in pain following treatment, novel therapeutic approaches are an urgent priority [2]. One tactic for treating neuropathic pain is through the inhibition of specific protein targets involved in the initiation or maintenance of pain sensitization within the spinal cord.

Surgically-induced rodent models of neuropathic pain cause the levels of various inflammatory proteins such as cytokines and, in particular, certain matrix metalloproteinases (MMPs), to become elevated within the spinal nerves [3–9]. MMPs are zinc-dependent endopeptidases belonging to a larger family of proteases known as the metzincin superfamily. MMPs are involved in degrading the extracellular matrix as well as processing of growth factors, cytokines, chemokines, adhesion molecules, and a variety of other enzymes. Increased expression of MMPs or an imbalance of different MMPs has been observed in a variety of pathological conditions, including various neurodegenerative diseases [10,11].

It has been postulated that in injured dorsal root ganglion (DRG) primary sensory neurons, MMP-9 can induce early neuropathic pain via interleukin-1β cleavage and microglia activation, and MMP-2 induces delayed neuropathic pain via IL-1β cleavage and astrocyte activation [12]. MMP-9 also promotes Schwann cell-mediated myelin basic protein (MBP) degradation and macrophage infiltration in the spinal nerve and activation of astrocytes in the spinal cord [13]. Based upon this evidence, we sought to develop an orally active MMP-2 and MMP-9 inhibitor to treat neuropathic pain. Our first compound in this arena is AQU-118, a potent small molecule inhibitor of both MMP-2 and MMP-9 with an in vitro IC50 of 3 nM and 9 nM, respectively [14]. AQU-118 attenuates neuropathic pain behaviors in the SNL and chronic constriction injury of the infraorbital nerve (CCI-IoN) rodent models as well as in the naloxone precipitated morphine withdrawal rodent model [14,15]. While these models demonstrated the analgesic efficacy of AQU-118 in neuropathic pain, they did not address the mechanistic or biochemical correlates associated with this activity. The present study analyzes the effects of AQU-118 on mechanical allodynia in the SNL-rat model and then identifies underlying molecular effects of oral dosing with AQU-118 on various inflammatory biomarkers associated with neuropathic pain.

Although many biomarkers are associated with neuropathic pain, we focused on those whose expression and/or protein levels have been reported to change within the dorsal root ganglion (DRG) or dorsal horn following spinal nerve injuries [12,13,16–18]. We examined transcript expression levels of MMP-2, MMP-9, IL-1β, IL-6 and protein levels of MBP (20 kDa and 15 kDa molecular weight bands), the pro and active forms of IL-1β, IL-6 and caspase-3 from rat DRG samples. We first determined if any protein or mRNA expression changes were related to the spinal nerve ligation (SNL) surgery by comparing DRG samples from rats who received the SNL with control animals that underwent a sham surgery. Any biomarker(s) that were observed to significantly change in the DRG after SNL-surgery were then examined further to determine if they would return to pre-SNL levels (i.e., sham) upon oral dosing with AQU-118 and its corresponding reduction in mechanical allodynia. The ultimate goals were to better understand the mechanism of action for the antiallodynic effects of AQU-118 and to find biomarkers that could be used in a clinical setting to help monitor the effectiveness of AQU-118. This study presents for the first time biomarker data for an orally administered, dual active MMP-2/MMP-9 inhibitor in the SNL rat model for neuropathic pain. These results further validate the oral effectiveness of AQU-118 and support its continued preclinical development and future clinical testing as a novel treatment for general neuropathic pain.

#### **2. Results**

#### *2.1. Attenuation of von Frey Mechanical Allodynia by Oral Administration of AQU-118 in the Spinal Nerve Ligation (SNL)-Rat Model*

Due to the need for sufficient spinal tissue (i.e., DRG) to perform both the gene expression and protein biomarker work, the SNL-rat model studies were designed to incorporate higher numbers of rodents among certain groups than would typically be needed for measuring only mechanical allodynia. Except for the positive control (*n* = 10, gabapentin, 100 mg/kg, 0.58 mmoles/kg), a larger number of rodents were used for the sham (*n* = 20), vehicle (0.5% methyl cellulose, *n* = 40) and drug groups (*n* = 20, AQU-118, 160 mg/kg, 0.34 mmoles/kg) (Table 1). Two weeks after surgery, rats with L-5 spinal nerve ligation displayed significant mechanical allodynia as compared to pre-operative testing (Figure 1). Oral dosing of AQU-118 beginning on day 1 caused an increase in the paw withdrawal threshold (PWT) as compared to the vehicle control group (Figure 1). No statistically significant effect on contralateral PWT was observed with oral dosing of AQU-118 which was comparable to both the vehicle and positive control (gabapentin) arms (Figure 2).

**Table 1.** Protocol for SNL study using male Sprague-Dawley rats.

1 Once per day dosing via gastric gavage (P.O.), 60 min before von-Frey testing. 2 NA not applicable. 3 0.5% methyl cellulose at a dose volume equivalent to the test compound (5 mL/kg). 4 Gabapentin was dissolved in saline and administered 60 min prior to von-Frey testing. 5 A base-line measure was taken via von-Frey before surgery and then two weeks after surgery (Day 0). The animals were then balanced and assigned to treatment groups based on their post-operative PWT values. Animals with a VF score over 4.5 g were excluded from the study.

**Figure 1.** Mechanical Response Threshold: Spinal Nerve Ligation (SNL) Chung Model (Chung), Ipsilateral (Ipsi) Paw Withdrawal. Paw withdrawal thresholds following SNL-surgery for ipsilateral hind paws. Data are presented as mean ± SEM. \* *p* < 0.05 vs. (SNL) Chung + Vehicle group on the same day. Post-Surgery Baseline was performed 15 days after SNL-surgery.