*Article* **The Diagnostic Usefulness of Circulating Profile of Extracellular Matrix Components: Sulfated Glycosaminoglycans (sGAG), Hyaluronan (HA) and Extracellular Part of Syndecan-1 (sCD138) in Patients with Crohn's Disease and Ulcerative Colitis**

**Alicja Derkacz 1 , Paweł Olczyk 2 , Agnieszka Jura-Półtorak 1 , Krystyna Olczyk 1 and Katarzyna Komosinska-Vassev 1, \***

**Citation:** Derkacz, A.; Olczyk, P.; Jura-Półtorak, A.; Olczyk, K.; Komosinska-Vassev, K. The Diagnostic Usefulness of Circulating Profile of Extracellular Matrix Components: Sulfated Glycosaminoglycans (sGAG), Hyaluronan (HA) and Extracellular Part of Syndecan-1 (sCD138) in Patients with Crohn's Disease and Ulcerative Colitis. *J. Clin. Med.* **2021**, *10*, 1722. https://doi.org/10.3390/ jcm10081722

Academic Editors: Konstantinos Papamichael and Emmanuel Andrès

Received: 22 March 2021 Accepted: 14 April 2021 Published: 16 April 2021

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**Abstract:** The described research focused on the diagnostic usefulness of sulfated glycosaminoglycans (sGAG), hyaluronan (HA), and extracellular part of syndecan-1 (sCD138) as new markers related to extracellular matrix (ECM) remodeling in the intestine during the two most common forms of inflammatory bowel diseases (IBD), i.e., ulcerative colitis (UC) and Crohn' disease (CD). Inflammatory markers belonging to ECM components were assessed in serum of patients with IBD using an immunoenzymatic method (HA and sCD138) and a method based on the reaction with dimethylmethylene blue (sulfated GAG). Measurements were carried out twice: at baseline and after one year of therapy with prednisone (patients with CD) or adalimumab (patients with UC). No quantitative changes were observed in serum sGAG, HA, and sCD138 concentrations between patients newly diagnosed with CD and the healthy group. In the case of patients with UC, the parameter which significantly differentiated healthy subjects and patients with IBD before biological therapy was HA. Significant correlation between serum HA level and inflammation activity, expressed as Mayo score, was also observed in patients with UC. Moreover, the obtained results have confirmed that steroid therapy with prednisone significantly influenced the circulating profile of all examined ECM components (sGAG, HA, and sCD138), whereas adalimumab therapy in patients with UC led to a significant change in only circulating sGAG levels. Moreover, the significant differences in serum HA levels between patients with UC and CD indicate that quantification of circulating HA may be useful in the differential diagnosis of CD and UC.

**Keywords:** inflammatory bowel diseases (IBD); Crohn's disease (CD); ulcerative colitis (UC); sulfated glycosaminoglycans (sGAG); hyaluronan (HA); soluble part of syndecan-1 (sCD138)

#### **1. Introduction**

In the last decades, global, dynamic growth in the frequency of chronic inflammatory bowel diseases (IBD) has been observed, further highlighting the role of environmental factors in this disease. Despite significant advances in treatment and its increased availability, the diseases still remain a challenge for modern medicine [1]. Among the inflammatory bowel diseases, the two most significant ones from a clinical standpoint are Crohn's disease (CD) and ulcerative colitis (UC). This group of diseases was most likely already recognized in ancient times, which is implied by the preserved descriptions. Thus, it can be assumed that their basis is not determined solely by the characteristics of modern lifestyle and environmental factors [2]. IBD etiology and pathogenesis is not yet completely understood, which leads to a lack of accurate methods for causal treatment. Difficulties in identifying

the factors responsible for provoking and aggravating inflammatory bowel diseases are the result of, among others, their varied genetical basis, multiple environmental factors, including stress and bacterial and viral infections, which often overlap on clinical images [3]. Furthermore, diagnosis IBD is a significant challenge, and the diagnostic process requires a joint analysis of data from medical history, physical examination, and laboratory tests. A key role in diagnosis is played by endoscopic examination along with mucous membrane biopsy followed by a histologic examination of the obtained sample [4,5]. In clinical practice, there is still a lack of non-invasive, reliable, and objective methods supporting diagnostic methods that allow differentiation, assessment of disease activity, and monitoring of treatment and prognosis of the disease course. Among them, biochemical markers related with the extracellular matrix (ECM) represent promising candidates that can support CD and UC diagnostics. Quantitative changes in the composition and structure of ECM components has been described as playing a significant role in the pathogenesis of inflammatory bowel diseases. Thus, determining the concentration of these macromolecules in body fluids, i.e., serum or urine, is finding increasingly more applications during clinical diagnostics, often being a marker of disease activity and correlating with the severity of the disease process. Hence, establishing the relationship between the level of the circulating matrix component and the progression of IBD might be an argument for the implementation of its determination in the assessment of the activity and progression of IBD [6–10].

Among the new markers related to the ECM remodeling in the intestine proteoglycans (PGs) and their sugar components, glycosaminoglycans (GAGs) are of particular interest due to their role in IBD progression. Sulfated GAGs, including chondroitin/dermatan sulfates, heparan sulfates, and keratan sulfates, integrate the structure of the extracellular matrix by interacting with its components [11]. They are also a reservoir for cytokines and growth factors, take part in regulating the progression of inflammatory processes, accelerate wound healing, and take part in morphogenesis [12,13]. The anticoagulative properties of heparan sulfate and heparin are a result of their ability to bind platelet factors and activate lipoprotein lipase. Due to these properties, GAGs play a significant role in the progression of immunological and inflammatory processes, including intestinal inflammation during IBD [10,13]. Apart from GAGs, the process of extracellular matrix remodeling throughout IBD also involves hyaluronan (HA), a non-sulfated glycosaminoglycan which actively participates in wound healing, proliferation, and migration of cells and modulation of the inflammatory process. Due to its ability to bind water within a polysaccharide net, it provides adequate hydration to the matrix, which allows, e.g., cell migration and substance diffusion [14]. During inflammatory conditions such as IBD, HA polymers are cleaved into fragments of low molecular weight that induce signaling of inflammatory responses via specific receptors. Moreover, during IBD, HA also forms complexes with trypsin interα-inhibitor, which are highly adhesive towards leukocytes and additionally exacerbate the inflammation [15].

Another significant component of the extracellular matrix, which influences the progression of inflammation processes including those associated with IBD, is syndecans–PGs belonging to the family of type 1 transmembrane heparan sulfate proteoglycans. The biological functions of syndecans, related to their structure, include cell adhesion, activation and binding of growth factors, and interaction with many ligands, including fibroblast growth factor, vascular endothelium growth factor, fibronectin, and antitrombin-1 [16,17]. Four syndecans have been recognized on the basis of their structure and origin: 1, 2, 3, and 4 [17]. The most interesting one in the context of IBD pathogenesis is syndecan-1 (CD138). It is expressed at high rates in vascular endothelium and circulatory cells. It can also be found in immature B cells, where it is regulated by IL-6 and bacterial lipopolysaccharides (LPS). Syndecan-1, as a co-receptor of multiple extracellular ligands including proinflammatory cytokines and growth factors, plays a significant role in inflammatory processes [17,18]. An extracellular part of syndecan-1 (sCD138) is proteolytically released from the cell surface. This process is activated in response to inflammation and pathogen infection [17].

It is therefore clearly implied that the presence of the extracellular domain of syndecan-1 in blood could be a precious marker of the activity of inflammatory processes [19–21]. For this reason, the aim of this article was to evaluate the diagnostic suitability of the serum profile of extracellular matrix components such as sulfated glycosaminoglycans (sGAG), hyaluronan (HA), and soluble syndecan-1 (sCD138) fragments in patients with inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis.

#### **2. Materials and Methods**

#### *2.1. Study Population*

The investigated biological material consisted of venous blood samples obtained from 87 subjects including 40 healthy individuals and 47 patients with inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis. The diagnosis was made on the basis of clinical symptoms, laboratory test and, in particular, the results of colonoscopy. Additionally, in the case of UC patients, assessment with the Mayo score was used.

#### *2.2. Patients with Ulcerative Colitis (UC)–Inclusion and Exclusion Criteria*

UC patients enrolled in this study included a group of 31 patients selected for a clinical research program involving biological treatment with adalimumab (Humira, Abbott GmbH, Wiesbaden, Federal Republic of Germany). Adalimumab bonds specifically with tumor necrosis factor (TNF), restricting its activity by blocking it from binding with p55 and p75 TNF receptors on the cell surface. This antibody also influences the biological response to TNF and impacts the concentrations of intercellular adhesion molecules responsible for leukocyte migration (ELAM-1, VCAM-1, and ICAM-1). In the case of patients with UC, the inclusion criteria included age between 18 and 75 during the control visit, diagnosed ulcerative colitis, diagnosed active form of ulcerative colitis confirmed through colonoscopy with biopsy, active form of ulcerative colitis confirmed through Patient Global Assessment (PGA) 2 or 3, and treatment with corticosteroids or immunosuppressive medicine (azathioprine (Imuran, Aspen Pharma Trading Ltd, Dublin, Ireland)) or 6-mercaptopurine (Mercaptopurinum VIS, Zakłady Chemiczno-Farmaceutyczne "VIS"®Spółka z o.o., Bytom, Poland)) which had not brought the intended results according to the practitioner's opinion.

The criteria for exclusion, in the case of patients with UC, were colectomy with ileorectostomy or colectomy with anastomosis, Kock pouch or ileostomy undertaken in the course or inflammatory bowel disease or before a planned bowel surgery, adalimumab treatment or former participation in clinical research involving adalimumab, exposure to infliximab or any other TNF inhibitor within 56 days from week 0, taking ciclosporin, tacrolimus, or mycophenolate mofetil within 30 days from week 0, intravenous corticosteroids within 14 days from the screening or throughout the screening, diagnosed fulminant colitis and/or toxic colitis, and taking any biological medicine which could potentially influence the disease under investigation. Adalimumab doses delivered to patients with UC were varied, and the patients were randomly assigned to one of two groups: the first received 160 mg of adalimumab in the initial dose, with a reduction to 80 mg and then to 40 mg, and there was a need for a faster response to treatment in these patients; the second group received 160 mg in the initial dose, with a reduction to 40 mg. The mean time from diagnosis was one year. Moreover, in patients with ulcerative colitis who were using prednisone (Encorton, Pabianickie Zakłady Farmaceutyczne Polfa S.A., Pabianice, Poland) at the time of qualifying for biological treatment after introducing biological drugs, the dose of prednisone was gradually reduced. The cigarette smoking status has not been established in patients with UC enrolled in this study.

#### *2.3. Patients with Crohn's Disease (CD)—Inclusion and Exclusion Criteria*

The next group of patients with IBD in the investigation included 16 people with CD, treated mainly with prednisone. The dosage of prednisolone was chosen on a case-by-case basis, depending on the response to treatment. Blood samples were obtained from patients at the point of diagnosis and again after a year of treatment. The following inclusion

criteria have been adapted for the volunteers: age above 18 and diagnosed with Crohn's disease (CD). The exclusion criteria were age under 18, diagnosed indeterminate colitis (IC), alcoholism, alcohol-related liver disease or any ongoing liver disease, acute infections of viral, fungal, or bacterial origin, mild or acute myocardial inefficiency, unstable coronary artery disease, chronic respiratory failure, chronic kidney disease, chronic liver disease, demyelinating disease, diabetes, pregnancy or breastfeeding, and diagnosed precancerous condition or cancerous comorbidities. The patients had been ill for twelve months with a period of activity and remission and did not take any additional medications during therapy. The cigarette smoking status has not been established in patients with CD enrolled in this study. None of the patients had a prior ileocolonic resection for CD and perianal fistulizing CD.

All patients were treated at the Department of Gastroenterology of St. Barbara's Regional Specialist Hospital in Sosnowiec. Reference number of the decision by the Bioethics Committee at the Medical University of Silesia in Katowice: KNW/0022/KB/309/15.

#### *2.4. Control Subjects*

The reference material for research consisted of blood samples obtained from 40 healthy people in properly sampled age groups. The group qualified for the research involved people who had not been hospitalized during the prior year, had not undergone surgical treatment, and had not been pharmacologically treated. Furthermore, the results of their routine laboratory tests (i.e., complete blood count, erythrocyte sedimentation rate test (ESR test), plasma fasting glucose, fasting lipid profile, creatinine, liver enzymes, rheumatoid factor (RF), and CRP) were within the reference values for their particular age group. Subjects were excluded from the study if they had been taking steroidal or non-steroidal anti-inflammatory drugs. None of the volunteers were cigarette smokers or had any history of drug or alcohol abuse.

#### *2.5. Biological Material for Research*

Blood sampled from the elbow vein, contained in test tubes with no added anticoagulant, was centrifuged for 10 min at 1500× *g* at 4 ◦C. The serum obtained through centrifugation was subjected to diagnostic examination as requested by the attending physician. The remaining part was frozen and stored at −80 ◦C until needed for biochemical analysis.

#### *2.6. Assessing the Serum Hyaluronan (HA) Concentration*

Hyaluronan concentration was determined using a using enzyme-linked immunosorbent assays test supplied by TECOmedicalGroup TE 1018-2, Sissach, Switzerland. The analytical sensitivity of the method for HA concentrations assessing was 2.7 ng/mL and the intra-run error was 6.4%.

#### *2.7. Assessing the Serum Soluble Part of Syndecan-1 (sCD138) Concentration*

The concentration of the extracellular part of syndecan-1 circulating in blood was evaluated with an immunoassay test supplied by Diaclone SAS, BesanconCedex, France. The analytical sensitivity of the method for sCD138 concentrations assessing was 4.94 ng/mL and the intra-run error was 6.2%.

#### *2.8. Assessing the Serum Sulfated Glycosaminoglycans (sGAG) Concentration*

Sulfated glycosaminoglycans were assessed using the Wieslab® sGAG quantitative test (GAG 201 RUO, Euro Diagnostica AB, Malmo, Sweden). The principle of the method is based on specific interaction between sulfated glycosaminoglycans (chondroitin sulfates, dermatan sulfates, keratan sulfates, heparan sulfates, and heparin) and the tetravalent cationic Alcian blue. The reagents for this kit, i.e. Alcian Blue stock solution (containing 0.1% H2SO4 and 0.4 M GuHCl), 8 M Guanidine-HCl, SAT solution (containing 0.3% H2SO4 and 0.75 Triton X-100), DMSO solution (containing 40% dimethylsulphoxide and 0.05 M

Mg2Cl2), Gu-Prop solution containing 4 M GuHCl, 33% 1-propanol and 0.25% Triton X-100, calibrators containing 1.1 mL chondroitin sulphate-6 were manufactured by Euro Diagnostica AB from Malmo, Sweden. The assay was performed at pH low enough to neutralize all carboxylic and phosphoric acid groups as well as at ionic strength large enough to eliminate ionic interactions other than those between Alcian blue and sulfated GAGs. Six calibrators containing chondroitin-6-sulfate at concentrations of 12.5, 25, 50, 100, 200, and 400 µg/mL were used. Then, 50 µL of the prepared standard solutions for control and test samples were added to the to the appropriate wells on the microplate. Then, 50 µL of 8 M guanidine-HCl solution (GuHCL) was added, the microplate was covered with sealing tape and incubated for 15 min at room temperature. In the next step, 50 µL of the SAT solution containing 0.3% H2SO4 and 0.75% Triton X-100 was added and incubated for 15 min at room temperature. Then, 750 µL of Alcian blue was added and incubated for 15 min at room temperature. The samples were then centrifuged for 15 min at 12,000× *g*. The next step was to remove the supernatant and add 500 µl of DMSO solution containing 40% dimethylsulfoxide and 0.05 M MgCl2 to the pellets. Prepared samples were incubated for 15 min at room temperature, followed by centrifugation for 15 min at 12,000× *g*. After removing the supernatant, 500 µL Gu-Prop solution containing 4 M guanidine-HCl, 33% 1-propanol and 0.25% Triton X-100 was added to the pellets and mixed for 15 min on a shaker. The absorbance of the standard and test samples was measured using a microplate reader at a wavelength of 620 nm.

#### *2.9. Statistical Analysis*

The statistical analysis of the obtained results was performed using the STATISTICA software, version 10.0, developed by StatSoft, Krakow, Poland. It involved verifying the normality of distribution of the variable using the Shapiro–Wilk test. The descriptive statistics for non-normally distributed variables included the median (Me) as a measure of position and the interquartile range—between the lower (Q1) and the upper (Q3) quartiles as a typical measure of variability. To verify the hypotheses related to the influence of treatment on the value of the investigated parameters, the pre- and post-treatment results were compared using the Wilcoxon test. The statistical significance of the difference in results between the affected group and the control group was determined using the Mann– Whitney U test. The pre- and post-treatment clinical parameters have been compared using the Wilcoxon test and the paired *t*-test. A significance level of *p* < 0.05 was used for all tests and statistical analyses.

#### **3. Results**

#### *3.1. Research Data*

The clinical characteristics of patients with CD and UC before treatment and after a year of treatment has been summarized in Tables 1 and 2.

**Table 1.** Clinical characteristics of patients with ulcerative colitis (UC).

Comparison of selected parameters before and after treatment. Data are presented as median, interquartile (25th–75th percentile) range or mean ± standard deviation (SD), or percentage (%). Data analyzed using the Wilcoxon test or paired *t*-test. *p* < 0.05, statistically significant. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CRP, C-reactive protein; PLT, platelets; WBC, white blood cells; UC, ulcerative colitis; UC0, patients with ulcerative colitis before adalimumab therapy; UC1, patients with ulcerative colitis after 1 year adalimumab therapy.

**Table 2.** Clinical characteristics of patients with Crohn's disease (CD).

Comparison of selected parameters before and after treatment. Data are presented as median, interquartile (25th–75th percentile) range or mean ± standard deviation (SD), or percentage (%). Data analyzed using the Wilcoxon test or paired *t*-test. *p* < 0.05, statistically significant. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CD, Crohn's disease, CD0, patients with Crohn's disease before prednisone therapy; CD1, patients with Crohn's disease after 1 year prednisone therapy; CDAI, The Crohn's disease activity index; CRP, C-reactive protein; PLT, platelets; WBC, white blood cells.

> It is interesting to note that the patients with CD had no change in standard markers of inflammation following intervention. The results of circulating levels of serum sulfated glycosaminoglycans (sGAG), hyaluronan (HA), and the soluble part of syndecan-1 (sCD138) in study individuals (control subjects, patients with CD, and patients with UC) are summarized and provided as supplementary material (Table S1).

#### *3.2. Quantitative Changes of Serum sGAG, HA, and sCD138 in Patients with Crohn's Disease*

The investigation has shown that sGAG and HA concentration in patients newly diagnosed with Crohn's disease is not significantly different from the concentration of these parameters in the serum of healthy people. A year-long prednisone treatment, on the other hand, caused a significant increase in both sGAG and HA concentrations in the serum of affected patients compared with pre-treatment values. The increase reached 42% for sGAG and 78% for HA. Furthermore, the differences in the concentration of sulfated GAGs and HA between patients with CD after a year of prednisone treatment and the control group were statistically significant. The increase in concentration reached 53% for sGAG and 41% for HA.

The quantitative evaluation of extracellular sydecan-1 (sCD138) component circulating in blood has shown a decrease in the concentration of this ECM component in the blood of patients newly diagnosed with CD who have not undergone treatment compared with the concentration in the blood of healthy people. The difference, however, was not statistically significant (*p* > 0.05). Moreover, a significant, almost 110% increase in circulating sCD138 was observed in patients with CD after a year-long treatment compared with pre-treatment concentrations.

#### *3.3. Quantitative Changes of Serum sGAG, HA, and sCD138 in Patients with Ulcerative Colitis*

In the case of patients with UC before implementing biological therapy, the changes to the serum profile of the analyzed ECM components were not statistically different from the concentrations of these components in healthy people, with the exception of HA. Before adalimumab treatment, patients with UC had significantly increased HA concentrations compared with the control group. A year-long treatment with biological medicine, on the other hand, only affected the circulating profile of sulfated GAGs. A statistically significant increase in sGAG concentration was observed in patients with UC compared with the pre-treatment concentration of these ECM components in the same patients. Moreover, patients with ulcerative colitis before therapy had significantly different HA concentration in the blood than patients with CD before steroid therapy implementation.

The results are shown in Figure 1.

#### *3.4. The Relationship between Serum ECM Components and Inflammatory Processes and Disease Activity*

The investigation has also revealed a relationship between disease activity and the circulating profile of ECM components only in the case of patients with UC. In these patients, a correlation between HA concentration in serum and the activity of inflammation, derived from the Mayo score, was observed both before (r = 0.45; *p* < 0.012), and after 12-month treatment (r = 0.42; *p* < 0.039). A correlation between sCD138 levels in serum and the Mayo score was also observed in patients before biological treatment with adalimumab (r = 0.44; *p* < 0.014). Results of the abovementioned correlation analysis are presented as a correlation plot and included in supplementary material (Figure S1).

The relationship between the concentration of the ECM components and a marker of inflammation process commonly used in clinical practice, the CRP protein, was also investigated. The investigation revealed significantly higher CRP concentrations in patients with CD compared to patients diagnosed with UC. A significant effect of 12-month adalimumab treatment inn lowering CRP concentration in the blood of patients with UC was also observed.

**Figure 1.** (**A**–**C**) Circulating total sulfated glycosaminoglycans (GAGs), hyaluronic acid (HA), and soluble syndecan-1 (sCD138) levels in healthy subjects and patients with inflammatory bowel diseases (IBD) before and after 12-month therapy. Note: Results are expressed as median, interquartile (25th–75th percentiles) range, and minimum and maximum of data in the groups. C: healthy subjects (control); UC-0: patients with ulcerative colitis before adalimumab therapy; UC-1: patients with ulcerative colitis after 12-month adalimumab therapy; CD-0: patients with Crohn's disease before prednisone therapy; CD-1: patients with Crohn's disease after 12-month prednisone therapy. HA, hyaluronic acid; sGAG, sulfated glycosaminoglycans; sCD138, soluble syndecan-1; \*\* *p* < 0.01, statistically significant.

#### **4. Discussion**

#### *4.1. Quantitative Changes of the ECM Components (sGAG, HA, and sCD138) in Serum of Patients Newly Diagnosed with CD or UC*

The constant effect of damaging factors during IBD progression leads to changes in the structure and function of the extracellular matrix [22,23]. Our investigation has shown that disturbances affecting the extracellular matrix in the connective tissue during IBD are reflected in quantitative changes of the composition of matrix components in the serum. In the case of patients newly diagnosed with CD, there was no difference in sGAG, HA, and sCD138 concentrations in their serum compared with that of healthy people. In patients with UC, the parameter which was significantly different for healthy people compared with patients before biological medicine treatment was the concentration of hyaluronan in the serum. During physiological states, hyaluronan plays a significant regulatory role in providing ECM integrity, while the depolymerized, small molecule HA fragments generated in IBD play a significant role as inflammatory factors [15]. Signaling through hyaluronan is a mechanism suggesting the regulatory role of ECM integrity failures in IBD pathogenesis. On the one hand, HA fragments promote wound healing; on the other hand, they also promote fibrotic processes, leading to the proliferation of fibroblasts and differentiating myofibroblasts [14,16,17,24]. The increased accumulation of HA fragments in the intestine is related to the inflammation which affects intestinal tissues during IBD progression. It has also been demonstrated that HA takes part in inducing the infiltration of leukocytes into the bowel and activates the immunological response [16,17]. These mechanisms explain the results of the investigation, which suggest increased concentration of HA in the blood of patients with UC before biological treatment. The research so far suggests that increased hyaluronan concentration in the blood and its accumulation in the intestines facilitates IBD progression by increasing the flow of leukocytes into the intestines [17]. Furthermore, HA binds coagulation factors and increases platelet recruitment, which facilitates and supports inflammation during IBD [10]. The research by Kessler et al. [24] points to a significant increase in the concentration of hyaluronanassociated protein (SHAP-HA) in the serum of patients with UC compared to patients with sustained remission, and this value was positively correlated with endoscopic damage. Importantly, the serum SHAP-HA level in patients with CD was correlated only with TNF-α [24]. A difference in HA turnover between UC and CD patients was also observed during our investigation. The significant difference between HA concentration in the serum of patients newly diagnosed with CD and the concentration of this glycan in the serum of patients with UC before treatment suggests the possibility of measuring HA levels during the differential diagnosis of these two very similar types of bowel inflammation, Crohn's disease and ulcerative colitis. Increased deposition of HA in bowel tissues and its increased transfer into circulation are active participants in the chronic inflammation typical for UC. Quantitative assessment of HA in blood may be an early marker of active disease. Another argument supporting this hypothesis is the fact that our results indicate that HA concentration in the blood of UC patients was correlated with the intensity of the disease as determined from the Mayo score.

#### *4.2. The Influence of Therapy with Prednisone (Patients with CD) or Biological Treatment with Adalimumab (Patients with UC) on the Circulating Profile of Analyzed ECM Components*

The performed analyses implied that year-long prednisone treatment applied to patients with CD caused an increase in the concentration of all investigated ECM components in the serum (sGAG, HA, and sCD138) compared with pre-treatment values. In the case of patients with UC, year-long treatment with a TNF-α inhibitor also caused a significant increase in sGAG concentration in serum; however, it did not influence the amount of circulating HA and sCD138. The obtained results confirmed that ECM components are an active part of the progression of the IBD disease process, which is possible to regulate by the type of pharmacological treatment. Sulfated GAGs proved to be the matrix components which undergo significant changes in response to the applied treatment both in CD and

UC patients. The effect of the observed changes, however, was much more noticeable in patients with CD. Moreover, the investigation revealed that sGAG levels in patients with IBD after a year of proper treatment were significantly higher in patients with CD than in patients with UC, which clearly differentiates the two groups of inflammatory bowel diseases. It has also been shown that steroid-based treatment significantly modulated the level of soluble syndecan-1 (sCD138), significantly increasing its levels in the blood. This is in accordance with the results obtained by other authors [21]. It is believed that releasing the extracellular domain of syndecan-1 from the intestinal epithelial cells may reduce the intensity of inflammatory bowel diseases and the transmigration of neutrophils by inactivating key inflammatory mediators and reducing the expression of proinflammatory cytokines. The protective effect of syndecan-1 extracellular domain circulating in the blood has been confirmed during research on mice lacking sCD138; when subjected to an experimental colitis, they have exhibited significantly increased mortality, impaired mucous membrane regeneration, and extended inflammatory cell recruitment. Furthermore, treating animals with a functional analog of the sCD138 ectodomain significantly affected the symptoms of inflammation [21].

#### *4.3. The Influence of Inflammatory Processes and Disease Activity on ECM Components in Patients with IBD*

Due to the fact that IBD are diseases involving an active inflammatory process, this article also analyzed the influence of treatment on the grade of inflammation, derived from the concentration of C-reactive protein in blood. The effect of treatment on disease activity, derived from the Mayo score, was also investigated. Even though CRP is not an innate marker of IBD, it may be an indicator of the intensity of the inflammatory process and help in monitoring the state of patients during the stages of remission and aggravation. The research by Mortensen et al. has shown a relationship between CRP and CD activity. The results indicated a significant increase in CRP levels in patients with an active variation of the disease [25]. The research by Chang et al. also revealed a significant correlation between increased CRP concentration and mild or acute clinical form of the disease [26]. In some studies, however, CRP levels were not associated with the disease activity in small intestinal lesions in patients with CD [27,28]. Solem et al. [27], through their analyses, observed that CRP concentration is within the reference range in 75% of patients with CD, in which endoscopic examinations have not confirmed changes to intestinal mucous membrane. In our investigation, pharmacologically treating patients with CD with prednisone also had no influence on changing the clinical activity of the disease, measured using the CDAI score, or on the inflammatory activity, derived from C-reactive protein concentration. On the other hand, significantly higher CRP concentrations have been observed in patients with CD when compared to patients diagnosed with UC; this is consistent with previous studies which showed that patients with CD generally have higher CRP and IL-6 production than patients with UC [29]. However, the mechanisms responsible for this phenomenon have not yet been fully elucidated. One possible mechanism involves the accumulation of mesenteric fat, a major site of IL-6 and TNF-α synthesis, in patients with CD [30]. In our research, the patients with Crohn's disease had no change in markers of inflammation after one year of treatment. This observation may indicate resistance to glucocorticoid treatment or the presence of fistulas. In the case of patients with ulcerative colitis, CRP concentrations observed during our investigation have therefore been—similarly to the results obtained by other authors—significantly lower than in patients with Crohn's disease, which suggests a potential role for CRP markers in differentiating these two diseases of similar symptomatology. We have also observed that 12-month adalimumab treatment applied to patients with UC decreased the activity of inflammatory processes within intestinal walls, which was reflected in lower CRP concentration and lower disease activity according to the Mayo score. This result was connected to reduced symptoms of the disease after 12-month treatment using a biological medicine. These observations are consistent with the results by Tursi et al. [31], who have researched the effectiveness and safety of treating patients with UC with adalimumab. They have noticed a similar decrease in CRP

concentration and reduced disease activity resulting from the biological treatment [31]. The data suggest that a year-long treatment with a biological medicine results in lower activity of the inflammatory process, which is reflected in the results of endoscopic examination. Furthermore, a correlation was observed between hyaluronan concentration in the blood of patients with UC, both before and after 12-month treatment, and the activity of the disease derived from the Mayo score, which supports the hypothesis that hyaluronan is an active participant in the restructuring of the intestinal mucous membrane, related to chronic bowel inflammation. Hyaluronan, along with the enzymes and binding proteins responsible for its synthesis and degradation, may directly modulate the progression of the disease by controlling the recruitment of immune cells and releasing proinflammatory cytokines [19,24]. These attributes of hyaluronan allow its molecules to take part both in the progression and the remission of the disease. It makes HA molecules and, indirectly, the products of its degradation, perfect candidates for the role of UC progression markers.

#### **5. Conclusions**

The significant difference in HA concentration in the blood of UC and CD patients suggests the possibility of measuring HA levels during the differential diagnosis of these two very similar types of bowel inflammation. Increased deposition of HA in bowel tissues and its increased transfer into circulation are active participants in the chronic inflammation typical for UC. Quantitative changes of HA in blood may be an early marker of active disease. Moreover, our study confirmed that ECM components are an active part of the progression of the IBD disease process, which is possible to regulate according to the type of pharmacological treatment.

The results obtained during this investigation reveal a variety of mechanisms regulating the activity of the disease and the restructuring of extracellular matrix components as well as the potential usefulness of circulating ECM components as markers for evaluating ECM remodeling during IBD and for monitoring pharmacological treatment. Finding potentially useful diagnostic markers of changes occurring throughout IBD, determined in readily available biological material, may provide an aid to the clinical diagnosis and/or monitoring the progression of treatment. However, further research should be performed on a larger group of patients in order to confirm the obtained results. Further investigation could possibly lead to identifying a component of extracellular matrix that will meet the criteria of an ideal marker and will protect patients from invasive colonoscopy examination.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10081722/s1, Figure S1: Results of correlation analysis between disease activity expressed as the Mayo score and serum concentration of the hyaluronan (HA) or soluble syndecan-1 (sCD138) level in patients with ulcerative colitis both before, and after 12-month adalimumab therapy; Table S1: Serum concentrations of sulfated glycosaminoglycans (sGAG), hyaluronic acid (HA) and soluble syndecan-1 (sCD138) in patients with Crohn disease (CD) and ulcerative colitis (UC) at baseline and after one year of therapy.

**Author Contributions:** K.K.-V. and A.D.—conceptualization, investigation, performed the biochemical analysis, writing—original draft preparation; P.O.—conceptualization, investigation, review and editing; K.O.—writing-review and editing, supervision; A.J.-P.—conducting the analysis and contributing to the interpretation of data and the obtained results. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by the Medical University of Silesia in Katowice, Poland, grant No. PCN-007/N/0/I.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Bioethics Committee of the Medical University of Silesia in Katowice (protocol code KNW/0022/KB/309/15 and approval date 30 December 2015).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data is contained within the article or supplementary material.

**Acknowledgments:** The authors would like to thank Maciej Gonciarz from the Department of Gastroenterology, St. Barbara's Specialized Regional Hospital No. 5, Sosnowiec, Poland, for help and participation in the qualification of patients for this study. Our thanks also go to all department members who participated in this effort.

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

#### **References**


**3.1 Why framework-based approach?**

In general, a framework may be defined as a real or conceptual foundation, with a specified level of complexity that serves as a support or a guide for the building of a particular artifact or performing a particular activity by expanding and specializing the generic structure that specifies the family of interrelated products and/or procedures. Framework favors reusability by managing the overall control flow and orchestration of dynamically configured components in an inversion of control way. There are two major categories of contemporary framework: non-software empowered (usually represented as a set of structured and/or semi-structured

*The Foundation for Open Component Analysis: A System of Systems Hyper Framework Model DOI: http://dx.doi.org/10.5772/intechopen.103830*

documents) and software empowered (software supported collaborative/cooperative environments supporting the digital transformation of problem domain). The development of software empowered interoperability frameworks have usually been preceded by the intensive and time-consuming: specification; modeling; and metamodeling activities performed and managed within the scope of related projects and/ or portfolios.

### **3.2 Why interoperability-based approach?**

In the context of this chapter, cooperation and collaboration of independently developed or specified systems may be generally achieved through total homogenization; harmonization; adaptation; and orchestration interoperability principles. The total homogenization principle states that all of the related systems have to be designed or redesigned to achieve absolute compliance with the globally accepted and standardized template. Homogenization is the most radical, heavy-weight, approach. A harmonization principle is a lightweight approach that assumes the definition and standardization of interfaces that hide the internal characteristic of participating systems and enable their cooperation/collaboration over the unique communication protocol and through the standardized interfaces only. The participating systems need to be functionally complete while retaining the structural diversity. The adaptation interoperability principle assumes cooperation/collaboration of functionally and structurally incomplete systems where participating systems are homogenized up to the functional and structural completeness, in a virtual or real way (where homogenization payoff may substantially differ from system to system) and harmonized afterward. Adaptation interoperability may be seen as a middle-weight approach. It is more complex than the harmonization but, compared to the total homogenization, significantly more acceptable and achievable faster. The orchestration interoperability is the ability of the heterogeneous systems, with arbitrary functionality and topology, to interact toward the mutually beneficial dynamically configured mission, build through the functional and/or structural orchestration of participating systems features and/or resources. Each system retains and shares everything it can perform and/ or deliver and delegates and/or acquires everything that is beyond its scope, but available as a mutual benefit of a current configuration. The orchestration interoperability is an example of broker-based service-oriented dynamic architecture that may be formally described by the swarm intelligence concepts.

### **3.3 Why the interoperability framework approach?**

If carefully combined the best characteristics of two, previously discussed promising concepts, may result in an empowered solution capable of handling the growing complexity of SoS dynamic configurations associated with the multidimensional and multilevel LDOs. The development of interoperability framework generally requires a multi-stakeholder process and the long-term vision of a highly reusable generic solution that, in the context of SoS-HFM, may impact the overall ontology, configurable topology, state-driven behavior, and time and context-dependent LDOs: creation, processing, storing, retrieval and visualizing.

The starting point of SoS and POS paradigms integration is the formulation of a hybrid system meta-model that combines multidimensionality and context-based multilevel features. The meta-concept (MetaConcept) of SoS-HFM is modeled as a typed composite MetaElement presented in **Figure 3**.


**3. Chiral-induced spin selectivity (CISS) effect for hybrid materials**

By the way, as a chirality phenomenon that can still cross the hierarchy, "chiralinduced spin selectivity (CISS)" proposed by Professor Ron Naaman is one of the most notable concepts at present [2]. The information gained through the experiment will be meaningful to the science, though this description is merely a "proposal" level.

As mentioned below, injecting electrons into oxidoreductase through oligopeptide which works as a spin filter is the first ever experiment, and it is crucial to cooperate with Dr. Naaman for this research. They believe several years of experience on metal complexes and profound knowledge about the CISS Dr. Naaman has can lead to a promising outcome. One of results they already expect to have is that if they detect the change of chiral property affected by the subtle transformation of metal complex and oligopeptide, they can observe and analyze the electrochemical change.

Their cooperative research covers the fields of the surface physics and the biochemistry, and their results of fixing the electron transfer pathway between electrode and enzyme, and of control of magnetism that metal complex has by the external magnetic field, would be applied to other fields. Also, the CISS effect will offer new possibilities for a better understanding of process of spin selection in the biology and spin electronics applications.

The injection of electrons into laccase using the CISS effect is the first trial of its kind. If this experiment was conducted successfully, the results would contribute to other fields including the spin electronics.

Then they were planning to apply the CISS effect to metal complex to magnetize spins from outside to control them while performing electrochemical measurement. The CISS effect is that in chiral molecules only spins which are orientated in a certain direction can be transferred smoothly. This effect has been studied a lot in recent years with Dr. Naaman, as the leading researcher. With Dr. Naaman's cooperation, who is the leading researcher of this effect, it is particularly noteworthy that difference in chirality shows different spin polarization, which suggests that this effect is applicable to a system where laccase is used (**Figure 2**).

Prepare a measuring device described, then inject spin polarized electrons into laccase where metal complex is present. Place oligopeptide and the metal complex between the electrode and the laccase. They use the oligopeptide and the metal complex, which let spin polarized electrons be transferred smoothly, so that the increase of amperage should be expected. They also analyzed how spin polarized electrons affect the way electron-transfer to the enzyme is occurred. As

**Figure 2.** *Proposed of measuring system for CISS effect of redox proteins (laccase).*

a preliminary experiment, they conducted electrochemical measurement using the above method without the metal complex. The result shows that the chirality of peptide affects the electron-transfer to the laccase. Following this outcome, they expect the follows. If they insert the metal complex between the oligopeptide and laccase, with the metal complex being fixed inside the hydrophobic pockets of laccase, the metal complex would be fastened firmly to the laccase. Then the electron-transfer to the Cu-T1, which proceeds oxygen reduction reaction, can be carried out selectively. In addition to that, they will examine how the electron-transfer would change when creating an external magnetic field, and they will also analyze it by comparing the use of ferromagnetic or photoresponsive metal complexes.

Some reports have been submitted regarding the injection of electron spins only into membrane protein or oligopeptide in those days, but there has not been any report on the injection of the electron spins into oxidoreductase. The results from the preliminary experiments suggest there is a possibility of realizing the electrontransfer without being influenced by the external magnetic field. Their idea has an intension of examining the electron-transfer in depths from a new perspective by injecting the electron spins into laccase using the CISS effect to elucidate the difference electrochemically between other mediate complexes. The analysis about behavior of the electrons will contribute to the development of biochemistry and a field of spintronics device.

Unfortunately, actual research using laccase only has not yet been published to date.

#### **4. Concluding remarks**

In this way, "chirality" may be found in many fields of natural sciences and technology potentially. The main purpose of this plan about the CISS effect study was just to improve electron transfer between the electrode and the enzyme in enzymatic biofuel cell using the CISS effect. Along with that, they are also planning to reveal from a spin-polarized point of view how magnetic field and chiral property affect the electron transfer reactions. Also, if we achieve the implementation of the CISS effect, the result would be applied "chirality" for other areas such as energy materials science and medical biochemistry.

**5**

**Author details**

Takashiro Akitsu

Tokyo, Japan

Department of Chemistry, Faculty of Science, Tokyo University of Science,

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to<EMAIL_ADDRESS>
provided the original work is properly cited.

*Introductory Chapter: Chirality*

*DOI: http://dx.doi.org/10.5772/intechopen.98305*

*Introductory Chapter: Chirality DOI: http://dx.doi.org/10.5772/intechopen.98305*

*Current Topics in Chirality - From Chemistry to Biology*

*Proposed of measuring system for CISS effect of redox proteins (laccase).*

photoresponsive metal complexes.

field of spintronics device.

**4. Concluding remarks**

materials science and medical biochemistry.

to date.

**Figure 2.**

a preliminary experiment, they conducted electrochemical measurement using the above method without the metal complex. The result shows that the chirality of peptide affects the electron-transfer to the laccase. Following this outcome, they expect the follows. If they insert the metal complex between the oligopeptide and laccase, with the metal complex being fixed inside the hydrophobic pockets of laccase, the metal complex would be fastened firmly to the laccase. Then the electron-transfer to the Cu-T1, which proceeds oxygen reduction reaction, can be carried out selectively. In addition to that, they will examine how the electron-transfer would change when creating an external magnetic field, and they will also analyze it by comparing the use of ferromagnetic or

Some reports have been submitted regarding the injection of electron spins only into membrane protein or oligopeptide in those days, but there has not been any report on the injection of the electron spins into oxidoreductase. The results from the preliminary experiments suggest there is a possibility of realizing the electrontransfer without being influenced by the external magnetic field. Their idea has an intension of examining the electron-transfer in depths from a new perspective by injecting the electron spins into laccase using the CISS effect to elucidate the difference electrochemically between other mediate complexes. The analysis about behavior of the electrons will contribute to the development of biochemistry and a

Unfortunately, actual research using laccase only has not yet been published

In this way, "chirality" may be found in many fields of natural sciences and technology potentially. The main purpose of this plan about the CISS effect study was just to improve electron transfer between the electrode and the enzyme in enzymatic biofuel cell using the CISS effect. Along with that, they are also planning to reveal from a spin-polarized point of view how magnetic field and chiral property affect the electron transfer reactions. Also, if we achieve the implementation of the CISS effect, the result would be applied "chirality" for other areas such as energy

**4**


**5. Challenges and Future Perspective**

The nutritional value of plant-based food is dependent on the amount of proteins, the specific distribution of the amino acids, and, most importantly, the bioavailability of these amino acids. Reduced protein quality will compromise the nutritional value, therefore maintaining quality and stability upon processing of plant-base materials is of utmost importance. From nature, proteins are not equally digestible, as their proteolytic susceptibility varies due to different three-dimensional structures according to their origin. This inherent difference in digestibility across legume sources is mirrored into the effect of processing methods. The benefits of food processing covers preservation, palatability, functional properties, or added convenience, but the impact of processing are often disconnected to the nutritional status of the proteins. Surely, the ultimate goals are plant-based products with high protein quality to be enjoyed as part of the consumer's healthy diet and lifestyle. Assessing the quality of protein in relation to nutritional value means to determine the capacity of the proteins to satisfy the metabolic demand for amino acids. If perfect, a measure of the nutritional quality of dietary protein should provide the real protein digestibility and, thereby, enable the prediction of the overall efficiency of protein utilization. At present, various in vitro and in vivo protein digestibility methods exist, and neither of them can be used to accurately measure protein digestibility in absolute terms. An important conceptual difficulty is that proteins differ in their digestibility and the human GI tracts differ in their ability to digest proteins. Therefore, there is still much to learn and improve regarding methodologies for measuring and monitoring the protein digestibility before the "true" effect of processing on the important amino acids can be established. The golden goal for optimizing nutrition may be monitoring the individual protein digestive capacity in the individual body. However, even if food producers used the perfect PD method to optimize PD of a food product, the product must still appeal to consumers for success on the market. It is recognized that an increasing number of food companies are producing successful plant-based products providing variety, taste, and nutritional value to consumers.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/8/6/224/s1, Table S1. Protein digestibility (PD) of legumes using various processing methods.

**Author Contributions:** Conceptualization, V.O.; investigation, D.D.; writing—original draft preparation, D.D.; writing—review and editing, V.O.

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

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


**3. β-cell stress in type 1 diabetes**

### **3.1 Endoplasmic reticulum stress**

It has been suggested that endoplasmic reticulum (ER) stress is involved in β-cell destruction. Pancreatic -cells are specialised cells that rapidly synthesise and secrete insulin in response to fluctuations in blood glucose levels (Pirot et al., 2007). This imparts a heavy burden on the ER and, consequently, -cells are particularly susceptible to cellular conditions that impair the ER's ability to correctly fold nascent proteins. Under such conditions, the resultant accumulation of unfolded or damaged proteins within the ER lumen triggers the unfolded protein response (UPR), an adaptive signalling pathway that increases the folding capacity of the ER and restores homeostasis (Szegezdi et al., 2006). Although the initial UPR is a protective response, prolonged ER stress can lead to the initiation of apoptosis. Thus while under physiological conditions the UPR acts as a prosurvival mechanism in β-cells, chronic ER stress can lead to redirection of the UPR towards pro-apoptotic signalling.

Three ER-localized transmembrane proteins sense the accumulation of unfolded proteins in the ER lumen and initiate the UPR, PKR-like ER kinase (PERK), inositol-requiring enzyme 1 α (IRE1) and activating transcription factor 6 (ATF6). These proteins transduce information from the ER to the nucleus by activating transcription factors that control genes involved in restoring ER function (Szegezdi et al., 2006). The PERK arm of the UPR has been the main focus of studies with regard to β-cell stress in diabetes, therefore this chapter will focus on PERK signalling in more detail. Upon accumulation of unfolded proteins, PERK is activated and induces a translational block by phosphorylating eukaryotic initiation factor 2 α (eIF2). Phosphorylation of eIF2α by PERK leads to inhibition of cap-dependent protein synthesis. This reduces the protein load of the ER while allowing cap-independent translation to persist and leads to preferential translation of the transcription factor ATF4. One target gene induced by ATF4 (in conjunction with ATF6) is C/EBP homologous protein CHOP, a transcription factor that is known to promote apoptosis (Zinszner et al., 1998).

#### **3.1.1 The role of PERK in β-cell function**

The PERK signalling branch of the UPR appears to be essential for the regulation of β-cell function. Stimulation of insulin production in mouse pancreatic islets leads to dephosphorylation of eIF2α (P. Zhang et al., 2002) reversing the translational block caused by PERK signalling and allowing for increased biosynthesis of insulin. Studies with knockout mice showed that PERK is essential for β-cell function and survival (Harding et al., 2001, P. Zhang et al., 2002). Pancreatic β-cells of PERK-/- mice degenerated within the first four weeks after birth, and a diabetic phenotype could be observed (Harding et al.,

regulation of DP5 in β-cells is mediated by the transcription factor STAT-1 which is regulated by IFNγ (Moore et al., 2011). In addition, inflammatory cytokines led to upregulation of the pro-apoptotic BH3-only protein PUMA in primary rat β-cells as well as in human islets through a pathway involving NF-κB signalling, iNOS activation and ER stress (Gurzov et al., 2010). Furthermore, down-regulation of the anti-apoptotic multidomain Bcl-2 family member Mcl-1 turned out to be critically involved in the cytokine-induced apoptosis of the rat insulinoma cell line INS-1E (Allagnat et al., 2011). In summary, exposure to cytokines leads to alterations in expression of several Bcl-2 family members in β-cells in a

It has been suggested that endoplasmic reticulum (ER) stress is involved in β-cell destruction. Pancreatic -cells are specialised cells that rapidly synthesise and secrete insulin in response to fluctuations in blood glucose levels (Pirot et al., 2007). This imparts a heavy burden on the ER and, consequently, -cells are particularly susceptible to cellular conditions that impair the ER's ability to correctly fold nascent proteins. Under such conditions, the resultant accumulation of unfolded or damaged proteins within the ER lumen triggers the unfolded protein response (UPR), an adaptive signalling pathway that increases the folding capacity of the ER and restores homeostasis (Szegezdi et al., 2006). Although the initial UPR is a protective response, prolonged ER stress can lead to the initiation of apoptosis. Thus while under physiological conditions the UPR acts as a prosurvival mechanism in β-cells, chronic ER stress can lead to redirection of the UPR towards

Three ER-localized transmembrane proteins sense the accumulation of unfolded proteins in the ER lumen and initiate the UPR, PKR-like ER kinase (PERK), inositol-requiring enzyme 1 α (IRE1) and activating transcription factor 6 (ATF6). These proteins transduce information from the ER to the nucleus by activating transcription factors that control genes involved in restoring ER function (Szegezdi et al., 2006). The PERK arm of the UPR has been the main focus of studies with regard to β-cell stress in diabetes, therefore this chapter will focus on PERK signalling in more detail. Upon accumulation of unfolded proteins, PERK is activated and induces a translational block by phosphorylating eukaryotic initiation factor 2 α (eIF2). Phosphorylation of eIF2α by PERK leads to inhibition of cap-dependent protein synthesis. This reduces the protein load of the ER while allowing cap-independent translation to persist and leads to preferential translation of the transcription factor ATF4. One target gene induced by ATF4 (in conjunction with ATF6) is C/EBP homologous protein CHOP, a

The PERK signalling branch of the UPR appears to be essential for the regulation of β-cell function. Stimulation of insulin production in mouse pancreatic islets leads to dephosphorylation of eIF2α (P. Zhang et al., 2002) reversing the translational block caused by PERK signalling and allowing for increased biosynthesis of insulin. Studies with knockout mice showed that PERK is essential for β-cell function and survival (Harding et al., 2001, P. Zhang et al., 2002). Pancreatic β-cells of PERK-/- mice degenerated within the first four weeks after birth, and a diabetic phenotype could be observed (Harding et al.,

transcription factor that is known to promote apoptosis (Zinszner et al., 1998).

manner that favours activation of the intrinsic pathway of apoptosis.

**3. β-cell stress in type 1 diabetes 3.1 Endoplasmic reticulum stress**

pro-apoptotic signalling.

**3.1.1 The role of PERK in β-cell function**

2001, P. Zhang et al., 2002). β-cell loss was associated with damaged rough ER and high levels of apoptosis in the pancreas (P. Zhang et al., 2002). However, a subsequent study discovered that the onset of diabetes in PERK-/- mice is due to developmental defects during β-cell proliferation and differentiation leading to a reduction in β-cell mass (W. Zhang et al., 2006). At the molecular level, down-regulation of PERK in rat β-cells was shown to induce deregulation of ER chaperones Grp78 and ERp72 and disruption of ER function leading to reduced insulin production and reduced cell proliferation (Feng et al., 2009).

#### **3.1.2 Involvement of the UPR in cytokine-induced β-cell death**

There is some evidence to suggest that cytokines induce β-cell apoptosis by stimulating proapoptotic signalling of the UPR. Ca2+ levels in the ER are about four times higher than in the cytosol as high Ca2+ levels are required for ER function in aiding protein folding and posttranslational processing. Disruption of Ca2+ homeostasis causes severe ER stress resulting in accumulation of unfolded proteins in the ER and activation of the UPR. It was shown that cytokine-exposure leads to elevated basal cytosolic Ca2+ levels selectively in mouse pancreatic β-cells compared to glucagon-secreting -cells and this was associated with cytokine-induced apoptosis (L. Wang et al., 1999). In line with these results, it was shown that increased production of NO in rodent β-cells leads to depletion of ER Ca2+ levels (Oyadomari et al., 2001). Furthermore, overexpression of the ER-located Ca2+-binding protein, calreticulin increased levels of Ca2+ in the ER and made cells more resistant to NOinduced apoptosis (Oyadomari et al., 2001). This suggests that NO-induced apoptosis in rodent β-cells is at least partly caused by ER stress induced by NO-mediated Ca2+ depletion. Some evidence suggests that NO may regulate Ca2+ levels in β-cells through downregulation of the sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) (Cardozo et al., 2005). SERCA pumps Ca2+ from the cytoplasm into the ER thus maintaining ER Ca2+ levels. Rodent and human islet cells have been reported to express the isoforms SERCA2b and SERCA3 (Varadi et al., 1996). Treatment of rodent pancreatic β-cells with a combination of IL-1β and IFNγ induced transcriptional down-regulation of SERCA2b and this was partially prevented by inhibition of iNOS. Furthermore, after inhibition of SERCA, the effect of cytokine exposure on ER Ca2+ was abolished (Cardozo et al., 2005). This suggests that NOinduced depletion of ER Ca2+ is at least in part mediated by SERCA down-regulation. The SERCA isoform SERCA2a has been shown to be specifically inactivated by peroxynitrite (ONOO-)-mediated nitration of a tyrosine residue within the channel-like domain *in vitro* (Viner et al., 1999). Peroxynitrite is produced in cells by a reaction between NO and the free radical superoxide (Pacher et al., 2007). SERCA2a differs from SERCA2b only in regions of the C-terminus (Dode et al., 1998) and it could be hypothesised that cytokine-induced NO production inhibits SERCA2b Ca2+-ATPase activity by peroxynitrite-mediated nitration in the same way. Another possible mechanism by which NO might mediate reduction of ER Ca2+ levels is via activation of the ryanodine receptor-2. Ryanodine receptor-2 is a calcium channel located in the ER membrane that releases Ca2+ from the ER into the cytosol and has been reported to be expressed in mouse pancreatic β-cells (Islam et al., 1998). NO-induced poly-S-nitrosylation enhances the activity of this calcium channel (Xu et al., 1998) but whether this mechanism is relevant to cytokine-exposed β-cells remains to be determined.

Treatment of rodent β-cells with a combination of IL-1β and IFNγ induces the expression of CHOP in an NO-dependent manner (Fig. 4). This is in line with a number of other reports (Cardozo et al., 2001b, Cardozo et al., 2005). Inhibition of iNOS by N5-(1-iminoethyl)-Lornithine (L-NIO) or NG-methyl-L-arginine (LMA) blocked cytokine-induced NO

Cytokine-Induced -Cell Stress and Death in Type 1 Diabetes Mellitus

death may be partly mediated by induction of CHOP.

conditions of cytokine-induced β-cell stress.

**3.2 Oxidative stress**

Tiedge et al., 1998).

**4. Therapeutic strategies**

autoimmunity (Kort et al., 2011).

that CHOP is not required for β-cell death and the development of diabetes in NOD mice (Satoh et al., 2011). However, CHOP-/- NOD mice showed delayed production of insulin autoimmune antibodies (Satoh et al., 2011) suggesting a role for CHOP in the early onset of the autoimmune reaction leading to β-cell destruction. Therefore, cytokine-induced β-cell

While a functional UPR (at least the PERK branch) appears to be essential for β-cell function and development, its downstream target CHOP has been associated with cytokine-induced β-cell destruction suggesting that PERK signalling regulates β-cell function and survival under physiological conditions but may switch towards pro-death signalling under

Cytokines induce multiple stress pathways in β-cells. Oxidative stress is induced by increased production of ROS and an imbalanced, low level of antioxidant enzymes (Sies, 1997). IL-1β, TNFα and IFNγ induce the production of ROS and NO by inducing iNOS (Rabinovitch & Suarez-Pinzon, 1998). Free oxygen and nitrogen radicals generated can react to form peroxynitrite, which is a very strong oxidant. Oxygen free radicals, nitrogen free radicals as well as the radical peroxynitrite can react with and damage a range of cellular proteins and in this way block metabolic functions and induce β-cell death (Azevedo-Martins et al., 2003). β-cells have been shown to be especially susceptible to such oxidative stress because they have particularly low levels of antioxidant enzymes (Lenzen et al., 1996,

The number of people affected by T1DM is approximately 20 million worldwide and is rapidly rising (Chabot, 2002). While exogenous administration of insulin is an effective treatment for acute hyperglycaemia in T1DM, it does not prevent secondary complications (White et al., 2008) and can in some cases lead to hypoglycaemia (Kort et al., 2011). Alternative therapeutic strategies include pancreas transplantation and islet transplantation. While whole pancreas transplantation is an invasive surgical method associated with major complications, islet transplantation is less invasive and associated with significantly lower morbidity and mortality. Successful islet transplantation would result in insulin independence, protection from hypoglycaemia, improvement of microvascular complications, improved patient survival and enhanced quality of life (Kort et al., 2011). The method is currently in clinical trials and has been used to treat around 1,000 individuals worldwide (Kort et al., 2011). Islet transplantation has many limitations, including limited availability of suitable islet graft donors, high cost and high rate of partial or total graft failure. Islet graft failure can be caused by allorejection, toxicity of immunosuppressive drugs that are required to reduce immune rejection, glucotoxicity, and recurrence of

An approach to reduce β-cell death in islet grafts is the transfer of therapeutically useful genes into islet cells prior to transplantation (McCabe et al., 2006). The development of gene therapy techniques that can protect β-cells from autoimmune destruction may not only improve outcomes after islet transplantation but may also lead to preventive therapies for

patients at high risk of developing T1DM (McCabe et al., 2006).

225

production and expression of CHOP. In addition to CHOP, the UPR marker proteins Grp78 and phosphorylated eIF2 were also found to be up-regulated after cytokine treatment, without affecting expression of spliced X-box binding protein 1 (sXBP-1) which is induced downstream of IRE1α (Fig. 4). Overexpression of iNOS alone was sufficient for CHOP expression (Fig. 4) and treatment with the NO-donor molecule S-nitroso-N-acetyl-D,Lpenicillamine (SNAP) induced expression of Grp78 and CHOP (Oyadomari et al., 2001). This suggests that NO is sufficient to activate the UPR in rodent pancreatic β-cells. Pancreatic islet cells from CHOP-/- mice were shown to be resistant to cytokine- and NOmediated apoptosis compared to cells from CHOP+/+ and CHOP+/- mice (Oyadomari et al., 2001). Together these results suggest that the apoptotic effects of cytokine-induced NO are mediated by activation of CHOP.

Fig. 4. IL-1β/IFNγ induced the expression of the pro-apoptotic transcription factor CHOP and other UPR markers. (A) A time-course cytokine treatment (IL-1β and IFNγ, 60 U/ml of each) was carried out and samples assayed for NO production. (B) The same samples were then analysed by Western blotting for iNOS and CHOP expression. This data demonstrates an increase in NO production, iNOS expression and CHOP induction occurring at 6 h post cytokine treatment, although CHOP is not strongly expressed until 9 h. (C) Samples were assayed for CHOP expression following cytokine treatment in the presence and absence of the iNOS inhibitor L-NIO. Cytokine-induced CHOP expression was decreased in the presence of L-NIO indicating that this is an NO-dependent process. (D) Alterations in the expression of ER stress-associated proteins after cytokine treatment were analysed by Western blotting. The expression of UPR markers Grp78 and phosphorylated eIF2 (peIF2were up in response to cytokine treatment. (E) Production of spliced XBP-1 mRNA after cytokine treatment was determined by RT-PCR. Thapsigargin (Tg) treatment was used as a positive control. Cytokine treatment did not show an effect on the level of spliced XBP-1 mRNA. The images presented are representative of three independent experiments.

Conversely, another study suggested that although cytokine signalling induces ER stress as demonstrated by activation of PERK and JNK, induction of CHOP is not required for -cell death in rodents (Åkerfeldt et al., 2008). In support of these findings, a recent study suggests that CHOP is not required for β-cell death and the development of diabetes in NOD mice (Satoh et al., 2011). However, CHOP-/- NOD mice showed delayed production of insulin autoimmune antibodies (Satoh et al., 2011) suggesting a role for CHOP in the early onset of the autoimmune reaction leading to β-cell destruction. Therefore, cytokine-induced β-cell death may be partly mediated by induction of CHOP.

While a functional UPR (at least the PERK branch) appears to be essential for β-cell function and development, its downstream target CHOP has been associated with cytokine-induced β-cell destruction suggesting that PERK signalling regulates β-cell function and survival under physiological conditions but may switch towards pro-death signalling under conditions of cytokine-induced β-cell stress.

### **3.2 Oxidative stress**

224 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

production and expression of CHOP. In addition to CHOP, the UPR marker proteins Grp78 and phosphorylated eIF2 were also found to be up-regulated after cytokine treatment, without affecting expression of spliced X-box binding protein 1 (sXBP-1) which is induced downstream of IRE1α (Fig. 4). Overexpression of iNOS alone was sufficient for CHOP expression (Fig. 4) and treatment with the NO-donor molecule S-nitroso-N-acetyl-D,Lpenicillamine (SNAP) induced expression of Grp78 and CHOP (Oyadomari et al., 2001). This suggests that NO is sufficient to activate the UPR in rodent pancreatic β-cells. Pancreatic islet cells from CHOP-/- mice were shown to be resistant to cytokine- and NOmediated apoptosis compared to cells from CHOP+/+ and CHOP+/- mice (Oyadomari et al., 2001). Together these results suggest that the apoptotic effects of cytokine-induced NO are

Fig. 4. IL-1β/IFNγ induced the expression of the pro-apoptotic transcription factor CHOP and other UPR markers. (A) A time-course cytokine treatment (IL-1β and IFNγ, 60 U/ml of each) was carried out and samples assayed for NO production. (B) The same samples were then analysed by Western blotting for iNOS and CHOP expression. This data demonstrates an increase in NO production, iNOS expression and CHOP induction occurring at 6 h post cytokine treatment, although CHOP is not strongly expressed until 9 h. (C) Samples were assayed for CHOP expression following cytokine treatment in the presence and absence of the iNOS inhibitor L-NIO. Cytokine-induced CHOP expression was decreased in the presence of L-NIO indicating that this is an NO-dependent process. (D) Alterations in the expression of ER stress-associated proteins after cytokine treatment were analysed by Western blotting. The expression of UPR markers Grp78 and phosphorylated eIF2 (peIF2were up in response to cytokine treatment. (E) Production of spliced XBP-1 mRNA after cytokine treatment was determined by RT-PCR. Thapsigargin (Tg) treatment was used as a positive control. Cytokine treatment did not show an effect on the level of spliced XBP-1

mRNA. The images presented are representative of three independent experiments.

Conversely, another study suggested that although cytokine signalling induces ER stress as demonstrated by activation of PERK and JNK, induction of CHOP is not required for -cell death in rodents (Åkerfeldt et al., 2008). In support of these findings, a recent study suggests

mediated by activation of CHOP.

Cytokines induce multiple stress pathways in β-cells. Oxidative stress is induced by increased production of ROS and an imbalanced, low level of antioxidant enzymes (Sies, 1997). IL-1β, TNFα and IFNγ induce the production of ROS and NO by inducing iNOS (Rabinovitch & Suarez-Pinzon, 1998). Free oxygen and nitrogen radicals generated can react to form peroxynitrite, which is a very strong oxidant. Oxygen free radicals, nitrogen free radicals as well as the radical peroxynitrite can react with and damage a range of cellular proteins and in this way block metabolic functions and induce β-cell death (Azevedo-Martins et al., 2003). β-cells have been shown to be especially susceptible to such oxidative stress because they have particularly low levels of antioxidant enzymes (Lenzen et al., 1996, Tiedge et al., 1998).