pregnancy - associated plasma protein - a ( papp - a ) is a metzincin metalloproteinase primarily produced by the placental syncytiotrophoblast during pregnancy .
papp - a is also synthesized by fibroblasts , osteoblasts , vascular smooth muscle cells ( vsmcs ) , and endothelial cells ( ecs ) . in vitro ,
papp - a functions to cleave insulin - like growth factor - binding protein 4 ( igfbp-4 ) , an inhibitory igfbp , consequently increasing igf bioavailability for receptor activation [ 14 ] . in vivo ,
several studies have shown a similar role for papp - a in modulating site- and event - specific igf signaling during injury repair processes .
recent studies have indicated that papp - a is a novel biomarker for plaque instability and inflammation useful in early diagnosis , risk stratification , and prognostic prediction in patients with acute coronary syndrome ( acs ) [ 5 , 6 ] .
papp - a was found abundantly expressed in ruptured and eroded human atherosclerotic plaques , colocalized with activated smooth muscle cells and macrophages [ 7 , 8 ] . since plaque - derived papp - a is being considered as a new biomarker that may potentially play a role in the development of atherosclerotic lesions [ 9 , 10 ]
. a better understanding of its cellular source and regulation is important to the future development and implementation of therapeutics utilizing this biomarker .  
previous studies have indicated that proinflammatory cytokines , interleukin- ( il- ) 1 , and tumor necrosis factor- ( tnf- )  were potent stimulators of papp - a expression in cultured human fibroblasts , osteoblasts , coronary artery smooth muscle cells , and endothelial cells ( ecs ) [ 9 , 11 ] . despite these significant findings , little
is known about the effect of c - reactive protein ( crp ) and tnf- on papp - a expression in human peripheral blood monocytes ( pbmcs ) .
the current study investigates the ability of crp and tnf- to induce papp - a expression in the pbmcs of healthy volunteers .
furthermore , inhibitor experiments have been designed to explore the underlying intracellular signaling pathways involved in papp - a expression .
the focus of these studies is nuclear factor- ( nf- ) b pathways , a major pathway associated with cytokine stimulation in various cell types .
peripheral blood was collected from the forearm vein of healthy volunteers enrolled in the current study . for each experiment ,
30  ml samples were freshly collected from 6 healthy subjects for use in pbmc preparations .
each of these 6 subjects provided 4 blood donations over the course of the study .
the study was approved by the ethics committee of the beijing friendship hospital and conforms to the principles outlined in the declaration of helsinki .
human pbmcs were isolated from 30  ml fresh blood samples obtained from healthy volunteers by ficoll - paque ( amersham bioscience , uppsala , sweden ) centrifugation .
resultant cells were washed 3 times with pbs and subsequently resuspended in rpmi 1640 ( gibcobrl , grand island , ny , usa ) supplemented with 10% fetal calf serum ( gibcobrl , grand island , ny , usa ) , penicillin ( 100  u / ml ) , and streptomycin ( 100  g / ml ) . cells were then cultured for 24 hours in plastic dishes at 37c in a humidified atmosphere of 5% co2 . upon observation of subconfluent growth , the medium was replaced with fresh medium .
all nonadherent lymphocytes were discarded during the medium change , reserving only healthy adherent monocytes .
after 24 hours , trypan blue exclusion indicated that 95% of cultured pbmcs were living .
the pharmacological agents recombinant human tnf- ( perprotech , rocky hill , ct , usa ) , crp , actinomycin d , and bay11 - 7082 ( sigma chemicals , deisenhofen , germany ) were dissolved into solution according to the manufacturer 's instructions .
resultant solutions were added to cultured pmbcs at defined time intervals ( 2 , 8 , 16 , 24 hours ) and concentrations ( crp : 5 , 10 , or 20  mg / l ; tnf- : 25 , 50 , or 100  ng / ml ; bay11 - 7082 : 20  m ) in the absence or presence of actinomycin d ( 1  g / ml ) , as further described in the following sections .
individual controls were determined for each experiment , as described in figures 14 .   briefly , total rna was isolated using trizol ( invitrogen , calsbad , ca , usa ) according to the manufacturer 's instructions .
reverse transcription - generating cdna was performed using the superscript iii first - strand synthesis system ( invitrogen , calsbad , ca , usa ) .
papp - a cdna was amplified using forward ( 5-ata tct cac gtg acc gag ga-3 ) and reverse ( 5-aga tga tgg tgc tgg aag tc-3 ) primers , which produce a 529  bp product .
amplification was performed at 94c for 2  min for preheating , followed by 30 cycles of 94c for 45  s , 65c for 45  s , 72c for 60  s , and a final extension of 72c for 10  min .
-actin was amplified using forward ( 5-gca tgg agt cct gtg gca t-3 ) and reverse ( 5-cta gaa gca ttt gcg gtg g-3 ) primers , which produce a 320  bp product .
amplification was performed at 94c for 2  min for preheating , followed by 28 cycles of 94c for 30  s , 60c for 30  s , 72c for 30  s , and a final extension of 72c for 20  min .  
the resulting bands were photographed under ultraviolet light and analyzed using a gel imaging system ( gel doc2000 , bio - rad , hercules , ca , usa ) .
the relative intensity of bands of interest was expressed as the ratio to -actin mrna bands . for cell lysates , cells were washed twice with ice - cold pbs and lysed in ripa buffer .
total protein was quantified using the bca assay ( pierce , rockford , il , usa ) .
equal amounts of protein ( 40  g ) were separated by sds - page in 14% tris - glycine gels ( tefco , tokyo , japan ) .
after electrophoresis , the proteins were blotted onto a nitrocellulose membrane and blocked with 5% skim milk powder diluted in tris - buffered saline ( tbs ) with 0.05% tween 20 .
rabbit polyclonal antibodies against human papp - a ( 1  :  1000 , abcam systems , cambridge , usa ) were used as the primary antibody .
membranes were incubated with diluted antibody preparations overnight at 4c . after washing the next day
, membranes were incubated with horseradish peroxidase- ( hrp- ) conjugated affinity - purified goat anti - rabbit igg antibody ( 1  :  3000 , santa cruz .
papp - a levels were determined using the ultrasensitive elisa kit ( diagnostic systems laboratories , webster , tx ) .
the assay was calibrated using recombinant papp - a calibrated against the world health organization 's international reference preparation 78/610 for pregnancy - associated proteins , by definition containing a papp - a concentration of 100 
minimum sensitivity was 0.24  miu / l with intra- and interassay coefficients of variation of 4.7% and 4.2% , respectively .
all statistical analyses were carried out using the spss statistical package , version 13.0 ( spss inc . ,
p - values less than 0.05 were considered statistically significant ( p < 0.05 ) .
the time course of papp - a mrna expression in pbmcs under basal and cytokine - stimulated conditions is presented in figure 1(a ) .
little papp - a expression was observed in pbmc cultures under basal conditions after 24 hours .
treatment with crp ( 20  mg / l ) or tnf- ( 100  ng / ml ) significantly increased paap - a mrna expression at all time points ( 2 , 8 , 16 , 24 hours ) .
papp - a mrna levels increased 2 hours after stimulation with crp ( 20  mg / l ) and remained elevated by approximately 3.7-fold up to 24 hours .
pappp - a mrna expression , however , rapidly increased and peaked at approximately 6.8-fold 2 hours after tnf- ( 100  ng / ml ) stimulation . a subsequent decrease
was then observed , though levels remained elevated at approximately 4.5-fold up to 24 hours .
maximal papp - a protein expression in pbmcs and concentrations in culture supernatants were achieved with crp stimulation by 24 hours and tnf- stimulation by 8 hours ( figures 1(b ) and 1(c ) ) , reflecting the changes in papp - a mrna expression . as shown in figure 2 , dose - response experiments confirmed crp or tnf- treatment elicited dose - dependent increases in papp - a mrna expression , protein expression in pbmcs , and secretion in the supernatant after 24 hours .
crp showed half - maximal effectiveness at approximately 5  mg / l , with maximal effectiveness at approximately 20  mg / l ( figure 2 ) .
tnf- showed half - maximal effectiveness at approximately 25  ng / ml , with maximal effectiveness at approximately 100  ng / ml . the dependence of papp - a expression on mrna synthesis was explored in the following three experiments .  
figure 3 showed that the effects of these proinflammatory cytokines appeared to be at the level of transcription , as the dna - directed rna polymerase inhibitor , actinomycin d , completely prevented crp or tnf- induction of papp - a mrna expression , protein expression , and concentrations in culture supernatants .
these results showed that crp or tnf- was responsible for new protein synthesis of the papp - a protein .
furthermore , papp - a protein was actively secreted into the supernatant . as indicated in our previous experiments , treatment of human pbmcs with crp (
20  mg / l ) or tnf- ( 100  ng / ml ) significantly increased papp - a mrna expression , protein expression , and concentrations in culture supernatants . to confirm the role of nfb activation , bay11 - 7082 , which inhibits inducible phosphorylation of ib , was shown to effectively inhibited crp and tnf--stimulated papp - a expression ( figures 4(a ) , 4(b ) , and 4(c ) ) .
it is well known that the nfb pathway is the critical mediator of prooxidant stimuli , such as inflammatory cytokines .
the basic mechanism by which nfb is activated is through phosphorylation of intrinsic inhibitors , with the ib , subsequently freeing nfb to translocate into the nucleus where it regulates gene expression .
novel markers of coronary artery disease progression have been confirmed in recent years , with circulating levels of papp - a standing out as one of the most prominent indicators of this profile .
the current study indicates that papp - a expression in human pbmcs may be regulated by crp and tnf- through the nf-b pathway , a mechanism that may play a critical role in increases in serum papp - a levels during acute coronary syndrome ( acs ) .
these findings are consistent with previous reports indicating that papp - a is a marker of atheromatous plaque instability as well as extent and prognosis of cardiovascular disease [ 1315 ] .
serum papp - a levels increase in patients with acs , indicating that papp - a may also be a marker of adverse events [ 1618 ] .
moreover , in chronic stable angina ( csa ) patients , papp - a is an independent predictor for the extent of vessel stenosis , where it has been shown to correlate with the presence of vulnerable coronary artery stenosis [ 19 , 20 ] .
thus , papp - a levels have demonstrated a firm association with angiographic plaque complexity in csa patients .
the stimulation of papp - a expression by tnf- has been observed previously in human fibroblasts , osteoblasts , vsmcs , and ecs [ 4 , 11 , 22 ] . using specific monoclonal antibodies , bayes - genis et al .
reported that papp - a was abundantly expressed in both eroded and ruptured plaques , but was only minimally expressed in stable plaques .
moreover , in plaques with large lipid cores and cap rupture , staining for papp - a occurred mostly in the inflammatory shoulder region , in areas surrounding the lipid core , and in areas with localized cd68-positive cells .
thus , papp - a levels have been associated with inflammation in regions of atherosclerotic plaques , potentially contributing to progression and poor outcomes in patients .  
most evidence of monocyte involvement of papp - a expression has been completed through analysis of atherosclerotic plaques , where monocytes are the predominant leukocyte contributing to the development , progression , and instability of atherosclerotic lesions , they often contain high levels of papp - a . because samples of plaques may contain a mixture of leukocytes and circulating compounds , these tests have left the source of papp - a observed in these monocytes a point of debate among researchers .
double immunofluorescence confocal microscopy ( icm ) has been used to characterize cell types expressing papp - a , suggesting that monocytes are the primary source of papp - a in plaques and the target cells for cytokines .
it has also been speculated that papp - a may be produced by activated monocytes or macrophage cells in unstable plaques and released into the extracellular matrix and circulation .
conversely , conover et al . suggested that in vivo macrophages actually failed to produce papp - a , but instead internalized the compound through their membranes , thus accounting for the accumulation of circulating papp - a produced by other sources in macrophage cells associated with plaques .
the current study , however , indicates that the specific mrna expression associated with papp - a production increases in macrophages in vitro , resulting in expression and supernatant secretion of papp - a .
these findings indicate that papp - a production occurs in macrophages rather than being internalized through the membrane , as suggested by conover et al .
further in vivo studies will be required to assess the affect of circulating papp - a levels on macrophage papp - a expression and secretion , which may account for the discrepancies between these two studies .
cumulatively , the findings of the current study indicate that macrophages in cultured human pbmcs can synthesize and secrete papp - a .  
furthermore , crp and tnf- were indicated to be potent stimulators of papp - a gene expression and protein secretion in human pbmcs , suggesting a link between the increase in local inflammatory cytokine production and papp - a during acs .
while further studies of the in vivo effects in acs patients will be required to confirm these results , these findings suggest that serum papp - a , hscrp , and tnf- levels may be significantly higher in acs patients than in patients with stable angina pectoris and that increasing papp - a mrna levels in patients with acs may also have a positive association with serum hscrp and tnf- mrna expression .
exploration of these effects is a topic being explored in our current research based on the initial positive findings of the current research . furthermore , the time course of stimulation by crp and tnf- differed , with papp - a expression increasing 2 hours after stimulation with crp , peaking at approximately 3.7-fold by 24 hours , whereas a rapid increase to approximately 6.8-fold was seen in only 2 hours with tnf- stimulation .
the current study provides significant evidence for papp - a production in pmbcs and stimulation by the cytokines crp and tnf- ; however , further in vivo studies will be required to verify these findings and assess the effects of circulating papp - a on pmbc papp - a production .
actinomycin d was observed to complete block the induction of papp - a mrna expression by crp and tnf- , indicating potential regulation at the level of transcription .
the rapid increase in papp - a mrna levels after treatment with these cytokines further confirms the hypothesis of transcriptional regulation .
as expected , increases in papp - a protein expression and secretion into the supernatants were paralleled by increases in gene expression .
based on this observation , it is possible that the biological consequence of crp- and tnf--induced papp - a expression in human pbmcs was enhanced igf - i bioactivity mediated by papp - a proteolysis of igfbp-4 , thus contributing to the progression of both coronary atherosclerosis and restenosis .
it is well known that the free fraction of circulating and locally synthesized igf - i stimulates vsmc proliferation , migration , and extracellular matrix synthesis . in macrophages
, igf - i also promotes excess ldl cholesterol uptake , production of proinflammatory cytokines , and chemotaxis [ 26 , 27 ] .
crp and tnf- were also identified as potential regulators of papp - a expression , operating through a mechanism involving the activation of the nfb system in pbmcs of healthy volunteers .
nfb is a ubiquitous transcription factor that is activated by inflammatory cytokines , infection , oxidative stress , and shear stress .
ritchie reported that nfb was activated in peripheral monocytes using an electromobility shift assay in patients suffering from unstable angina .
the nfb family of transcription factors plays a critical role in coordinating and regulating the expression of a wide variety of inflammatory genes that have been linked to the pathologies of acs .
some studies have even demonstrated that crp induces nfb activity in various cell types , including peripheral monocytes , saphenous vein endothelial cells , and human aortic endothelial cells [ 23 , 33 , 34 ] .
moreover , resch et al . reported that tnf- induced ib degradation and papp - a - regulated expression in human fibroblasts by tnf- was mediated by nfb activation .
the current study also demonstrated that bay11 - 7082 was a potent inhibitor of both crp- and tnf--stimulated papp - a expression in human pbmcs .
the view of nfb as a transcription factor for papp - a gene expression , however , will require further study to identify the promoter region and validate these findings .
the current study provides a variety of evidence to support the expression on papp - a by leukocytes in pmbcs , including increased papp - a - associated mrna expression , papp - a expression , and papp - a secretion into the supernatant of fresh in vitro samples collected from healthy subjects .
furthermore , the cytokines crp and tnf- were shown to stimulate papp - a expression in these cells .
based on these findings and the previously observed association between pmbcs and acs , it is likely that activated monocytes or macrophages in pmbcs surrounding developing artherosclerotic plaques may secrete proinflammatory cytokines , thus stimulating the expression and secretion of papp - a .
further studies will , however , be required to assess the effects of elevated papp - a concentrations on autocrine and paracrine mechanisms for the exacerbation of atherosclerosis procession and plaque rupture through igfbp-4 cleavage and enhanced local igf - i bioavailability .
the current results , however , provide the fundamental mechanistic groundwork for further understanding of the entire mechanism associated with cytokine regulation of papp - a expression and igf bioavailability .
this understanding may lead to the future development of novel therapeutic targets for the treatment of acs .
 
 objective . the effects of c - reactive protein ( crp ) and tumor necrosis factor- ( tnf- ) on pregnancy - associated plasma protein - a ( papp - a ) expression in human peripheral blood mononuclear cells ( pbmcs ) require further investigation . methods . 
 the papp - a levels in culture supernatants , papp - a mrna expression , and cellular papp - a expression were measured in human pbmcs isolated from fresh blood donations provided by 6 healthy volunteers ( 4 donations per volunteer ) . 
 analyses were conducted by ultrasensitive elisa , western blotting , and rt - pcr following stimulation with crp or tnf- cytokines . 
 results . 
 papp - a mrna and protein levels after crp stimulation peaked at 24 hours , whereas peak papp - a mrna and protein levels were achieved after tnf- stimulation at only 2 and 8 hours , respectively . 
 these findings indicate the dose - dependent effect of crp and tnf- stimulation . 
 actinomycin d treatment completely prevented crp and tnf- induction of papp - a mrna and protein expression . 
 additionally , nuclear factor- ( nf- ) b inhibitor ( bay11 - 7082 ) potently inhibited both crp and tnf- stimulated papp - a mrna and protein expression . conclusions . 
 human pbmcs are capable of expressing papp - a in vitro , expression that may be regulated by crp and tnf- through the nf-b pathway . 
 this mechanism may play a significant role in the observed increase of serum papp - a levels in acute coronary syndrome ( acs ) . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion 