Medical Research Journal:
Versican and coronary artery spasm
Department of Pediatrics Nephrology, Ziegler's group, MHH, OE 6720, Carl-Neuberg-Strae 1, D-30625 Hannover, Germany
Correpondence to Mohamed Mohamed, Department of Pediatrics Nephrology, Ziegler's group, MHH, OE 6720, Carl-Neuberg-Strae 1 D-30625 Hannover, Germany Tel: +49-511-532-7806; fax: +49-511-532-8073; e-mail: Mohamed.firstname.lastname@example.org
Received January 14, 2012
Accepted February 13, 2012
Objective: Coronary artery spasm is a temporary, sudden narrowing of one of the coronary arteries. The spasm slows or stops blood flow through the artery and deprives part of the heart of oxygen-rich blood. A contraction of muscles in the artery wall causes this spasm. The coronary artery may appear normal during angiography, but it does not function normally. Versican (VCAN) is synthesized by vascular smooth muscle cells, accumulates during arterial atherosclerosis and restenosis, and has been shown to bind low-density lipoproteins. VCAN is important for the proliferation of smooth muscle cells as well as for the inhibition of elastogenesis. The aim of this work was to assess VCAN gene expression in coronary spasm patients.
Methods: Blood samples were collected, with consent, from 20 angiographically documented coronary spasm patients over 4 years from the NHI (Egypt). The VCAN gene expression in the leukocytes of the cases was compared with its expression in the leukocytes of 20 age-matched and sex-matched control individuals who have never complained of any cardiac or angina symptoms. Blood samples were collected in PAXgene Blood RNA tubes and stored at −70° till RNA extraction according to the PAXgene Blood RNA Kit. This was followed by the assessment of VCAN expression by quantitative real-time PCR using TaqMan gene expression assay probes for total VCAN, V0, V1, V2, V3, and cyclophilin A for normalization.
Results: The most abundant expression in both cases and controls was for total VCAN, followed by V1, V2, V0, and V3. There was no significant difference between cases and controls in the expression of total VCAN or any VCAN isoform (P values>0.05).
Conclusion: Although VCAN appears to play an important role in vasospastic angina, the molecular expression does not vary significantly between cases and controls. This may require more invasive studies or may be related to various gene–environment interactions.
Coronary artery vasospasm, or smooth muscle constriction of the coronary artery, is a major cause of chest pain syndromes that can lead to myocardial infarction, ventricular arrhythmias, and sudden death. It also plays a key role in the development of myocardial infarction subsequent to atherosclerotic lesions. In 1959, Prinzmetal et al. 1 reported a syndrome of nonexertional chest pain with ST-segment elevation.
Unlike patients with typical angina, exercise tolerance in these patients was characteristically normal and pain patterns tended to be cyclical, with most episodes occurring in the early morning hours without regard to cardiac workload. This syndrome became known as Prinzmetal’s or variant angina 1 and was believed to be because of vasospasm in coronary arteries without obstructive lesions. Subsequently, Maseri et al. 2 described the clinical, ECG, and angiographic features of 138 patients with variant angina and concluded that the syndrome is considerably more polymorphic than initially reported by Prinzmetal et al. 1.
The pathophysiological mechanisms leading to coronary artery vasospasm are incompletely understood at present. Coronary arterial tone varies normally through physiological mechanisms, but the degree of vasoconstriction can range along a spectrum from undetectable constriction to complete arterial occlusion. In some patients with partial vasoconstriction, symptoms can arise with activities that exceed a threshold of myocardial demand 3. In other patients, constriction may be so severe that myocardial ischemia develops at rest 4. Interestingly, certain behavioral traits (such as type A personality, panic disorder, and severe anxiety) have also been described in association with coronary artery vasospasm 4.
Versican (VCAN) is a chondroitin sulfate proteoglycan (CSPG) that is present in the extracellular matrix (ECM) of normal blood vessels and increases markedly in all forms of vascular disease 5,6. A number of reports over the last few years have documented a significant involvement of VCAN in lesions of atherosclerosis and restenosis and these observations, coupled with those showing that ECM proteoglycan regulates many of the events that contribute to the formation of atherosclerotic and restenotic lesions, highlight the critical importance of VCAN in the pathogenesis of vascular disease 6. VCAN is one of many proteoglycans identified in vascular tissue or synthesized by vascular cells 7 and, together with biglycan, decorin, and perlecan, constitute the bulk of the proteoglycans found in the interstitial space 6. VCAN interacts with hyaluronan, a long-chain high-molecular-weight glycosaminoglycan (GAG) that is also present in the ECM of blood vessels and increases as VCAN in vascular disease 8.
VCAN exists in at least four different isoforms created by alternative splicing of mRNA from a single gene 9. RNA splicing occurs in the two large exons, 7 and 8, that encode the GAG attachment sites, yielding V0, V1, V2, and V3 forms of VCAN 10. These variants differ in the length of the core proteins and the number of attached GAGs 11. The expression of the VCAN gene is regulated by a promoter that harbors a typical TATA box and potential binding sites for several transcription factors such as AP2, CCAAT enhancer protein, and cAMP-responsive elements 12.
VCAN is the principal CSPG in blood vessels 13 and immunohistochemistry shows that VCAN is prominent in the intima and adventitia of most arteries and veins 14. It is likely that the accumulation of VCAN in the normal arterial intima is mainly responsible for the proteoglycan-rich nature of this layer 15. VCAN interacts with hyaluronan and links protein to form high-molecular-weight stable aggregates (http://circres.ahajournals.org/content/94/9/1158.full-ref-13) 16–18 that fill the ECM space not occupied by the other fibrous proteins such as collagens and elastic fibers. These complexes create a reversibly compressive compartment and provide a swelling pressure within the ECM that is offset by collagen and elastic fibers (http://circres.ahajournals.org/content/94/9/1158.full20-20ref-16) 19,20. Early biochemical studies of bovine (http://circres.ahajournals.org/content/94/9/1158.full20-20ref-18) 21,22 and human aorta (http://circres.ahajournals.org/content/94/9/1158.full20-20ref-20) 23 have identified a large CSPG that resembled the principal CSPG synthesized by cultured arterial smooth muscle cells (ASMCs) (http://circres.ahajournals.org/content/94/9/1158.full20-20ref-21) 24,25 and later identified as VCAN 5. Recent studies have identified specific cleavage products of VCAN in the human aorta that contain the G1 domain of VCAN. Further studies have identified the enzymes that cleaved aortic VCAN as members of the ADAMS-TS family (http://circres.ahajournals.org/content/94/9/1158.full20-20ref-23) 26,27. Other proteases in arteries that are known to degrade VCAN include matrilysin and plasmin 28. These findings are consistent with the presence of multiple sizes of VCAN and VCAN fragments within vascular tissue. Versican is important for the proliferation of smooth muscle cells as well as for the inhibition of elastogenesis 29.
The aim of this work was to assess VCAN gene expression in coronary spasm patients.
Materials and methods
Blood samples were collected, with consents, from 20 angiographically documented coronary spasm patients (angina patients with ST-segment elevation and normal-to-mild atherosclerotic coronaries with a positive provocation test) over 4 years from the NHI (Egypt). The VCAN gene expression in the leukocytes of the cases was compared with its expression in the leukocytes of 20 age-matched and sex-matched control individuals who have never complained of any cardiac or angina symptoms. All patients and controls were smokers, nondiabetic, and nonhypertensive, with normal plasma lipid profiles.
Blood samples were collected in PAXgene Blood RNA tubes and stored at −70° until RNA extraction according to the PAXgene Blood RNA Kit. This was followed by the assessment of VCAN expression by quantitative real-time PCR using TaqMan gene expression assay probes for total VCAN, V0, V1, V2, V3, and cyclophilin A for normalization.
Quantitative real-time PCR was performed to evaluate VCAN mRNA expression in the leukocytes of cases and controls. cDNA was generated from 15 ng of total RNA using the WT-Ovation RNA amplification system for RT-PCR (NuGen Technologies, San Carlos, California, USA). Q-RT-PCR was performed using the 7900HT fast RT-PCR system and TaqMan gene expression assay probes (Applied Biosystems, Carlsbad, California, USA) for total VCAN (Hs01007940_m1), V0 (Hs 01007944_m1), V1 (Hs 01007937_m1), V2 (Hs 01007943_m1), V3 (Hs 01007941_m1), and cyclophilin (NM_0211303). The relative VCAN isoform level of each sample was normalized with endogenous cyclophilin A. The statistical analysis of the results was carried out using statistical package for the social sciences (SPSS) program version 16 (SPSS Inc., Chicago, Illinois, USA), yielding the arithmetic mean and the SD (Student’s t-test). P values 0.05 or less were considered significant 30.
This study included 20 cases and 20 controls. Each group included five men and 15 women. There was no significant difference between both groups in the age and the sex distribution (Table 1).
There was no significant difference between the cases and the controls in any of the parameters of the lipid profile (Table 2).
The results of VCAN expression are shown in Table 3.
The differential VCAN expression in the cases and the controls is shown in Fig. 1.
The most abundant expression in both cases and controls was for total VCAN, followed by V1, V2, V0, and V3. There was no significant difference between cases and controls in the expression of total VCAN or any VCAN isoform (P values>0.05, Table 3).
The current study was carried out on the basis of the concept that increased VCAN expression leads to increased proliferation of ASMCs and inhibition of elastogenesis 29. The bulk of information on the involvement of VCAN in vascular diseases of human and experimental animals comes from immunohistochemical studies 31–37. The development of an atherosclerotic lesion involves the migration and proliferation of ASMCs from the media to the intima 38. VCAN is present in early intimal thickenings that characterize developing atherosclerotic lesions and are primarily associated with ASMCs. In a gene array study of neointimal formation in graft healing, VCAN was one of only 13 genes selectively upregulated and found to accumulate in early graft lesions 38. All the above-mentioned findings support the basic hypothesis of the current study. In addition, VCAN tends to accumulate in human vessels susceptible to atherosclerosis such as the coronary arteries and saphenous veins used for grafting when compared with those resistant such as internal mammary and radial arteries 14. Whereas VCAN appears to contribute to intimal expansion and lesion progression, VCAN degradation may be part of lesion regression. In a high blood flow model of intimal atrophy in polytetrafluoroethylene aortoiliac grafts, extracts of 4-day high-flow intimas degraded more vascular VCAN than similar extracts from 4-day low-flow intimas. This activity was shown to be because of elevated plasmin levels 29.
VCAN, with its unique structure and multiple interactions, is one of several proteoglycans that contribute to the pathogenesis of vascular disease in general. It is well placed to play a central role in the control or the modulation of key events in atherogenesis and restenosis. Although further evidence is required to confirm causative roles for VCAN, there is sufficient evidence to place it at the center of many key processes in vascular pathology 39. In moderate amounts, and in association with hyaluronan, VCAN provides a hydrated viscoelastic ECM able to accommodate cyclic mechanical forces exerted on vessels. Elevated levels, however, shift the balance in favor of pathological changes. Increased amounts, especially in the intima, contribute physically to increased intimal thickness and to the creation of a pericellular and ECM that supports cell migration and proliferation and inflammatory cell adhesion 39. Concomitantly, VCAN enrichment of the pericellular matrix decreases the residence time for cell-associated receptors involved in ECM assembly, such as elastin-binding protein, thereby decreasing elastic fiber assembly and influencing ASMC proliferation and the mechanical properties of the expanding intima 39. The most damaging, however, is the alteration in the chondroitin sulfate chains of VCAN, caused by specific growth factors and cytokines, that results in increased retention and accumulation of lipoproteins 39. Finally, the presence of VCAN at the plaque thrombus interface implicates this ECM component in one of the final phases of atherosclerosis and vessel occlusion. This encompassing hypothesis has yet to be tested, but accumulating evidence strongly suggests that VCAN is a key molecule in atherosclerosis and restenosis and a potential target for therapeutic intervention. Unfortunately, VCAN knockout is lethal because of the failure of heart and blood vessels to develop normally 40. Therefore, future directions will be to develop molecular genetic models in which the expression of VCAN and its variants can be selectively controlled so that the impact of removing this component from the developing vascular pathology can be assessed directly and the molecular mechanisms responsible for the effects on vascular cell phenotype can be determined.
There are no published studies that link vasospastic angina and VCAN expression. Thus, this study is considered a leading one in this respect, although its results are not positive. However, nonsignificant differences between patients and controls in this study may be because of the small sample size of each group (only 20 cases and 20 controls), which again can be attributed to the relative rareness of the disease. Much larger samples may yield more precise results. Another possible explanation for these results is that, in the current study, the VCAN expression was assessed in leukocytes of patients and controls, whereas the study of such a vascular disease requires the assessment of VCAN expression within the vascular wall in correlation to the wall histology. The aim of this study was to search for a noninvasive marker for this disease; thus, an assessment of expression in leukocytes was carried out. However, the study of arterial biopsies may ultimately yield more accurate results.
The finding that leukocytes express the extracellular matrix gene VCAN, which is known to be mainly expressed by ASMCs, was surprising. However, this was in agreement with the result obtained by Zheng and colleagues and Toeda and colleagues in two different studies. Zheng et al. 41 detected VCAN and its degraded C-terminal G3 fragments in human plasma and observed that the VCAN G3 domain promoted blood coagulation. Toeda and colleagues examined VCAN expression in a rat model of myocardial infarction. Northern blot analysis indicated increased expression of VCAN mRNA. Quantitative real-time RT-PCR analysis showed that VCAN mRNA began to increase as early as 6 h and reached its maximal level 2 days after coronary artery ligation. VCAN mRNA then decreased gradually, whereas the mRNA of decorin, another small proteoglycan, increased thereafter. VCAN mRNA was localized in monocytes, as indicated by CD68-positive staining, around the infarct tissue. Toeda and colleagues then isolated human peripheral blood mononuclear cells and stimulated them with granulocyte/macrophage colony-stimulating factor. The stimulation of mononuclear cells with granulocyte/macrophage colony-stimulating factor increased the expression of VCAN mRNA as well as cytokine induction 42.
Although VCAN appears to play an important role in vasospastic angina, the molecular expression does not vary significantly between cases and controls. This may require more invasive studies or may be related to various gene–environment interactions. Documentation of such gene–environment interactions is important if genotype information is ever to be used in a clinical or a diagnostic setting. Future studies should be designed specifically to examine VCAN genetic polymorphisms and the interactions between these polymorphisms and acquired risk factors of vasospastic angina.
Conflicts of interest
There are no conflicts of interest.
1. Prinzmetal M, Kennamer R, Merliss R, Wada T, Bor N. Angina pectoris I. A variant form of angina pectoris. Preliminary report. Am J Med. 1959;27:375–388
2. Maseri A, Severi S, Nes M, L’Abbate A, Chierchia S, Marzilli M, et al. ‘Variant’ angina: one aspect of a continuous spectrum of vasospastic myocardial ischemia. Pathogenetic mechanisms, estimated incidence and clinical and coronary arteriographic findings in 138 patients. Am J Cardiol. 1978;42:1019–1035
3. Yasue H, Omote S, Takizawa A. Circadian variation of exercise capacity in patients with Prinzmetal’s variant angina: role of exercise-induced coronary arterial spasm. Circulation. 1979;59:938–948
4. Yasue H, Nakagawa H, Itoh T, Harada E, Mizuno Y. Coronary artery spasm – clinical features, diagnosis, pathogenesis and treatment. J Cardiol. 2008;51:2–17
5. Yao LY, Moody C, Schonherr E, Wight TN, Sandell LJ. Identification of the proteoglycan versican in aorta and smooth muscle cells by DNA sequence analysis, in situ hybridization and immunohistochemistry. Matrix Biol. 1994;14:213–225
6. Wight TFuster V, Ross R, Topol EJ. The vascular extracellular matrix. Atherosclerosis and coronary artery disease. 1996 New York, USA Raven Press
7. Järveläinen H, Wight TGarg HG. Vascular proteoglycans. Proteoglycans in lung disease. 20021st ed. Florida, USA CRC Press:291–321
8. Toole BP, Wight TN, Tammi MI. Hyaluronan-cell interactions in cancer and vascular disease. J Biol Chem. 2002;277:4593–4596
9. Zimmermann DR, Ruoslahti E. Multiple domains of the large fibroblast proteoglycan, versican. EMBO J. 1989;8:2975–2981
10. Ito K, Shinomura T, Zako M, Ujita M, Kimata K. Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing. J Biol Chem. 1995;270:958–965
11. Wight TN. Versican: a versatile extracellular matrix proteoglycan in cell biology. Curr Opin Cell Biol. 2002;14:617–623
12. Naso MF, Zimmermann DR, Iozzo RV. Characterization of the complete genomic structure of the human versican gene and functional analysis of its promoter. J Biol Chem. 1994;269:32999–33008
13. Theocharis AD, Tsolakis I, Hjerpe A, Karamanos NK. Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V0. Atherosclerosis. 2001;154:367–376
14. Merrilees MJ, Beaumont B, Scott LJ. Comparison of deposits of versican, biglycan and decorin in saphenous vein and internal thoracic, radial and coronary arteries: correlation to patency. Coron Artery Dis. 2001;12:7–16
15. Stary HC. The sequence of cell and matrix changes in atherosclerotic lesions of coronary arteries in the first forty years of life. Eur Heart J. 1990;11(Suppl E):3–19
16. Radhakrishnamurthy B, Jeansonne N, Tracy RE, Berenson GS. A monoclonal antibody that recognizes hyaluronic acid binding region of aorta proteoglycans. Atherosclerosis. 1993;98:179–192
17. Levesque H, Girard N, Maingonnat C, Delpech A, Chauzy C, Tayot J, et al. Localization and solubilization of hyaluronan and of the hyaluronan-binding protein hyaluronectin in human normal and arteriosclerotic arterial walls. Atherosclerosis. 1994;105:51–62
18. Binette F, Cravens J, Kahoussi B, Haudenschild DR, Goetinck PF. Link protein is ubiquitously expressed in non-cartilaginous tissues where it enhances and stabilizes the interaction of proteoglycans with hyaluronic acid. J Biol Chem. 1994;269:19116–19122
19. Comper WD, Laurent TC. Physiological function of connective tissue polysaccharides. Physiol Rev. 1978;58:255–315
20. Comper WD, Zamparo O. Hydrodynamic properties of connective-tissue polysaccharides. Biochem J. 1990;269:561–564
21. Oegema TR Jr, Hascall VC, Eisenstein R. Characterization of bovine aorta proteoglycan extracted with guanidine hydrochloride in the presence of protease inhibitors. J Biol Chem. 1979;254:1312–1318
22. Kapoor R, Phelps CF, Wight TN. Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta. Biochem J. 1986;240:575–583
23. Salisbury BGJ, Wagner WD. Isolation and preliminary characterization of proteoglycans dissociatively extracted from human aorta. J Biol Chem. 1981;256:8050–8057
24. Wight TN, Hascall VC. Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture. J Cell Biol. 1983;96:167–176
25. Chang Y, Yanagishita M, Hascall VC, Wight TN. Proteoglycans synthesized by smooth muscle cells derived from monkey (Macaca nemestrina
) aorta. J Biol Chem. 1983;258:5679–5688
26. Sandy JD, Westling J, Kenagy RD, Iruela Arispe ML, Verscharen C, Rodriguez Mazaneque JC, et al. Versican V1 proteolysis in human aorta in vivo occurs at the Glu 441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4. J Biol Chem. 2001;276:13372–13378
27. Somerville RPT, Longpre JM, Jungers KA, Engle JM, Ross M, Evanko S, et al. Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans
GON-1. J Biol Chem. 2003;278:9503–9513
28. Halpert I, Sires UI, Roby JD, Potter Perigo S, Wight TN, Shapiro SD, et al. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci USA. 1996;93:9748–9753
29. Kenagy RD, Fischer JW, Davies MG, Berceli SA, Hawkins SM, Wight TN, et al. Increased plasmin and serine proteinase activity during flow-induced intimal atrophy in baboon PTFE grafts. Arterioscler Thromb Vasc Biol. 2002;22:400–404
30. Levesque R SPSS programming and data management: a guide for SPSS and SAS users. 20074th ed. Chicago SPSS Inc.
31. Gutierrez P, O’Brien KD, Ferguson M, Nikkari ST, Alpers CE, Wight TN. Differences in the distribution of versican, decorin and biglycan in atherosclerotic human coronary arteries. Cardiovasc Pathol. 1997;6:271–278
32. Lin H, Wilson JE, Roberts CR, Horley KJ, Winters GL, Costanzo MR, et al. Biglycan, decorin and versican protein expression patterns in coronary arteriopathy of human cardiac allografts: distinctness as compared to native atherosclerosis. J Heart Lung Transpl. 1996;15:1233–1247
33. Lin H, Ignatescu M, Wilson JE, Roberts CR, Horley KJ, Winters GL, et al. Prominence of apolipoproteins B, (a) and E in the intimae of coronary arteries in transplanted human hearts: geographic relationship to vessel wall proteoglycans. J Heart Lung Transpl. 1996;15:1223–1232
34. Evanko SP, Raines EW, Ross R, Gold LI, Wight TN. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics and the proximity of platelet-derived growth factor and transforming growth factor-β. Am J Pathol. 1998;152:533–546
35. O’Brien KD, Olin KL, Alpers CE, Chiu W, Ferguson M, Hudkins K, et al. Comparison of apolipoprotein and proteoglycan deposits in human coronary atherosclerotic plaques: colocalization of biglycan with apolipoproteins. Circulation. 1998;98:519–527
36. Kolodgie FD, Burke AP, Farb A, Weber DK, Kutys R, Wight TN, et al. Differential accumulation of proteoglycans and hyaluronan in culprit lesions: Insights into plaque erosion. Arterioscler Thromb Vasc Biol. 2002;22:1642–1648
37. Luttun A, Lupu F, Storkebaum E, Hoylaerts MF, Moons L, Crawley J, et al. Lack of plasminogen activator inhibitor-1 promotes growth and abnormal matrix remodeling of advanced atherosclerotic plaques in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2002;22:499–505
38. Geary RL, Wong JM, Rossini A, Schwartz SM, Adams LD. Expression profiling identifies 147 genes contributing to a unique primate neointimal smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol. 2002;22:2010–2016
39. Wight TN, Merrilees MJ. Proteoglycans in atherosclerosis and restenosis: key roles for versican. Circ Res. 2004;94:1158–1167
40. Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR. The Cspg2 gene, disrupted in the HDF mutant, is required for right cardiac chamber and endocardial cushion formation. Dev Biol. 1998;202:56–66
41. Zheng PS, Reis M, Sparling C, Lee DY, La Pierre DP, Wong CKA, et al. Versican G3 domain promotes blood coagulation through suppressing the activity of tissue factor pathway inhibitor-1. J Biol Chem. 2006;281:8175–8182
42. Toeda K, Nakamura K, Hirohata S, Hatipoglu OF, Demircan K, Yamawaki H, et al. Versican is induced in infiltrating monocytes in myocardial infarction. Mol Cell Biochem. 2005;280:47–56
© 2012 Medical Research Journal
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