Yonsei Med J.  2017 Mar;58(2):305-311. 10.3349/ymj.2017.58.2.305.

VEGF Promoter Polymorphism Confers an Increased Risk of Pulmonary Arterial Hypertension in a Chinese Population

• Zhuo Y
• Zeng Q
• Zhang P
• Li G
• Xie Q
• Cheng Y

• KMID: 2427118
• DOI: http://doi.org/10.3349/ymj.2017.58.2.305

Abstract

PURPOSE
Evidence on the contribution of genes to the hereditary predisposition to pulmonary arterial hypertension (PAH) is limited.
MATERIALS AND METHODS
In this study, we hypothesized that single nucleotide variants in vascular endothelial growth factor (VEGF) gene may alter gene function and expression and may be associated with PAH risk. Five putatively functional loci (rs699947C>A and rs833061T>C in the promoter, rs3025040C>T, rs10434G>A and rs3025053G>A in the 3'-UTR) in the VEGF gene were genotyped and analyzed in a retrospective study of 587 patients with PAH and 736 healthy subjects from southern China.
RESULTS
We found that the rs833061T>C polymorphism was significantly associated with PAH risk, while the other single nucleotide polymorphisms were not. Compared to carriers with TT genotype, those with rs833061C variant genotype (CT/CC) had an increased risk of PAH (odds ratio=1.47, 95% confidence interval=1.18-1.83, p=0.001). Functional assays indicated that CT/CC variant genotype had significantly higher mRNA levels of VEGF in peripheral blood mononuclear cells than TT genotype (p=0.021). Luciferase reporter assay indicated that having a C allele conferred a significantly higher transcription activity than that with a T allele.
CONCLUSION
Our findings suggest that the functional polymorphism rs833061T>C in VEGF gene promoter modulates VEGF expression and may be a valuable biomarker for predicting PAH susceptibility.

Keyword

VEGF; polymorphism; pulmonary arterial hypertension; susceptibility

MeSH Terms

Alleles
Asian Continental Ancestry Group/*genetics
Case-Control Studies
China/epidemiology
Familial Primary Pulmonary Hypertension/ethnology/*genetics
Female
*Genetic Predisposition to Disease
Genotype
Humans
Leukocytes, Mononuclear
Male
Middle Aged
Odds Ratio
*Polymorphism, Single Nucleotide
*Promoter Regions, Genetic
RNA, Messenger
Retrospective Studies
Risk
Vascular Endothelial Growth Factor A/*genetics
RNA, Messenger
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Effects of rs833061T>C SNP on VEGF expression. (A) Graphic illustration of the reporter constructs comprising the VEGF gene promoter with 2475 bp containing rs833061T or C allele. (B) Luciferase expression levels of different VEGF promoter constructs in PASMCs. The Renilla luciferase activity of each reporter was normalized against the internal control of firefly luciferase. Columns, mean from three separate assays; bars, standard deviation. (C) Comparison of relative mRNA levels in PBMCs between patients and healthy controls. (D) Relative mRNA expression of the VEGF gene by rs833061T>C genotypes. VEGF, vascular endothelial growth factor; PASMCs, pulmonary artery smooth muscle cells; PBMCs, peripheral blood mononuclear cells; SNP, single nucleotide polymorphism.

Reference

1. Sitbon O, Morrell N. Pathways in pulmonary arterial hypertension: the future is here. Eur Respir Rev. 2012; 21:321–327.

2. Wilcox SR, Kabrhel C, Channick RN. Pulmonary hypertension and right ventricular failure in emergency medicine. Ann Emerg Med. 2015; 66:619–628.

3. McLaughlin V. Managing pulmonary arterial hypertension and optimizing treatment options: prognosis of pulmonary artery hypertension. Am J Cardiol. 2013; 111:8 Suppl. 10C–15C.

4. Park YM, Chung WJ, Choi DY, Baek HJ, Jung SH, Choi IS, et al. Functional class and targeted therapy are related to the survival in patients with pulmonary arterial hypertension. Yonsei Med J. 2014; 55:1526–1532.

5. Evans I. An overview of VEGF-mediated signal transduction. Methods Mol Biol. 2015; 1332:91–120.

6. Guru SK, Pathania AS, Kumar S, Ramesh D, Kumar M, Rana S, et al. Secalonic acid-D represses HIF1α/VEGF-mediated angiogenesis by regulating the Akt/mTOR/p70S6K signaling cascade. Cancer Res. 2015; 75:2886–2896.

7. Massena S, Christoffersson G, VÅgesjö E, Seignez C, Gustafsson K, Binet F, et al. Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans. Blood. 2015; 126:2016–2026.

8. Fearnley GW, Bruns AF, Wheatcroft SB, Ponnambalam S. VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration. Biol Open. 2015; 4:731–742.

9. Khayati F, Pérez-Cano L, Maouche K, Sadoux A, Boutalbi Z, Podgorniak MP, et al. EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF. Oncotarget. 2015; 6:9766–9780.

10. Smadja DM, Levy M, Huang L, Rossi E, Blandinières A, Israel-Biet D, et al. Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells. Thromb Haemost. 2015; 114:735–747.

11. Seyfarth HJ, Sack U, Gessner C, Wirtz H. [Angiogenin, bFGF and VEGF: angiogenic markers in breath condensate of patients with pulmonary hypertension]. Pneumologie. 2015; 69:207–211.

12. Vannay A, Vásárhelyi B, Környei M, Treszl A, Kozma G, Györffy B, et al. Single-nucleotide polymorphisms of VEGF gene are associated with risk of congenital valvuloseptal heart defects. Am Heart J. 2006; 151:878–881.

13. Smedts HP, Isaacs A, de Costa D, Uitterlinden AG, van Duijn CM, Gittenberger-de Groot AC, et al. VEGF polymorphisms are associated with endocardial cushion defects: a family-based case-control study. Pediatr Res. 2010; 67:23–28.

14. Han X, Liu L, Niu J, Yang J, Zhang Z, Zhang Z. Association between VEGF polymorphisms (936c/t, -460t/c and -634g/c) with haplotypes and coronary heart disease susceptibility. Int J Clin Exp Pathol. 2015; 8:922–927.

15. Sitbon O, Humbert M, Jaïs X, Ioos V, Hamid AM, Provencher S, et al. Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation. 2005; 111:3105–3111.

16. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3:1101–1108.

17. Uh HW, Mertens BJ, Jan van der Wijk H, Putter H, van Houwelingen HC, Houwing-Duistermaat JJ. Model selection based on logistic regression in a highly correlated candidate gene region. BMC Proc. 2007; 1:Suppl 1. S114.

18. Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, et al. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol. 2004; 43:12 Suppl S. 13S–24S.

19. Soubrier F, Chung WK, Machado R, Grünig E, Aldred M, Geraci M, et al. Genetics and genomics of pulmonary arterial hypertension. J Am Coll Cardiol. 2013; 62:25 Suppl. D13–D21.

20. de Jesus Perez VA. Molecular pathogenesis and current pathology of pulmonary hypertension. Heart Fail Rev. 2016; 21:239–257.

21. Guignabert C, Tu L, Girerd B, Ricard N, Huertas A, Montani D, et al. New molecular targets of pulmonary vascular remodeling in pulmonary arterial hypertension: importance of endothelial communication. Chest. 2015; 147:529–537.

22. Morin-Brureau M, Lebrun A, Rousset MC, Fagni L, Bockaert J, de Bock F, et al. Epileptiform activity induces vascular remodeling and zonula occludens 1 downregulation in organotypic hippocampal cultures: role of VEGF signaling pathways. J Neurosci. 2011; 31:10677–10688.

23. Zhang J, Silva T, Yarovinsky T, Manes TD, Tavakoli S, Nie L, et al. VEGF blockade inhibits lymphocyte recruitment and ameliorates immune-mediated vascular remodeling. Circ Res. 2010; 107:408–417.

24. Li Y, Zhu H, Klausen C, Peng B, Leung PC. Vascular endothelial growth factor-A (VEGF-A) mediates activin A-induced human trophoblast endothelial-like tube formation. Endocrinology. 2015; 156:4257–4268.

25. Lee CG, Link H, Baluk P, Homer RJ, Chapoval S, Bhandari V, et al. Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung. Nat Med. 2004; 10:1095–1103.

26. Gnudi L, Benedetti S, Woolf AS, Long DA. Vascular growth factors play critical roles in kidney glomeruli. Clin Sci (Lond). 2015; 129:1225–1236.

27. Kieran MW, Kalluri R, Cho YJ. The VEGF pathway in cancer and disease: responses, resistance, and the path forward. Cold Spring Harb Perspect Med. 2012; 2:a006593.

28. Baker GJ, Yadav VN, Motsch S, Koschmann C, Calinescu AA, Mineharu Y, et al. Mechanisms of glioma formation: iterative perivascular glioma growth and invasion leads to tumor progression, VEGF-independent vascularization, and resistance to antiangiogenic therapy. Neoplasia. 2014; 16:543–561.

29. Métais C, Li J, Li J, Simons M, Sellke FW. Effects of coronary artery disease on expression and microvascular response to VEGF. Am J Physiol. 1998; 275(4 Pt 2):H1411–H1418.

30. Bull TM, Coldren CD, Geraci MW, Voelkel NF. Gene expression profiling in pulmonary hypertension. Proc Am Thorac Soc. 2007; 4:117–120.

31. Hojo Y, Ikeda U, Zhu Y, Okada M, Ueno S, Arakawa H, et al. Expression of vascular endothelial growth factor in patients with acute myocardial infarction. J Am Coll Cardiol. 2000; 35:968–973.

32. García-Closas M, Malats N, Real FX, Yeager M, Welch R, Silverman D, et al. Large-scale evaluation of candidate genes identifies associations between VEGF polymorphisms and bladder cancer risk. PLoS Genet. 2007; 3:e29.

33. Jung YW, Kim JO, Rah H, Kim JH, Kim YR, Lee Y, et al. Genetic variants of vascular endothelial growth factor are associated with recurrent implantation failure in Korean women. Reprod Biomed Online. 2016; 32:190–196.

34. Nazir N, Siddiqui K, Al-Qasim S, Al-Naqeb D. Meta-analysis of diabetic nephropathy associated genetic variants in inflammation and angiogenesis involved in different biochemical pathways. BMC Med Genet. 2014; 15:103.