Bioinformatics analysis for bicuspid aortic valve with ascending aorta dilation_West China Medical Journal

Authors：

LEI Wenhua ^1,2 , HUANG Fangyang ¹ , LIAO Yanbiao ¹ , LI Changming ¹ , LI Junli ² ,  YANG Yiqing ³ ,  CHEN Mao ^1,2

1. Department of Cardiology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Laboratory of Heart Valve Disease, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
3. Department of Cardiology, Shanghai Fifth People’s Hospital, Fudan University, Shanghai 200240, P. R. China;

Corresponding author：

YANG Yiqing, Email: dryyq@tongji.edu.cn; CHEN Mao, Email: hmaochen@vip.sina.com

Keywords：

Bicuspid aortic valve; ascending aorta dilation; bioinformatics; differentially expressed gene; immune infiltration

DOI：

10.7507/1002-0179.202206098

Video：

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Objective To explore the key genes, pathways and immune cell infiltration of bicuspid aortic valve (BAV) with ascending aortic dilation by bioinformatics analysis. Methods The data set GSE83675 was downloaded from the Gene Expression Omnibus database (up to May 12th, 2022). Differentially expressed genes (DEGs) were analyzed and gene set enrichment analysis (GSEA) was conducted using R language. STRING database and Cytoscape software were used to construct protein-protein interaction (PPI) network and identify hub genes. The proportion of immune cells infiltration was calculated by CIBERSORT deconvolution algorithm. Results There were 199 DEGs identified, including 19 up-regulated DEGs and 180 down-regulated DEGs. GSEA showed that the main enrichment pathways were cytokine-cytokine receptor interaction, pathways in cancer, regulation of actin cytoskeleton, chemokine signaling pathway and mitogen-activated protein kinase signaling pathway. Ten hub genes (EGFR, RIMS3, DLGAP2, RAPH1, CCNB3, CD3E, PIK3R5, TP73, PAK3, and AGAP2) were identified in PPI network. CIBERSORT analysis showed that activated natural killer cells were significantly higher in dilated aorta with BAV. Conclusions These identified key genes and pathways provide new insights into BAV aortopathy. Activated natural killer cells may participate in the dilation of ascending aorta with BAV.

Citation： LEI Wenhua, HUANG Fangyang, LIAO Yanbiao, LI Changming, LI Junli, YANG Yiqing, CHEN Mao. Bioinformatics analysis for bicuspid aortic valve with ascending aorta dilation. West China Medical Journal, 2022, 37(8): 1203-1212. doi: 10.7507/1002-0179.202206098 Copy

1.	Siu SC, Silversides CK. Bicuspid aortic valve disease. J Am Coll Cardiol, 2010, 55(25): 2789-2800.
2.	Michelena HI, Khanna AD, Mahoney D, et al. Incidence of aortic complications in patients with bicuspid aortic valves. JAMA, 2011, 306(10): 1104-1112.
3.	Verma S, Siu SC. Aortic dilatation in patients with bicuspid aortic valve. N Engl J Med, 2014, 370(20): 1920-1929.
4.	Ferencik M, Pape LA. Changes in size of ascending aorta and aortic valve function with time in patients with congenitally bicuspid aortic valves. Am J Cardiol, 2003, 92(1): 43-46.
5.	Beroukhim RS, Kruzick TL, Taylor AL, et al. Progression of aortic dilation in children with a functionally normal bicuspid aortic valve. Am J Cardiol, 2006, 98(6): 828-830.
6.	Davis S, Meltzer PS. GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor. Bioinformatics, 2007, 23(14): 1846-1847.
7.	Hirata Y, Aoki H, Shojima T, et al. Activation of the AKT pathway in the ascending aorta with bicuspid aortic valve. Circ J, 2018, 82(10): 2485-2492.
8.	Wickham H. ggplot2: elegant graphics for data analysis. 2nd ed. New York: Springer, 2016: 189-201.
9.	Tang Y, Horikoshi M, Li W. Ggfortify: unified interface to visualize statistical results of popular R packages. R J, 2016, 8(2): 474-485.
10.	Ritchie ME, Phipson B, Wu D, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res, 2015, 43(7): e47.
11.	Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A, 2005, 102(43): 15545-15550.
12.	Gennady K, Vladimir S, Nikolay B, et al. Fast gene set enrichment analysis. (2016-06-20)[2021-02-01]. https://www.biorxiv.org/content/10.1101/060012v3.
13.	Wu T, Hu E, Xu S, et al. ClusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation (Camb), 2021, 2(3): 100141.
14.	Newman AM, Steen CB, Liu CL, et al. Determining cell type abundance and expression from bulk tissues with digital cytometry. Nat Biotechnol, 2019, 37(7): 773-782.
15.	LeMaire SA, Wang X, Wilks JA, et al. Matrix metalloproteinases in ascending aortic aneurysms: bicuspid versus trileaflet aortic valves. J Surg Res, 2005, 123(1): 40-48.
16.	de Sa M, Moshkovitz Y, Butany J, et al. Histologic abnormalities of the ascending aorta and pulmonary trunk in patients with bicuspid aortic valve disease: clinical relevance to the ross procedure. J Thorac Cardiovasc Surg, 1999, 118(4): 588-594.
17.	Fedak PW, de Sa MP, Verma S, et al. Vascular matrix remodeling in patients with bicuspid aortic valve malformations: implications for aortic dilatation. J Thorac Cardiovasc Surg, 2003, 126(3): 797-806.
18.	Messner B, Bernhard D. Bicuspid aortic valve-associated aortopathy: where do we stand?. J Mol Cell Cardiol, 2019, 133: 76-85.
19.	Holbro T, Hynes NE. ErbB receptors: directing key signaling networks throughout life. Annu Rev Pharmacol Toxicol, 2004, 44: 195-217.
20.	Barrick CJ, Roberts RB, Rojas M, et al. Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice. Am J Physiol Heart Circ Physiol, 2009, 297(1): H65-H75.
21.	Schreier B, Rabe S, Schneider B, et al. Loss of epidermal growth factor receptor in vascular smooth muscle cells and cardiomyocytes causes arterial hypotension and cardiac hypertrophy. Hypertension, 2013, 61(2): 333-340.
22.	Krause M, Leslie JD, Stewart M, et al. Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Dev Cell, 2004, 7(4): 571-583.
23.	Li H, Wu X, Hou S, et al. Phosphatidylinositol-3, 4-bisphosphate and its binding protein lamellipodin regulate chemotaxis of malignant B lymphocytes. J Immunol, 2016, 196(2): 586-595.
24.	Brazzo JA, Biber JC, Nimmer E, et al. Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation. J Cell Sci, 2021, 134(12): jcs257709.
25.	Vehlow A, Soong D, Vizcay-Barrena G, et al. Endophilin, lamellipodin, and mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis. EMBO J, 2013, 32(20): 2722-2734.
26.	Nguyen TB, Manova K, Capodieci P, et al. Characterization and expression of mammalian cyclin b3, a prepachytene meiotic cyclin. J Biol Chem, 2002, 277(44): 41960-41969.
27.	Davis MM. A new trigger for T cells. Cell, 2002, 110(3): 285-287.
28.	Borroto A, Lama J, Niedergang F, et al. The CD3ε subunit of the TCR contains endocytosis signals. J Immunol, 1999, 163(1): 25-31.
29.	Vadas O, Dbouk HA, Shymanets A, et al. Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs). Proc Natl Acad Sci U S A, 2013, 110(47): 18862-18867.
30.	Voigt P, Brock C, Nürnberg B, et al. Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase gamma. J Biol Chem, 2005, 280(6): 5121-5127.
31.	Fernandez-Alonso R, Martin-Lopez M, Gonzalez-Cano L, et al. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling. Cell Death Differ, 2015, 22(8): 1287-1299.
32.	Sabapathy K. p73: a positive or negative regulator of angiogenesis, or both?. Mol Cell Biol, 2015, 36(6): 848-854.
33.	Maeso-Alonso L, López-Ferreras L, Marques MM, et al. p73 as a tissue architect. Front Cell Dev Biol, 2021, 9: 716957.
34.	Hooper C, Tavassoli M, Chapple JP, et al. TAp73 isoforms antagonize Notch signalling in SH-SY5Y neuroblastomas and in primary neurones. J Neurochem, 2006, 99(3): 989-999.
35.	Rufini A, Niklison-Chirou MV, Inoue S, et al. TAp73 depletion accelerates aging through metabolic dysregulation. Genes Dev, 2012, 26(18): 2009-2014.
36.	Billaud M, Phillippi JA, Kotlarczyk MP, et al. Elevated oxidative stress in the aortic media of patients with bicuspid aortic valve. J Thorac Cardiovasc Surg, 2017, 154(5): 1756-1762.
37.	Balint B, Yin H, Nong Z, et al. Seno-destructive smooth muscle cells in the ascending aorta of patients with bicuspid aortic valve disease. EBioMedicine, 2019, 43: 54-66.
38.	Gao Z, Zhong M, Ye Z, et al. PAK3 promotes the metastasis of hepatocellular carcinoma by regulating EMT process. J Cancer, 2022, 13(1): 153-161.
39.	Navarro-Corcuera A, Ansorena E, Montiel-Duarte C, et al. AGAP2: modulating TGFβ1-signaling in the regulation of liver fibrosis. Int J Mol Sci, 2020, 21(4): 1400.
40.	Navarro-Corcuera A, López-Zabalza MJ, Martínez-Irujo JJ, et al. Role of AGAP2 in the profibrogenic effects induced by TGFβ in LX-2 hepatic stellate cells. Biochim Biophys Acta Mol Cell Res, 2019, 1866(4): 673-685.
41.	Zhu Y, Wu Y, Kim JI, et al. Arf GTPase-activating protein AGAP2 regulates focal adhesion kinase activity and focal adhesion remodeling. J Biol Chem, 2009, 284(20): 13489-13496.
42.	Ahn JY, Rong R, Kroll TG, et al. PIKE (phosphatidylinositol 3-kinase enhancer)-A GTPase stimulates Akt activity and mediates cellular invasion. J Biol Chem, 2004, 279(16): 16441-16451.
43.	Wang Y, Südhof TC. Genomic definition of RIM proteins: evolutionary amplification of a family of synaptic regulatory proteins. Genomics, 2003, 81(2): 126-137.
44.	Jiang-Xie LF, Liao HM, Chen CH, et al. Autism-associated gene Dlgap2 mutant mice demonstrate exacerbated aggressive behaviors and orbitofrontal cortex deficits. Mol Autism, 2014, 5: 32.
45.	Björck HM, Du L, Pulignani S, et al. Altered DNA methylation indicates an oscillatory flow mediated epithelial-to-mesenchymal transition signature in ascending aorta of patients with bicuspid aortic valve. Sci Rep, 2018, 8(1): 2777.
46.	Forester ND, Cruickshank SM, Scott DJ, et al. Increased natural killer cell activity in patients with an abdominal aortic aneurysm. Br J Surg, 2006, 93(1): 46-54.

1. Siu SC, Silversides CK. Bicuspid aortic valve disease. J Am Coll Cardiol, 2010, 55(25): 2789-2800.
2. Michelena HI, Khanna AD, Mahoney D, et al. Incidence of aortic complications in patients with bicuspid aortic valves. JAMA, 2011, 306(10): 1104-1112.
3. Verma S, Siu SC. Aortic dilatation in patients with bicuspid aortic valve. N Engl J Med, 2014, 370(20): 1920-1929.
4. Ferencik M, Pape LA. Changes in size of ascending aorta and aortic valve function with time in patients with congenitally bicuspid aortic valves. Am J Cardiol, 2003, 92(1): 43-46.
5. Beroukhim RS, Kruzick TL, Taylor AL, et al. Progression of aortic dilation in children with a functionally normal bicuspid aortic valve. Am J Cardiol, 2006, 98(6): 828-830.
6. Davis S, Meltzer PS. GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor. Bioinformatics, 2007, 23(14): 1846-1847.
7. Hirata Y, Aoki H, Shojima T, et al. Activation of the AKT pathway in the ascending aorta with bicuspid aortic valve. Circ J, 2018, 82(10): 2485-2492.
8. Wickham H. ggplot2: elegant graphics for data analysis. 2nd ed. New York: Springer, 2016: 189-201.
9. Tang Y, Horikoshi M, Li W. Ggfortify: unified interface to visualize statistical results of popular R packages. R J, 2016, 8(2): 474-485.
10. Ritchie ME, Phipson B, Wu D, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res, 2015, 43(7): e47.
11. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A, 2005, 102(43): 15545-15550.
12. Gennady K, Vladimir S, Nikolay B, et al. Fast gene set enrichment analysis. (2016-06-20)[2021-02-01]. https://www.biorxiv.org/content/10.1101/060012v3.
13. Wu T, Hu E, Xu S, et al. ClusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation (Camb), 2021, 2(3): 100141.
14. Newman AM, Steen CB, Liu CL, et al. Determining cell type abundance and expression from bulk tissues with digital cytometry. Nat Biotechnol, 2019, 37(7): 773-782.
15. LeMaire SA, Wang X, Wilks JA, et al. Matrix metalloproteinases in ascending aortic aneurysms: bicuspid versus trileaflet aortic valves. J Surg Res, 2005, 123(1): 40-48.
16. de Sa M, Moshkovitz Y, Butany J, et al. Histologic abnormalities of the ascending aorta and pulmonary trunk in patients with bicuspid aortic valve disease: clinical relevance to the ross procedure. J Thorac Cardiovasc Surg, 1999, 118(4): 588-594.
17. Fedak PW, de Sa MP, Verma S, et al. Vascular matrix remodeling in patients with bicuspid aortic valve malformations: implications for aortic dilatation. J Thorac Cardiovasc Surg, 2003, 126(3): 797-806.
18. Messner B, Bernhard D. Bicuspid aortic valve-associated aortopathy: where do we stand?. J Mol Cell Cardiol, 2019, 133: 76-85.
19. Holbro T, Hynes NE. ErbB receptors: directing key signaling networks throughout life. Annu Rev Pharmacol Toxicol, 2004, 44: 195-217.
20. Barrick CJ, Roberts RB, Rojas M, et al. Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice. Am J Physiol Heart Circ Physiol, 2009, 297(1): H65-H75.
21. Schreier B, Rabe S, Schneider B, et al. Loss of epidermal growth factor receptor in vascular smooth muscle cells and cardiomyocytes causes arterial hypotension and cardiac hypertrophy. Hypertension, 2013, 61(2): 333-340.
22. Krause M, Leslie JD, Stewart M, et al. Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Dev Cell, 2004, 7(4): 571-583.
23. Li H, Wu X, Hou S, et al. Phosphatidylinositol-3, 4-bisphosphate and its binding protein lamellipodin regulate chemotaxis of malignant B lymphocytes. J Immunol, 2016, 196(2): 586-595.
24. Brazzo JA, Biber JC, Nimmer E, et al. Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation. J Cell Sci, 2021, 134(12): jcs257709.
25. Vehlow A, Soong D, Vizcay-Barrena G, et al. Endophilin, lamellipodin, and mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis. EMBO J, 2013, 32(20): 2722-2734.
26. Nguyen TB, Manova K, Capodieci P, et al. Characterization and expression of mammalian cyclin b3, a prepachytene meiotic cyclin. J Biol Chem, 2002, 277(44): 41960-41969.
27. Davis MM. A new trigger for T cells. Cell, 2002, 110(3): 285-287.
28. Borroto A, Lama J, Niedergang F, et al. The CD3ε subunit of the TCR contains endocytosis signals. J Immunol, 1999, 163(1): 25-31.
29. Vadas O, Dbouk HA, Shymanets A, et al. Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs). Proc Natl Acad Sci U S A, 2013, 110(47): 18862-18867.
30. Voigt P, Brock C, Nürnberg B, et al. Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase gamma. J Biol Chem, 2005, 280(6): 5121-5127.
31. Fernandez-Alonso R, Martin-Lopez M, Gonzalez-Cano L, et al. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling. Cell Death Differ, 2015, 22(8): 1287-1299.
32. Sabapathy K. p73: a positive or negative regulator of angiogenesis, or both?. Mol Cell Biol, 2015, 36(6): 848-854.
33. Maeso-Alonso L, López-Ferreras L, Marques MM, et al. p73 as a tissue architect. Front Cell Dev Biol, 2021, 9: 716957.
34. Hooper C, Tavassoli M, Chapple JP, et al. TAp73 isoforms antagonize Notch signalling in SH-SY5Y neuroblastomas and in primary neurones. J Neurochem, 2006, 99(3): 989-999.
35. Rufini A, Niklison-Chirou MV, Inoue S, et al. TAp73 depletion accelerates aging through metabolic dysregulation. Genes Dev, 2012, 26(18): 2009-2014.
36. Billaud M, Phillippi JA, Kotlarczyk MP, et al. Elevated oxidative stress in the aortic media of patients with bicuspid aortic valve. J Thorac Cardiovasc Surg, 2017, 154(5): 1756-1762.
37. Balint B, Yin H, Nong Z, et al. Seno-destructive smooth muscle cells in the ascending aorta of patients with bicuspid aortic valve disease. EBioMedicine, 2019, 43: 54-66.
38. Gao Z, Zhong M, Ye Z, et al. PAK3 promotes the metastasis of hepatocellular carcinoma by regulating EMT process. J Cancer, 2022, 13(1): 153-161.
39. Navarro-Corcuera A, Ansorena E, Montiel-Duarte C, et al. AGAP2: modulating TGFβ1-signaling in the regulation of liver fibrosis. Int J Mol Sci, 2020, 21(4): 1400.
40. Navarro-Corcuera A, López-Zabalza MJ, Martínez-Irujo JJ, et al. Role of AGAP2 in the profibrogenic effects induced by TGFβ in LX-2 hepatic stellate cells. Biochim Biophys Acta Mol Cell Res, 2019, 1866(4): 673-685.
41. Zhu Y, Wu Y, Kim JI, et al. Arf GTPase-activating protein AGAP2 regulates focal adhesion kinase activity and focal adhesion remodeling. J Biol Chem, 2009, 284(20): 13489-13496.
42. Ahn JY, Rong R, Kroll TG, et al. PIKE (phosphatidylinositol 3-kinase enhancer)-A GTPase stimulates Akt activity and mediates cellular invasion. J Biol Chem, 2004, 279(16): 16441-16451.
43. Wang Y, Südhof TC. Genomic definition of RIM proteins: evolutionary amplification of a family of synaptic regulatory proteins. Genomics, 2003, 81(2): 126-137.
44. Jiang-Xie LF, Liao HM, Chen CH, et al. Autism-associated gene Dlgap2 mutant mice demonstrate exacerbated aggressive behaviors and orbitofrontal cortex deficits. Mol Autism, 2014, 5: 32.
45. Björck HM, Du L, Pulignani S, et al. Altered DNA methylation indicates an oscillatory flow mediated epithelial-to-mesenchymal transition signature in ascending aorta of patients with bicuspid aortic valve. Sci Rep, 2018, 8(1): 2777.
46. Forester ND, Cruickshank SM, Scott DJ, et al. Increased natural killer cell activity in patients with an abdominal aortic aneurysm. Br J Surg, 2006, 93(1): 46-54.

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Bioinformatics analysis for bicuspid aortic valve with ascending aorta dilation

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