Abstract
We tested the hypothesis that endothelial capillary tube formation in 3D cultures in basement membrane extract (BME) is secondary to the altered DNA promoter methylation and mRNA expression in human brain micro endothelial cells (HBMECs). We conducted a whole-genome transcriptomic and methylation microarray and CRISPR/Cas9-mediated gene knockdown to test our hypothesis. The data demonstrated that with angiogenic transformation 1318 and 1490 genes were significantly (p < 0.05) upregulated and downregulated, respectively. We compared our gene expression data with the published databases on GEO and found several genes in common. PTGS2, SELE, ID2, HSPA6, DLX2, HEY2, FOSB, SMAD6, SMAD7, and SMAD9 showed a very high level of expression during capillary tube formation. Among downregulated gene were ITGB4, TNNT1, PRSS35, TXNIP, IGFBP5. The most affected canonical pathways were ATM signaling and cell cycle G2/M DNA damage checkpoint regulation. The top upstream regulators of angiogenic transformation were identified to be VEGF, TP53, HGF, ESR1, and CDKN1A. We compared the changes in gene expression with the change in gene methylation and found hypomethylation of the CpG sites was associated with upregulation of 515 genes and hypermethylation was associated with the downregulation of 31 genes. Furthermore, the silencing of FOSB, FZD7, HEY2, HSPA6, NR4A3, SELE, PTGS2, SMAD6, SMAD7, and SMAD9 significantly inhibited angiogenic transformation as well as cell migration of HBMECs. We conclude that the angiogenic transformation is associated with altered DNA methylation and gene expression changes.
Highlights
Cerebral angiogenesis is an essential mechanism for restoring cerebral perfusion under various physiological and pathological conditions
To elucidate the pathways involved in angiogenesis, we decided to examine the gene expression changes in human brain micro endothelial cells (HBMECs) with angiogenic transformation in 3-D cultures in basement membrane extract (BME)
Using Ingenuity Pathway Analysis Program (IPA), we found 44 upstream regulatory molecules to be significantly altered with HBMEC angiogenic transformation
Summary
Cerebral angiogenesis is an essential mechanism for restoring cerebral perfusion under various physiological and pathological conditions. Previous studies have analyzed gene expression changes as a consequence of in vitro capillary tube formation in human umbilical vein endothelial cells (HUVEC). We provide a comparative analysis of the changes in gene expression in HUVEC versus human brain micro endothelial cell (HBMEC). To elucidate the pathways involved in angiogenesis, we decided to examine the gene expression changes in HBMECs with angiogenic transformation in 3-D cultures in BME. This is regarded as one of the most specific tests and is reasonably close to the in vivo situations (Madri et al, 1988). The role of DNA methylation and the resulting transcriptional control of gene expression during angiogenesis are not completely known. The present study is an attempt to elucidate some of the mechanistic events involved during this complicated process
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