Glomerular endothelial cell (GEnC) dysfunction is important in the pathogenesis of glomerular sclerotic diseases, including Focal Segmental Glomerulosclerosis (FSGS) and overt diabetic nephropathy (DN). GEnCs form the first cellular barrier in direct contact with cells and factors circulating in the blood. Disturbances in these circulating factors can induce GEnC dysfunction. GEnC dysfunction occurs in early stages of FSGS and DN, and is characterized by a compromised endothelial glycocalyx, an inflammatory phenotype, mitochondrial damage and oxidative stress, aberrant cell signaling, and endothelial-to-mesenchymal transition (EndMT). GEnCs are in an interdependent relationship with podocytes and mesangial cells, which involves bidirectional cross-talk via intercellular signaling. Given that GEnC behavior directly influences podocyte function, it is conceivable that GEnC dysfunction may culminate in podocyte damage, proteinuria, subsequent mesangial activation, and ultimately glomerulosclerosis. Indeed, GEnC dysfunction is sufficient to cause podocyte injury, proteinuria and activation of mesangial cells. Aberrant gene expression patterns largely contribute to GEnC dysfunction and epigenetic changes seem to be involved in causing aberrant transcription. This review summarizes literature that uncovers the importance of cross-talk between GEnCs and podocytes, and GEnCs and mesangial cells in the context of the development of FSGS and DN, and the potential use of GEnCs as efficacious cellular target to pharmacologically halt development and progression of DN and FSGS.

Endothelial–mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFβ1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.

IFNγ enhances allograft immunogenicity and facilitates T-cell mediated rejection. This may cause interstitial fibrosis and tubular atrophy (IFTA), contributing to chronic allograft loss. We assessed if inhibition of T-cell activation by N-octanoyl dopamine (NOD) impairs adherence of activated T-cells to endothelial cells and the ability of activated T-cells to produce IFNγ. We also assessed if NOD affects IFNγ mediated gene expression in endothelial cells. The presence of NOD during T-cell activation significantly blunted their adhesion to unstimulated and cytokine stimulated HUVEC. Supernatants of these T-cells displayed significantly lower concentrations of TNFα and IFNγ and were less capable to facilitate T-cell adhesion. In the presence of NOD VLA-4 (CD49d/CD29) and LFA-1 (CD11a/CD18) expression on T-cells was reduced. NOD treatment of IFNγ stimulated HUVEC reduced the expression of MHC class II transactivator (CIITA), of MHC class II and its associated invariant chain CD74. Since IFTA is associated with T-cell mediated rejection and IFNγ to a large extent regulates immunogenicity of allografts, our current data suggest a potential clinical use of NOD in the treatment of transplant recipients. Further in vivo studies are warranted to confirm these in vitro findings and to assess the benefit of NOD on IFTA in clinically relevant models.

Introduction Endothelial cells play a pivotal role in the formation of neointimal lesions by the acquisition of a fibro-proliferative phenotype through endothelial-to-mesenchymal transition (EndMT). Uniform laminar shear stress activates the mitogen-activated protein kinase 7 (MAPK7) which suppresses EndMT. It is elusive how MAPK7 activity is regulated in fibroproliferative disease. We recently found in intimal hyperplasia the signaling activity of MAPK7 is rapidly lost through the activation microRNA-374b. The histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2), which is the catalytic subunit of the Polycomb Repressive Complex 2, plays a pivotal role in endothelial dysfunction. EZH2 trimethylates lysine 27 on histone 3, which silences gene expression and is elevated in endothelial cells in atherosclerotic lesions. Here, we found the reciprocity that exists between MAPK7 and EZH2 in the regulation of EndMT and in human coronary artery stenosis. Materials and results In endothelial cells, activation of MAPK7 increases the expression of microRNA-101, which represses the expression of EZH2. Reciprocally, the loss of EZH2 coincides with a decreased expression of the Dual Specificity Phosphatase (DUSP)-1 and DUSP-6 – the phosphatases responsible for the dephosphorylation of MAPK7 - which facilitates the activation of MAPK7. H3K27Me3, the repressive histone mark placed by EZH2, is abundantly present in the promoter regions of the miR-200b/a/429 and miR-200c/141 gene clusters, which are responsible for the loss of DUSP-1 and DUSP-6 expression. Endothelial cells deficient in EZH2 have reduced levels of H3K27Me3 at these gene promoters, which associates with the increased expression of miR-200b and miR-200c and concurrent increased MAPK7 activation. In endothelial cells with constitutively active MAPK7 signaling (MEK5D), the enrichment of H3K27Me3 at the promoter regions of miR-200b/a/429 (1.6-fold, p=0.034) and miR-200c/141 (1.9-fold, p=0.035) is decreased, suggesting that MAPK7 activation results in a decreased EZH2 activity. Disbalances in this reciprocal signaling circuit culminate in the induction of EndMT and associate to the severity of human coronary artery stenosis. Conclusion In summary, we show that in endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and that disturbances in this reciprocal signaling circuit associate with the induction of EndMT and severity of human coronary artery stenosis. The reciprocity between MAPK7 and EZH2 is governed by a complex mechanism involving microRNAs and the phosphatases DUSP-1 and DUSP-6. Our study contributes to a better understanding of the molecular and epigenetic cascades that underlie EndMT during coronary artery stenosis and might identify novel targets for therapy. Acknowledgement/Funding Mongolian Government Scholarship #621

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