Figure 1. A) Ventricularis layer experiences laminar shear stress profile whereas the fibrosa layer experiences oscillatory shear stress. B) Shear stress regulates gene transcription regulation which lead to the synthesis of mechanosensitive miRNAs that repress the expression of specific targets in the endothelium.
miR-1237-3p is increased in laminar unidirectional flow in HAVECS and in the fibrosa of porcine aortic valves
Figure 2. A) Fold change of miRNAs between fibrosa HAVECs under oscillatory flow (FO) and ventricularis HAVECs under laminar flow (VL) obtained from microarray and qPCR (Holliday et al, 2011). n = 6 *p<0.05, +p<0.1.B) Validation of miRNA expression in sheared HAVECs quantified by qPCR. Three of the miRNAs found in the array were validated and found to be significantly upregulated in LS condition in HAVECs. n=6,**p<0.01. C) Expression of miR-1237-3p in endothelial-enriched RNA in each side of porcine aortic valves. Expression of miR-1237-3p is upregulated in ventricularis layer (side of the valve that experiences LS) compared to the fibrosa layer (side of the valve that experiences OS), (n=9, *p<0.05).
Loss of the mechano-sensitive miRNA-1237-3p in the Aortic Valve Endothelium causes endothelial inflammation leading to CAVD.
miR-1237-3p silencing increases monocyte binding in static and sheared HAVECs
Figure 3. A) Images of the monocyte binding assay in static HAVECs treated with either anti-miR-1237-3p or control anti-miR-scramble. B) Bound monocytes were counted in static HAVECs treated with anti-miR-scramble or anti-miR-1237-3p. n=6,*p<0.05,**p<0.01. C) Images of the monocyte binding assay in HAVECs sheared with LS treated with either anti-miR-scramble or Anti-miR-1237-3p for 24 hours. D) Quantification of the number of bound monocytes in sheared HAVECs treated with anti-miR-Scramble or anti-miR-1237-3p. n=3,*p<0.05. In both experiments silencing of miR-1237-3p in HAVECs induced a pro-inflammatory phenotype in HAVECs.
miR-1237-3p modulation regulates inflammatory cytokines in HAVECs
Figure 4. A) Expression of miR-1237-3p in HAVECs transfected with miR-1237-3p mimic for 48hours (n=4). B) Expression of endothelial inflammatory cytokines in HAVECs transfected with miR-mimic-1237-3p for 48 hours. NF-κB, IL-6 and IL-1β are significantly downregulated when miR-1237-3p is overexpressed (n=4). C) Expression of miR-1237-3p in HAVECs transfected with anti-miR-1237-3p for 48hours (n=3). D) Expression of endothelial inflammatory cytokines in HAVECs transfected with anti-miR-1237-3p for 48 hours. VCAM-1, IL-6 and IL-1β are significantly upregulated when miR-1237-3p is silenced (n=3-9). *p<0.05.
miR-1237-3p regulates critical targets of endothelial dysfunction such as CxCL2, CxCL12, NOX4 or THBS1
Figure 5. A) In silico analysis where we compare shear-sensitive gene targets in our HAVEC mRNA array (downregulated in LS conditions) with predicted gene targets of miR-1237-3p (http://mirdb.org/miRDB/). From the 78 genes that were found to meet this criteria we have chosen to study genes related to regulatory pathways of endothelial dysfunction. B) Expression of the gene targets chosen from the in silico analysis in HAVECs treated with anti-miR-1237-3p. CxCL2, CxCL12 , NOX4 and THBS1 are significantly upregulated when miR-1237-3p is silenced in static HAVECs. N=3-6, *p<0.05.