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Role of miRNA-483-3p in Valvular Endothelial Dysfunction

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§Calcific Aortic Valve Disease (CAVD) is a significant cause of mortality in aging population with only one current viable treatment: surgical valve replacement.
§CAVD is defined by pathological changes in the valve which begin with aortic valve sclerosis that progress to aortic valve stenosis.
§Calcification preferentially occurs on the aortic side of the valve (fibrosa), where the endothelial cells are subjected to disturbed blood flow (OS), and exhibit an inflammatory phenotype.
§The ventricularis side experience laminar shear stress (LS) and exhibit an anti-inflammatory phenotype and  it is rarely found to be calcified.
§miRNAs are small nucleotide sequences that bind to the 3’-untranslated region (UTR) of mRNA leading to the degradation of that mRNA or inhibition of protein translation.
§We seek to determine miRNAs and genes that are differentially expressed in the valve in response to shear stress and their potential as therapeutic targets to prevent aortic valve calcification. 

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.

§In vitro shear stress experiments were carried out using our cone-and-plate shear system. LS was induced by parallel force of 20 dynes/cm2, and OS was induced at ± 5 dynes/cm2. After 24 hours, cell alignment as shown in the first image above was confirmed in laminar shear stress (LS).
§Endothelial-enriched RNA is collected from porcine valves by placing an isopropanol-soaked nitrocellulose membrane over the endothelial sides of the valve.
§miRNA overexpression and inhibition was achieved using pre-miR-mimics and LNA-anti-miRs from Life technologies and Exiqon respectively at a concentration of 5-100nM, according to observed efficacy.
§Monocyte binding assays were conducted by incubating HAVECs with the human monocytic line. 10 images from each well were taken and the average was used as the value for that sample.

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.

§miRNA-1237-3p is a novel shear-sensitive miRNA in HAVECs as well as side-specific in porcine aortic valves.
§miR-1237-3p is an anti-inflammatory miRNA that regulates inflammation in HAVECs as shown by monocyte binding assay and inflammatory cytokine expression.
§We have found CxCL2, CxCL12, NOX4 and THBS1 to be shear-sensitive in HAVECs as well as targets of miR-1237-3p. These genes are key regulators of endothelial dysfunction and may provide insight in the role of miR-1237-3p in valvular endothelium dysfunction.
§miR-1237-3p may be a novel therapeutic target for CAVD by inhibiting shear-dependent endothelium inflammation.

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