MiR-30b Is A Multifunctional Regulator Of Aortic Valve Interstitial Cells

Calcific aortic valve disease (CAVD) is a major health problem threatening agingsocieties. Once hypothesized to be the result of passively introduced degenerativecalcification, CAVD pathogenesis is now appreciated to be a complex and positiveprocess characterized by a multiple pathological changes such as osteogenisis andimmflamation. Valve interstitial cells (VICs) are the most prevalent cells in the heartvalve that maintain normal valve structure and function. In diseased valves, however,VICs are induced to acquire an osteoblastic phenotype during valve repair andremodeling. Thus, prevention of VICs transformation, either by blocking celldifferentiation or apoptosis, promises to be beneficial. However, until recently,regulation of VIC function has been poorly understood.MicroRNAs (miRNAs), binding to the3’ untranslated region (3’UTR) of specifictarget genes and regulate their expression by promoting degradation of transcribedmRNAs or by inhibiting their translation. Recent studies indicate that miRNAs playan important role in osteogenic differentiation. Previous experiments in MC3T3-E1cells have shown that miR-30family members function as negative regulators ofosteoblastic differentiation by targeting the master osteogenic transcription factorsSmad1and Runx2. However, specific miRNAs and their target genes in theregulation of human VICs calcification have not been well characterized.In the present study, we determined that, among the miR-30family members,miR-30b was most dramatically downregulated both in pathology samples and inBMP-2-stimulated aortic VICs. Our gain-and loss-of-function experimentsdemonstrated that, by binding to multiple sites in the3’-UTR of Runx2, Smad1andcaspase3, miR-30b acts as an endogenous attenuator of these genes in VICs toantagonize heart vlave calcification. Objective: To study the correlation among the calcification related factors, miR-30family and degenerative calcified aortic valves.Methods: Degenerative calcified aortic valves which were surgically removedduringm aortic valve replacement serves as experimental group. Softy aortic valvestissues adjacent to the calcification nodules was selected as the control. HEstaining was performed to indicate the structure of valves. Von kossa staining wasused to show calcium deposit in diseased valves. Apoptosis nodules were labled byterminal-deoxynucleotidyl transferase mediated nick end labeling method (TUNEL).Osteoblast related factors Runx2, Smad1, osteocalcin and caspase-3were stained byimmunohistochemistry. Expression of Runx2, Smad1, osteocalcin, caspase-3mRNAand miR-30family (miR-30s) were measured via real time reverse transcriptionPCR (RT-PCR).Results: HE staining showed fibroplasias and mess collagen array. Von Kossastaining confirmed the presence of calcification deposit in calcified valves. Sporadicapoptosis bodies were marked by TUNEL. Immunohistochemistry showeddistribution of Runx2, Smad1, osteocalcin and caspase-3. RT-PCR revealed theincreased mRNA levels of Runx2, Smad1, osteocalcin and caspase-3. To thecontrary miR-30s fell dramatically in calcified valves. The down-regulating trend ofmiR-30b was the most significant.Conclusion: Interstitial cells in calcified valves were activate to show a osteoblastphenotype manifesting Runx2, Smad1, osteocalcin and caspase-3,A dramatic descrease of miR-30b indicats that the interstitial cell activation level isprobabalely regulated by the miR-30b. Objective: To establish a simple and feasible protocol of isolating and culturinghuman aortic valve interstitial cells in vitro.Methods: Aortic valve interstitial cells were digested from newly resected humanaortic valves by collagenase digestion. Cellular morphologic change was observedunder inverted microscope. Cell proliferation was detected by direct count andshown as growth curve. Immunocytochemical staining and Immunophenotypicanalysis of cell surface markers were used to identify the purities of cell populations.Results: Enzymatic digestion methods yielded human aortic interstitial in vitrosuccessfully. Cellular monolayer was formed after1-2weeks of culture. Monolayersof valve interstitial cells were spindle-shaped and overlapped layers of parallel.Once reaches confluence, valve interstitial cells displayed a cobblestone morphology.The growth curve showed that the active growth phase was during the2rd–4thdayafter passage for interstitial cells. Valve interstitial cells were positive stained forα-SMA and vimentin, negative stained for vWF.Conclusion: The results of these studies provided recommendations for theimproved protocol for isolating and culturing human interstitial cells from nativeaortic valves. Objective: To construct an induced calcification model of aortic valves byinducting osteogenisis in human aortic valve interstitial cells (VICs) in vitro.Methods: VICs were used for experiments after3-7passages. Cells were culturedin osteogenic media or in normal media (α-MEM supplemented with FBS, BMP-2,dexamethasone, ascorbic acid, β-glycerophosphate)After1week, calcified nodules were quantified by direct counting calcium depositunder microscope.which was stained and measured by von Kossa staining. Activityof alkaline phosphatase (ALP) was measured. mRNA expression of bone formationrelated factors such as Runx2, Smad1, osteocalcin and caspase-3were quantitatedby real time reverse transcription PCR (RT-PCR). Changes of up mentiond genes inprotein level were determined by immunoblotting.Results: VICs could calcify after1weeks of osteogenic induction under thecondition of low serum with calcified nodules formation. Quantification ofcalcified nodules and calcification deposit were higher in experimental group than incontrol group (51.20±14.31/well VS3.60±1.82/well，P<0.05). Activity of ALPrised in experimental group compared with control group (0.0266±0.0063unit/μgprotein vs0.1298±0.0316unit/μg protein, p<0.05). RT-PCR indicated thatexpression of calcification related markers like Runx2、Smad1、osteocalcin andcaspase-3was much higher in BMP-2stimulated group than that in control group(P<0.05).Conclusion: VICs were activated during progress of calcification with phenotypeshifting to contraction and ossification, which might be the pathological basis ofvalvular calcification. Objective: Calcific aortic valve disease (CAVD) is an active process involving awide range of pathologic changes. Valve interstitial cells (VICs) are the mostprevalent cells in the heart valve that maintain normal valve structure and function.MicroRNAs are essential post-transcriptional modulators of gene expression, andmiRNA-30b is a known repressor of bone morphogenetic protein-2(BMP-2)-mediated osteogenesis. We hypothesized that miRNA-30b is amultifunctional regulator of aortic VICs during calcification.Methods: To determine the role of miRNA-30b in CAVD, microRNA expressionwas evaluated in human calcific aortic valve leaflets obtained intra-operatively.Furthermore, human valvular interstitial cells were isolated, transfected withmiRNA-30b mimics or miRNA-30b inhibitors, stimulated with BMP-2andsubjected to analysis by quantitative reverse transcriptase polymerase chain reaction(qRT-PCR), western blotting, flow cytometry and alkaline phosphatase (ALP)assays.Results: In this study, we demonstrate that miR-30b negatively regulatesBMP-2-induced osteoblast differentiation by targeting Runx2, Smad1and caspase-3.Overexpression of miR-30b led to a decrease in ALP activity and expression ofRunx2, Smad1and caspase-3. Furthermore, dual-luciferase reporter assay confirmedthat Runx2, Smad1and caspase-3are direct targets of miR-30b.Conclusions: We demonstrate a remarkable role of miRNA-30b in calcific aorticvalve disease. We report a completely novel role of miRNA-30b as a regulator ofhuman aortic valvular calcification and apoptosis by its direct targeting of Runx2,Smad1and caspase-3. Targeting of miRNA-30b could serve as a novel therapeuticstrategy to limit progressive calcification in aortic stenosis.