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An epigenetic regulatory loop controls pro-osteogenic activation by TGF-β1 or bone morphogenetic protein 2 in human aortic valve interstitial cells.

ABSTRACT: Calcific aortic valve disease (CAVD) is common in the elderly population, but pharmacological interventions for managing valvular calcification are unavailable. Transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) induce pro-osteogenic activation of human aortic valve interstitial cells (AVICs) that play an important role in valvular calcification. However, the molecular mechanism underlying pro-osteogenic activation in AVICs is incompletely understood. Here, we investigated an epigenetic regulatory mechanism in human AVIC pro-osteogenic activation induced by TGF-β1 and BMP-2. Microarray and real-time PCR analyses revealed that microRNA (miR)-486 up-regulation and miR-204 down-regulation were characteristic changes in TGF-β1- and BMP-2-stimulated normal AVICs and in AVICs from calcified valves. Both TGF-β1 and BMP-2 down-regulated miR-204 through Smad pathways. Interestingly, an miR-486 antagomir diminished the effect of TGF-β1 and BMP-2 on miR-204 levels and calcium deposit formation. Furthermore, the miR-486 antagomir increased the expression of Smurf2, a Smad inhibitor, in the presence or absence of TGF-β1 or BMP-2 stimulation, whereas a miR-486 mimic reduced Smurf2 expression. Smurf2 knockdown augmented TGF-β1- or BMP-2-induced miR-204 down-regulation and resulted in increased expression of the osteoblastic biomarkers Osx and Runx2. In summary, we found that TGF-β1 and BMP-2 up-regulate miR-486 and down-regulate miR-204 in human AVICs to promote pro-osteogenic activity and that miR-486 inhibits Smurf2 expression to augment the miR-204 down-regulation. We conclude that the miR-486-Smurf2-Smad loop plays an important role in regulating AVIC pro-osteogenic activation in response to TGF-β1 or BMP-2. Targeting this regulatory loop may have therapeutic potential for suppressing aortic valve calcification.

SUBMITTER: Song R

PROVIDER: S-EPMC5448094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Aortic valve calcification is an active proliferative process, where interstitial cells of the valve transform into either myofibroblasts or osteoblast-like cells causing valve deformation, thickening of cusps and finally stenosis. This process may be triggered by several factors including inflammation, mechanical stress or interaction of cells with certain components of extracellular matrix. The matrix is different on the two sides of the valve leaflets. We hypothesize that inflammation and mechanical stress stimulate osteogenic differentiation of human aortic valve interstitial cells (VICs) and this may depend on the side of the leaflet. Methods: Interstitial cells isolated from healthy and calcified human aortic valves were cultured on collagen or elastin coated plates with flexible bottoms, simulating the matrix on the aortic and ventricular side of the valve leaflets, respectively. The cells were subjected to 10% stretch at 1 Hz (FlexCell bioreactor) or treated with 0.1 μg/ml lipopolysaccharide, or both during 24 h. Gene expression of myofibroblast- and osteoblast-specific genes was analyzed by qPCR. VICs cultured in presence of osteogenic medium together with lipopolysaccharide, 10% stretch or both for 14 days were stained for calcification using Alizarin Red. Results: Treatment with lipopolysaccharide increased expression of osteogenic gene bone morphogenetic protein 2 (BMP2) (5-fold increase from control; p = 0.02) and decreased expression of mRNA of myofibroblastic markers: α-smooth muscle actin (ACTA2) (50% reduction from control; p = 0.0006) and calponin (CNN1) (80% reduction from control; p = 0.0001) when cells from calcified valves were cultured on collagen, but not on elastin. Mechanical stretch of VICs cultured on collagen augmented the effect of lipopolysaccharide. Expression of periostin (POSTN) was inhibited in cells from calcified donors after treatment with lipopolysaccharide on collagen (70% reduction from control, p = 0.001), but not on elastin. Lipopolysaccharide and stretch both enhanced the pro-calcific effect of osteogenic medium, further increasing the effect when combined for cells cultured on collagen, but not on elastin. Conclusion: Inflammation and mechanical stress trigger expression of osteogenic genes in VICs in a side-specific manner, while inhibiting the myofibroblastic pathway. Stretch and lipopolysaccharide synergistically increase calcification.

Project description:Elevated level of blood phosphate (Pi) associated with chronic kidney disease (CKD) is a risk factor of aortic valve calcification. Aortic valve interstitial cells (AVICs) display osteogenic responses to high Pi although the underlying mechanism is incompletely understood. Sox9 is a pro-chondrogenic factor and may play a role in ectopic tissue calcification. Circulating and kidney levels of Klotho are reduced in patients with CKD. We hypothesized that Sox9 mediates high Pi-induced osteogenic responses in human AVICs and that Klotho inhibits the responses. Treatment of human AVICs with high Pi increased protein levels of Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP), and a prolonged exposure to high Pi caused calcium deposition. High Pi induced Sox9 upregulation through PKD and Akt activation. Knockdown of Sox9 essentially abolished the effect of high Pi on the osteogenic responses. Lower Klotho levels were observed in calcified aortic valve tissues. Interestingly, high Pi decreased Klotho levels in AVICs from normal valves, and treatment with recombinant Klotho markedly reduced the effect of high Pi on the levels of Sox9, Runx2, and ALP and suppressed calcium deposition. We conclude that high Pi induces human AVIC osteogenic responses through Sox9. Human AVICs express Klotho, and its levels in AVICs are modulated by high Pi and valvular calcification. Importantly, Klotho suppresses the pro-osteogenic effect of high Pi on human AVICs. These novel findings indicate that modulation of Klotho may have therapeutic potential for mitigation of valvular calcification associated with CKD.Key messagesCAVD associated with chronic kidney disease is a significant clinical problem. High phosphate upregulates Sox9 through AKT and PKD in human AVICs. Calcified human aortic valves have lower levels of Klotho. Klotho suppresses Sox9 upregulation and intranuclear translocation. Klotho inhibits high phosphate-induced osteogenic activity in human AVICs.

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An epigenetic regulatory loop controls pro-osteogenic activation by TGF-β1 or bone morphogenetic protein 2 in human aortic valve interstitial cells.

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