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Inflammation and Mechanical Stress Stimulate Osteogenic Differentiation of Human Aortic Valve Interstitial Cells.

ABSTRACT: 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 ?g/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: ?-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.

SUBMITTER: Bogdanova M

PROVIDER: S-EPMC6256176 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description:BackgroundOsteogenic differentiation of aortic valve interstitial cells (AVICs) plays a key role in the calcific aortic valve disease progression. Extracellular vesicles (EVs)-derived from M1-polarized macrophages (M1-EVs) orchestrated intercellular communication by delivering non-coding RNAs such as tRNA-derived small RNAs (tsRNAs) is crucial for cardiovascular disease. However, the role and mechanism of M1-EVs tsRNAs in osteogenic differentiation of AVICs remains largely unclear.MethodsM1-EVs and PBS treated-RAW 264.7 cell-derived EVs (NC-EVs) were incubated with AVICs and subjected to small RNA sequencing. Candidate tsRNA in M1-EVs was silenced to explore their effects on AVIC osteogenic differentiation and mitophagy.ResultsDiI-labeled M1-EVs were internalized by AVICs, resulting in significantly increased calcium nodule formation and expression of osteogenesis-related genes in AVICs, including RUNX2, BMP2, osteopontin, and SPP1, compared with NC-EVs. Small RNA sequencing revealed that 17 tsRNAs were significantly up-regulated such as tsRNA-5006c, while 28 tsRNAs were significantly down-regulated in M1-EVs compared with NC-EVs. Intriguingly, tsRNA-5006c-deleted M1-EVs treatment significantly reduced calcium nodule formation and expression of osteogenesis-related genes in AVICs relative to control group. Moreover, target genes of tsRNA-5006c were mainly involved in autophagy-related signaling pathways, such as MAPK, Ras, Wnt, and Hippo signaling pathway. Hallmarks of mitophagy activation in AVICs including mitophagosome formation, TMRM fluorescence, expression of LC3-II, BINP3, and PGC1α, were significantly elevated in the M1-EVs group compared with NC-EVs group, whereas M1-EVs tsRNA-5006c inhibitor led to a significant reduction in these indicators.ConclusionM1-EVs carried tsRNA-5006c regulates AVIC osteogenic differentiation from the perspective of mitophagy, and we provide a new target for the prevention and treatment of aortic valve 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.

Project description:Calcific aortic valve disease (CAVD) is a common acquired valvulopathy, which carries a high burden of mortality. Chronic inflammation has been postulated as the predominant pathophysiological process underlying CAVD. So far, no effective medical therapies exist to halt the progression of CAVD. This review aims to outline the known pathways of inflammation and calcification in CAVD, focussing on the critical roles of mechanical stress and mechanosensing in the perpetuation of valvular inflammation. Following initiation of valvular inflammation, dysregulation of proinflammatory and osteoregulatory signalling pathways stimulates endothelial-mesenchymal transition of valvular endothelial cells (VECs) and differentiation of valvular interstitial cells (VICs) into active myofibroblastic and osteoblastic phenotypes, which in turn mediate valvular extracellular matrix remodelling and calcification. Mechanosensitive signalling pathways convert mechanical forces experienced by valve leaflets and circulating cells into biochemical signals and may provide the positive feedback loop that promotes acceleration of disease progression in the advanced stages of CAVD. Mechanosensing is implicated in multiple aspects of CAVD pathophysiology. The mechanosensitive RhoA/ROCK and YAP/TAZ systems are implicated in aortic valve leaflet mineralisation in response to increased substrate stiffness. Exposure of aortic valve leaflets, endothelial cells and platelets to high shear stress results in increased expression of mediators of VIC differentiation. Upregulation of the Piezo1 mechanoreceptor has been demonstrated to promote inflammation in CAVD, which normalises following transcatheter valve replacement. Genetic variants and inhibition of Notch signalling accentuate VIC responses to altered mechanical stresses. The study of mechanosensing pathways has revealed promising insights into the mechanisms that perpetuate inflammation and calcification in CAVD. Mechanotransduction of altered mechanical stresses may provide the sought-after coupling link that drives a vicious cycle of chronic inflammation in CAVD. Mechanosensing pathways may yield promising targets for therapeutic interventions and prognostic biomarkers with the potential to improve the management of CAVD.

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Inflammation and Mechanical Stress Stimulate Osteogenic Differentiation of Human Aortic Valve Interstitial Cells.

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