Responses In Aortic Valve Interstitial Cells Research Articles

Abstract 16687: Aging-Related Klotho Deficiency in Human Aortic Valve Interstitial Cells Exaggerates the Inflammatory Response Through Down-Regulation of Autophagy Activity

Calcific aortic valve disease (CAVD) is prevalent in the elderly. The inflammatory response of aortic valve interstitial cells (AVICs) contributes to the mechanism of CAVD progression. Our previous work demonstrated that the innate immunity mediated by Toll-like receptors (TLRs) in human AVICs up-regulates the production of inflammatory mediators and pro-osteogenic activity and that human AVICs express anti-aging protein Klotho, and Klotho deficiency is responsible for the enhanced osteogenic activity in diseased AVICs. Autophagy plays an important role in modulating cellular inflammatory response. Currently, the effect of Klotho on AVIC autophagy function and inflammatory response is unknown. We hypothesis that Klotho suppresses AVIC inflammatory response through maintaining cellular autophagy activity. Methods: Levels of autophagy proteins (LC3-I, and LC3-II), Klotho and ICAM-1 were determined in AVICs of normal valves from young (25.3±2.7 years) and older (61.5±4.3 years) donors by immunoblotting. Autophagy modulators (rapamycin, 2.0 μM; Autophinib, 100 nM and Bafilomysin A1, 10 nM) and recombinant Klotho (5.0 μg/ml) were applied to AVICs to assess their impact on inflammatory responses. Results: AVICs from older donors have lower levels of Klotho and autophagy activity, but produce higher levels of ICAM-1. TLR4 stimulation in AVICs from young donors reduces cellular levels of Klotho and autophagy activity, and increases ICAM-1 production. Further, recombinant Klotho enhances autophagy activity in AVICs from older donors through induction of PTEN and inhibition the Akt-mToR-p70S6K pathway, and it suppresses the inflammatory response in unstimulated AVICs from older donors and stimulated cells from young donors. Down-regulation of autophagy enhances, while up-regulation of autophagy reduces, the inflammatory response in AVICs. Conclusions: Autophagy plays an important role in suppression of human AVIC inflammatory response. Klotho deficiency in AVICs associated with aging exaggerates the inflammatory response through down-regulation of autophagy activity. Recombinant Klotho may have the therapeutic potential for suppression of valvular inflammation associated with CAVD progression.

MMP-12–Induced Pro-osteogenic Responses in Human Aortic Valve Interstitial Cells

BackgroundCalcific aortic valve disease (CAVD) is an age-related and slowly progressive valvular disorder. Overexpression of matrix metalloproteinase 12 (MMP-12) has been found in atherosclerosis, stiffed vascular tissue, and calcified aortic valves. We hypothesized that MMP-12 may induce the pro-osteogenic responses in human aortic valve interstitial cells (AVICs). MethodsHuman AVICs were isolated from normal and calcified aortic valves. Cells were treated with MMP-12. The pro-osteogenic marker Runt-related transcription factor 2 (RUNX-2), bone morphogenetic protein 2 (BMP-2), and alkaline phosphatase (ALP), as well as MMP-12–associated signaling molecules, were analyzed. ResultsHuman calcified aortic valves expressed significantly higher MMP-12 than normal human aortic valves. MMP-12–induced the expression of RUNX-2, BMP-2, ALP, and calcium deposit formation. Suppression of MMP-12 by its inhibitor decreased the expression of RUNX-2, BMP-2, and ALP. MMP-12–induced osteogenic responses were associated with higher levels of phosphorylation of p38 mitogen-activated protein kinases (MAPK), low density lipoprotein-related protein 6 (LRP-6), and β-catenin signaling molecules. Calcified aortic valves exhibited markedly higher levels of LRP-6 and β-catenin levels. Inhibition of either p38 MAPK or LRP-6 attenuated MMP-12–induced expression of RUNX-2, BMP-2, and ALP. Suppression of p38 MAPK abrogated MMP-12–induced activation of LRP-6 and β-catenin signaling pathways. ConclusionsMMP-12 induces pro-osteogenic responses in AVICs by activation of p38 MAPK-mediated LRP-6 and β-catenin signaling pathways. The study revealed that the potential role of MMP-12 in the pathogenesis of CAVD and therapeutically targeting MMP-12 may suppress the development of CAVD.

Rapamycin Decreases the Osteogenic Response in Aortic Valve Interstitial Cells Through the Stat3 Pathway

Calcific aortic valve disease (CAVD) is an age-related and slowly progressive valvular disorder. We have previously found that the increased inflammatory and osteogenic responses to Toll-like receptor 4 (TLR4) stimulation is correlated with lower signal transducer and activator of transcription 3 (Stat3) activity in aortic valve interstitial cells (AVICs). Rapamycin, a drug used clinically, induces feedback activation of Akt. Akt in turn may upregulate Stat3. Therefore we hypothesized that rapamycin will decrease TLR4-induced osteogenic response in human AVICs through modulation of Stat3 activity. AVICs were isolated from normal valves taken from the explanted hearts of patients undergoing transplantation. Cells were treated with TLR4 ligand lipopolysaccharide (LPS) or rapamycin, or both. The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and bone morphogenetic protein 2 (BMP-2), as well as activation ofStat3 and its associated signaling molecules, were analyzed. LPS induces the expression of RUNX2, ALP, and BMP-2. Rapamycin decreased both the baseline and LPS-induced expression of RUNX2, ALP, and BMP-2. Rapamycin also decreased calcium deposit formation. Rapamycin activated both Stat3 and Akt in AVICs. Suppression of Akt resulted in abolishment of Stat3 activation. Inhibition of Stat3 enhanced expression of RUNX2, ALP, and BMP-2 at baseline and in response to LPS. Rapamycin inhibits TLR4-induced osteogenic responses in AVICs by activation of Stat3 through Akt. Rapamycin may alleviate inflammation-induced initiation and progression of CAVD.

Ligation of ICAM-1 on human aortic valve interstitial cells induces the osteogenic response: A critical role of the Notch1-NF-κB pathway in BMP-2 expression

Calcific aortic valve disease (CAVD) is a chronic inflammatory condition and affects a large number of elderly people. Aortic valve interstitial cells (AVICs) occupy an important role in valvular calcification and CAVD progression. While pro-inflammatory mechanisms are capable of inducing the osteogenic responses in AVICs, the molecular interaction between pro-inflammatory and pro-osteogenic mechanisms remains poorly understood. This study tested the hypothesis that intercellular adhesion molecule-1 (ICAM-1) plays a role in mediating pro-osteogenic factor expression in human AVICs. AVICs were isolated from normal human aortic valves and cultured in M199 medium. Treatment with leukocyte function-associated factor-1 (LFA-1, an ICAM-1 ligand) up-regulated the expression of bone morphogenetic protein-2 (BMP-2) and resulted in increased alkaline phosphatase activity and formation of calcification nodules. Pre-treatment with lipopolysaccharide (LPS, 0.05μg/ml) increased ICAM-1 levels on cell surfaces and exaggerated the pro-osteogenic response to LFA-1, and neutralization of ICAM-1 suppressed this response. Further, ligation of ICAM-1 by antibody cross-linking also up-regulated BMP-2 expression. Interestingly, LFA-1 elicited Notch1 cleavage and NF-κB activation. Inhibition of NF-κB markedly reduced LFA-1-induced BMP-2 expression, and inhibition of Notch1 cleavage with a γ-secretase inhibitor suppressed LFA-1-induced NF-κB activation and BMP-2 expression. Ligation of ICAM-1 on human AVICs activates the Notch1 pathway. Notch1 up-regulates BMP-2 expression in human AVICs through activation of NF-κB. The results demonstrate a novel role of ICAM-1 in translating a pro-inflammatory signal into a pro-osteogenic response in human AVICs and suggest that ICAM-1 on the surfaces of AVICs contributes to the mechanism of aortic valve calcification.

Notch1 Promotes the Pro-Osteogenic Response of Human Aortic Valve Interstitial Cells via Modulation of ERK1/2 and Nuclear Factor-κB Activation

Calcific aortic valve disease is a leading cardiovascular disease in the elderly, and progressive calcification results in the failure of valvular function. Aortic valve interstitial cells (AVICs) from stenotic valves express higher levels of bone morphogenetic protein-2 in response to Toll-like receptor 4 stimulation. We recently found that Toll-like receptor 4 interacts with Notch1 in human AVICs. This study tests the hypothesis that Notch1 promotes the pro-osteogenic response of human AVICs. AVICs isolated from diseased human valves expressed higher levels of bone morphogenetic protein-2 and alkaline phosphatase after lipopolysaccharide stimulation. The augmented pro-osteogenic response is associated with elevated cellular levels of Notch1 and enhanced Notch1 cleavage in response to lipopolysaccharide stimulation. Inhibition or silencing of Notch1 suppressed the pro-osteogenic response in diseased cells, and the Notch 1 ligand, Jagged1, enhanced the response in AVICs isolated from normal human valves. Interestingly, extracellular signal-regulated protein kinases 1/2 (ERK1/2) and nuclear factor-κB phosphorylation induced by lipopolysaccharide was markedly reduced by inhibition or silencing of Notch1 and enhanced by Jagged1. Inhibition of ERK1/2 or nuclear factor-κB also reduced bone morphogenetic protein-2 and alkaline phosphatase expression induced by lipopolysaccharide. Notch1 mediates the pro-osteogenic response to Toll-like receptor 4 stimulation in human AVICs. Elevated Notch1 levels and enhanced Notch1 activation play a major role in augmentation of the pro-osteogenic response of AVICs of stenotic valves through modulation of ERK1/2 and nuclear factor-κB activation. These pathways could be potential therapeutic targets for prevention of the progression of calcific aortic valve disease.

Cross-Talk Between the Toll-Like Receptor 4 and Notch1 Pathways Augments the Inflammatory Response in the Interstitial Cells of Stenotic Human Aortic Valves

Calcific aortic stenosis is a chronic inflammatory disease, and aortic valve interstitial cells (AVIC) play an important role in valvular inflammation. Whereas AVIC from stenotic aortic valves exhibit an augmented response to Toll-like receptor 4 (TLR4) stimulation, the underlying mechanism is unclear. This study tested the hypothesis that an excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the inflammatory response to lipopolysaccharide (LPS) in AVIC of stenotic valves. Human AVIC were isolated from normal and stenotic leaflets. Nuclear factor kappa-B (NF-κB) activation and production of interleukin-8, monocyte chemoattactrant protein-1, and intercellular adhesion molecule-1 were analyzed after treatment with LPS. The role of Notch1 in the inflammatory response was determined using inhibitor, siRNA, and specific ligand. Cells from diseased valves produced greater levels of chemokines and intercellular adhesion molecule-1 that are associated with enhanced NF-κB activation. Interestingly, diseased cells exhibited augmented Jagged1 release and Notch1 activation after TLR4 stimulation. Inhibition and silencing of Notch1 each resulted in greater suppression of the TLR4-induced inflammatory response in diseased cells. Conversely, activation of Notch1 with a specific ligand, Jagged1, enhanced the LPS-induced inflammatory response in normal AVIC. Further, Notch1 intracellular domain was coimmunoprecipited with the inhibitor of NF-κB kinase after LPS stimulation, and inhibition of Notch1 abrogated the difference in the level of NF-κB activation between diseased and normal cells. Notch1 enhances the inflammatory response to TLR4 stimulation in human AVIC through modulating NF-κB activation. Excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the TLR4 response in AVIC of stenotic valves.

Abstract 486: Soluble Biglycan Induces the Osteogenic Response in Human Aortic Valve Interstitial Cells via TLR2 and Mediates the Pro-osteogenic Effect of OxLDL

Calcific aortic stenosis affects a large number of people 65 or older. Our previous studies found that stimulation of TLR2 or TLR4 in human aortic valve interstitial cells (AVICs) up-regulates the expression of osteogenic mediators. Identification of endogenous agents that activate TLR2 and TLR4 in AVICs could lead to pharmacological intervention of calcific aortic stenosis. While biglycan and oxLDL accumulate in diseased areas of aortic valves, their role in the development and progression of this valve disease is poorly understood. Soluble biglycan is recognized as an endogenous ligand for TLR2 and TLR4 in macrophages. We recently found that oxLDL induces bone morphogenetic protein-2 (BMP-2) expression in human coronary artery endothelial cells. This study tested the hypothesis that soluble biglycan induces the osteogenic response in human AVICs via TLR2/4 and mediates the pro-osteogenic effect of oxLDL. Methods and results: The expression and release of biglycan were greater in AVICs of stenotic valves. Stimulation of cells with biglycan (0.05-0.20 μg/ml) increased the expression of BMP-2 and alkaline phosphatase (ALP). Further, prolonged stimulation with biglycan caused accumulation of calcium deposits in AVIC culture. Silencing of TLR2, not TLR4, markedly reduced protein levels of BMP-2 and ALP following stimulation with biglycan. Co-immunoprecipitation revealed the interaction of biglycan with TLR2. Biglycan induced the phosphorylation of ERK1/2 and p38 MAPK, and inhibition of either ERK1/2 or p38 MAPK reduced biglycan-induced expression of BMP-2 and ALP. Stimulation of AVICs with oxLDL (20 and 40 μg/ml) up-regulated biglycan expression and release. Neutralization or silencing of biglycan reduced the effect of oxLDL on the expression of BMP-2 and ALP. Conclusions: Extracellular soluble biglycan induces the osteogenic response in AVICs via TLR2. The ERK1/2 and p38 MAPK pathways are involved in mediating the pro-osteogenic effect of soluble biglycan. OxLDL stimulates the synthesis and release of biglycan by AVICs, and biglycan contributes to the mechanism underlying the pro-osteogenic effect of oxLDL on AVICs. These findings provide evidence supporting a pathobiological role of soluble biglycan and oxLDL in calcific aortic stenosis.