CircZBTB44-Encoded Peptide ZBTB44-342aa Alleviates Aortic Valve Calcification Via cGAS-STING Inhibition.

Aortic valve interstitial cells (AVICs) have the potential to undergo calcification, which has been regarded as a critical issue during the pathology of calcific aortic valve disease (CAVD). In the past decade, epigenetics, in particular, DNA methylation dysregulation, has been reported to play a vital role in the occurrence and development of CAVD. In the present study, the expression of Notch1, which can inhibit the osteogenesis differentiation of valve interstitial cells, was downregulated whereas the expression of methyltransferases was upregulated in CAVD tissues, suggesting the potential role of DNA methylation in Notch1 expression and CAVD progression. As revealed by DNA extraction and bisulfite sequencing polymerase chain reaction (PCR), the methylation level in Notch1 promoter was much higher in CAVD tissues and human AVICs on Day 14 of osteogenesis differentiation induction. The silence of Notch1 intercellular domain (NICD) promoted while the treatment of demethylation agent, 5-Aza-dC, inhibited the osteogenesis differentiation. Moreover, NICD overexpression significantly suppressed the transcriptional activity of β-catenin on TCF4, and the expression of osteogenesis differentiation factors, indicating the involvement of Wnt/β-catenin signaling in Notch1 modulating the osteogenesis differentiation in human AVICs (hAVICs). Taken together, Notch1 promoter methylation leads to a decreased Notch1 expression and subsequent decreased release of NICD in the nucleus of hAVICs, therefore promoting the activation of Wnt/β-catenin signaling and the expression of osteogenesis differentiation factors, finally promoting the osteogenesis differentiation in hAVICs. DNA methylation might act as an important bridge to link epigenetic variation and CAVD progression.

Background: Calcific aortic valve disease (CAVD) is one of the most prevalent cardiovascular disorders in the elderly, and aortic valve interstitial cells (AVICs) play a major role in valvular calcification associated with CAVD progression. Recent studies found that pharmacological or genetic clearance of senescent cells attenuates aortic valve calcification in animal models, indicating an important role of senescent cells in promoting valvular calcification. However, the mechanism underlying cellular senescence in aortic valves is unclear. Our pilot work found that greater densities of senescent cells in AVIC isolates from diseased human aortic valves are associated with elevated levels of matrilin 2 and cathepsin D in AVICs. We tested the hypothesis that matrilin 2 and cathepsin D mediate the senescence and osteogenic responses in human AVICs. Methods and Results: AVICs were isolated from normal and diseased aortic valves. Levels of P16 were assessed by immunoblotting, and senescent changes is examined using senescence-associated β-galactosidase (SA-β-gal) staining. AVICs from diseased valves displayed higher levels of P16 and greater SA-β-gal staining. Both recombinant human matrilin 2 (2.0 μg/ml) and recombinant human cathepsin D (40 nM) promoted P16 expression and enhanced SA-β-gal staining in normal AVICs, leading to greater calcium deposition. Knockdown of cathepsin D reduced matrilin-2-induced P16 expression, SA-β-gal staining and calcium deposition in normal AVICs. Moreover, recombinant cathepsin D activated the M6PR-ERK1/2 pathway in normal AVICs. Blocking or knockdown of M6PR (M6P, 10 mM or siRNA, 1.0 nM) or inhibition of ERK1/2 (PD98059, 25 μM) attenuated the effect of cathepsin D on AVIC senescent change and associated osteogenic activation. Conclusion: AVICs of aortic valves from patients with CAVD have higher levels of senescence activity. Cathepsin D mediates AVIC senescence and associated osteogenic activation through the M6PR-ERK1/2 pathway and is involved in the mechanism by which soluble matrilin 2 promotes cellular senescence and osteogenic activity in human AVICs. Targeting the cathepsin D-M6PR-ERK1/2 cascade may suppress AVIC senescence activity and attenuate aortic valve calcification.

Calcific aortic valve disease (CAVD) is an active pathobiological process that takes place at the cellular and molecular levels. It involves fibrosis and calcification of aortic valve leaflets, which eventually contributes to heart failure. Galectin-3 (Gal-3), a β-galactoside-binding lectin, is involved in myocardial fibrosis and remodeling. Our study aimed to explore how Gal-3 promoted the osteogenic differentiation of human aortic valve interstitial cells (hVICs) along with elucidating the underlying molecular mechanisms. To determine the Gal-3 expression in this study, we included the blood samples and aortic valves (AVs) from patients with CAVD (n=20) and normal controls (n=20). The hVICs were stimulated by Osteogenic medium (OM) and were treated with or without recombinant human Gal-3. Calcified transformation of hVICs was assessed by Alizarin Red S staining and osteogenic gene/protein expression. RNA-sequencing was performed for all different treatments to investigate differentially expressed genes (DEGs) along with exploring the enriched pathways for potential molecular targets of Gal-3. The targets were further detected using Western blotting and immunofluorescence staining. Gal-3 levels were found to be significantly increased in CAVD patients. Treatment of valve interstitial cells (VICs) with Gal-3 led to a marked increase in Runx2 and ALP-mRNA/protein expression levels as well as calcification. Gene expression profiles of hVICs cultured with or without Gal-3 revealed 79 upregulated genes and 82 down-regulated genes, which were highly enriched in TNF and NF-κB signaling pathways. Furthermore, Gal-3 could activate the phosphorylation of IκBα and interfere with the translocation of p65 into the cell nucleus of hVICs. However, inhibition of this pathway can suppress the osteogenic differentiation by Gal-3. Gal-3 acts as a positive regulator of osteogenic differentiation by activating the NF-κB signaling pathway in hVICs. Our findings provide novel mechanistic insights into the critical role of Gal-3 in the CAVD progression.

Calcific aortic valve disease (CAVD) is a chronic, progressive inflammatory disease. Soluble extracellular matrix (ECM) proteins can function as damage-associated molecular patterns (DAMPs) and may play a role in the progression of CAVD. Matrilin-2 is an ECM protein and has been found to up-regulate the pro-osteogenic activity in human aortic valve interstitial cells (AVICs). Klotho is an anti-aging protein that is recently found to have an anti-inflammatory effect. The impact of matrilin-2 and Klotho on AVICs inflammatory response is unclear. This study is to test the hypothesis that matrilin-2 induces the inflammatory response in human AVICs and to explore the anti-inflammatory potential of Klotho for suppression AVIC inflammation. Methods and Results: Human AVICs isolated from normal valves were treated with recombinant matrilin-2 (2.0 μg/ml). Matrilin-2 caused NF-κB-dependent increase in the levels of ICAM-1, MCP-1 and IL-6. In addition, matrilin-2 induced rapid activation of PKR through Toll-like receptor (TLR) 2 and 4. Treatment with PKR inhibitors, 2-AP or C13H8N4OS, prior to matrilin-2 stimulation, abrogated NF-κB phosphorylation and intranuclear translocation. Inhibition of PKR abolished the production of inflammatory mediators induced by matrilin-2 in human AVICs. Further experiments using recombinant Klotho revealed that Klotho (0.5 μg/ml) suppressed the activation of PKR and NF-κB, and markedly reduced the production of inflammatory mediators in human AVICs exposed to matrilin-2. Conclusion: This study demonstrates that soluble matrilin-2 induces the inflammatory response in human AVICs through a TLR-PKR-NF-κB signaling cascade and that Klotho is capable of suppressing human AVICs inflammatory response to a soluble ECM protein. The novel findings of this study indicate that soluble ECM proteins may fuel the progression of CAVD by inducing aortic valve inflammation and that Klotho has the potential for suppression of such inflammation.

Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation and it is the target of widely used anti-inflammatory drugs. COX-2-inhibitor celecoxib was initially shown to reduce AV calcification in a murine model. However, in contrast to these findings, a recent retrospective clinical analysis found an association between AS and celecoxib use. In the present study, we investigated whether variations in COX-2 expression levels in human AVs may be linked to CAVD. We extracted total RNA from surgically explanted AVs from patients without CAVD or with CAVD. We found that COX-2 mRNA was higher in non-calcific AVs compared to calcific AVs (0.013 ± 0.002 vs. 0.006 ± 0.0004; p < 0.0001). Moreover, we isolated human aortic valve interstitial cells (AVICs) from AVs and found that COX-2 expression is decreased in AVICs from calcific valves compared to AVICs from non-calcific AVs. Furthermore, we observed that COX-2 inhibition with celecoxib induces AVICs trans-differentiation towards a myofibroblast phenotype, and increases the levels of TGF-β-induced apoptosis, both processes able to promote the formation of calcific nodules. We conclude that reduced COX-2 expression is a characteristic of human AVICs prone to calcification and that COX-2 inhibition may promote aortic valve calcification. Our findings support the notion that celecoxib may facilitate CAVD progression.

Aortic valve interstitial cells (AVIC) play a major role in the pathogenesis of calcific aortic valve disease (CAVD). Higher fibroblast growth factor 23 (FGF23) levels are associated with chronic kidney disease (CKD), an important risk factor of CAVD. The role of FGF23 in AVIC pathobiology associated with CAVD remains unclear. Klotho (KL) is an anti-aging protein present in membrane (mKL) and soluble (sKL) forms. While mKL is a coreceptor for FGF23, sKL may function as a FGF23 antagonist. We have found that KL inhibits osteogenic activity in AVIC exposed to CKD-associated high phosphate condition. We hypothesized that FGF23 mediates AVIC fibrocalcification, and sKL inhibits the effect of FGF23. Methods and Results: Protein levels of FGF23 and KL were analyzed in AVIC and aortic valves from CAVD patients. Diseased aortic valves and AVIC had higher FGF23 and lower KL levels than normal valves and AVIC, and diseased AVIC secreted greater amount of FGF23. Knockdown or neutralization of FGF23 in diseased AVIC suppressed their spontaneous fibrocalcification. Treatment of normal AVIC with recombinant FGF23 (40 ng/ml) for 72h enhanced the expression of collagens I and IV, Runx2, and alkaline phosphatase. Prolonged treatment of normal AVIC with FGF23 resulted in collagen and calcium deposition. Elevated levels of FGF receptor (FGFR) 1 and 4 were detected in normal AVIC after FGF23 stimulation. Inhibition of FGFR1 attenuated FGF23-induced fibrogenic response, while inhibition of FGFR4 abrogated FGF23-induced osteogenic response. Recombinant KL (0.5 μg/ml) reduced FGFR expression and fibrocalcification induced by FGF23. Further, KL attenuated FGF23-induced collagen and calcium deposition in normal AVIC. In vitro incubation of KL and FGF23 led to the formation of a complex. Conclusion: Diseased aortic valves and AVIC have higher levels of FGF23 and lower levels of KL. FGF23 is capable of inducing fibrocalcification in normal AVIC via FGFR1 and FGFR4. sKL suppresses FGF23 activities by inhibiting FGFR upregulation and direct interaction with FGF23. Our novel findings suggest that FGF23 may accelerate CAVD progression. sKL suppresses the pro-fibrocalcific effect of FGF23 and may offer therapeutic potential for slowing CAVD progression, especially in CKD patients.

Background: Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in the elderly, and elevated osteogenic activity of aortic valve interstitial cells (AVICs) play a critical role in CAVD progression to severe aortic valve stenosis. While soluble extracellular matrix (ECM) proteins, including biglycan and matrilin 2, have been found to up-regulate the osteogenic activity in human AVICs, the mechanism underlying the cellular and molecular mechanisms remain unclear. As greater densities of senescent AVICs were observed in human calcified aortic valves, we tested the hypothesis that soluble ECM protein induces senescence in AVICs to up-regulate valvular osteogenic activity. Methods and Results: Human AVICs were isolated from normal and CAVD aortic valves. Levels of P16 and P21 were assessed by immunoblotting, and senescent changes is identified using senescence-associated β-galactosidase (SA-β-gal) staining. AVICs from diseased valves displayed higher levels of P16 and P21, greater SA-β-gal staining and active calcium deposition. Soluble matrilin 2 (2.0 μg/ml) up-regulated the expression of P16 and P21, and enhanced SA-β-gal staining in normal AVICs, leading to greater calcium deposition. Further, knockdown of P16 markedly reduced calcium deposition in AVICs exposed to soluble matrilin 2. The impact of soluble matrlin 2 is associated with activation of the M6PR-ERK1/2 pathway. Blocking or knockdown of M6PR (M6P, 10 mM or siRNA, 1.0 nM), or inhibition of ERK1/2 (PD98059, 25 μM) attenuated the effect of soluble matrilin 2 on AVIC senescence and resultant up-regulation of osteogenic activity. Conclusion: AVICs from aortic valves of patients with CAVD have higher levels of senescence activity. Soluble matrilin 2, via the M6PR-ERK1/2 pathway, induces AVIC senescence to promote the osteogenic activity. Targeting the D-M6PR-ERK1/2 pathway may suppress AVIC senescence and attenuate aortic valve calcification associated with CAVD progression.

Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Currently, there is no non-invasive medical therapy for CAVD. Matrix metalloproteinases (MMPs) are upregulated in CAVD and play a role in its pathogenesis. Here, we evaluated the effect of doxycycline, a nonselective MMP inhibitor on CAVD progression in the mouse. Apolipoprotein (apo)E−/− mice (n = 20) were fed a Western diet (WD) to induce CAVD. After 3 months, half of the animals was treated with doxycycline, while the others continued WD alone. After 6 months, we evaluated the effect of doxycycline on CAVD progression by echocardiography, MMP-targeted micro single photon emission computed tomography (SPECT)/computed tomography (CT), and tissue analysis. Despite therapeutic blood levels, doxycycline had no significant effect on MMP activation, aortic valve leaflet separation or flow velocity. This lack of effect on in vivo images was confirmed on tissue analysis which showed a similar level of aortic valve gelatinase activity, and inflammation between the two groups of animals. In conclusion, doxycycline (100 mg/kg/day) had no effect on CAVD progression in apoE−/− mice with early disease. Studies with more potent and specific inhibitors are needed to establish any potential role of MMP inhibition in CAVD development and progression.

Calcific aortic valve disease (CAVD) is common in the elderly. Progressive valvular calcification is a characteristic pathobiology in CAVD, and chronic valvular inflammation is known to promote CAVD progression. Matrilin-2 is an extracellular matrix (ECM) protein. Soluble matrilin-2 can function as a damage - associated molecular pattern to induce the inflammatory and osteogenic responses in aortic valve interstitial cells (AVICs). Interleukin-38 (IL-38) is a novel anti-inflammatory cytokine. Lower IL-38 levels are associated with several chronic inflammatory diseases. Recent studies found that IL-38 limits skin inflammation and nephritis by reducing the inflammatory activity of immune cells. We hypothesized that IL-38 is capable of suppressing AVIC osteogenic activity. Methods and Results: We performed immunoblotting and ELISA to assess IL-38 protein levels in AVICs isolated from normal human aortic valves (normal AVICs) and diseased human aortic valves (diseased AVICs). Diseased AVICs had lower levels of IL-38 compared to normal AVICs. Interestingly, recombinant IL-38 inhibited spontaneous deposition of calcium in diseased AVICs. To determine the effect of recombinant IL-38 on AVIC osteogenic response to soluble matrilin-2, we treated normal AVICs with recombinant IL-38 (0, 1, 5 and 10 ng/ml) prior to an exposure to matrilin-2 for 24-72 hour. IL-38 at 10 ng/ml markedly reduced the expression of RUNX-2 and ALP in normal AVICs exposed to matrilin-2. Further, IL-38 suppressed calcium deposition in normal AVICs exposed to matrilin-2 for a prolonged period. Knockdown of IL-38 by shRNA enhanced the osteogenic responses and exaggerated the calcification deposit formation in normal AVICs exposed to matrilin-2. The anti-osteogenic effect of IL-38 is associated with down-regulation of Nod-like receptor protein 3 (NLRP3) inflammasomes, and inhibition of the NLRP3 inflammasomes also suppresses the osteogenic activity in normal AVICs exposed to matrilin-2. Conclusions: IL-38 suppresses the osteogenic activity in human AVICs by inhibiting NLRP3 inflammasomes. The novel findings of this study suggest that IL-38 may have therapeutic potential for prevention of CAVD progression.

Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

Background: Calcific aortic valve disease (CAVD) is a chronic inflammatory disease that manifests as progressive valvular fibrosis and calcification. An inflammatory milieu in valvular tissue promotes fibrosis and calcification. Aortic valve interstitial cell (AVIC) proliferation and the over-production of the extracellular matrix (ECM) proteins contribute to valvular thickening. However, the mechanism underlying elevated AVIC fibrogenic activity remains unclear. Recently, we observed that AVICs from diseased aortic valves express higher levels of neurotrophin 3 (NT3) and that NT3 exerts pro-osteogenic and pro-fibrogenic effects on human AVICs. Hypothesis: Pro-inflammatory stimuli upregulate NT3 production in AVICs to promote fibrogenic activity in human aortic valves. Methods and Results: AVICs were isolated from normal human aortic valves and were treated with lipopolysaccharide (LPS, 0.20 µg/mL). LPS induced TLR4-dependent NT3 production. This effect of LPS was abolished by inhibition of the Akt and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways. The stimulation of TLR4 in human AVICs with LPS resulted in a greater proliferation rate and an upregulated production of matrix metallopeptidases-9 (MMP-9) and collagen III, as well as augmented collagen deposition. Recombinant NT3 promoted AVIC proliferation in a tropomyosin receptor kinase (Trk)-dependent fashion. The neutralization of NT3 or the inhibition of Trk suppressed LPS-induced AVIC fibrogenic activity. Conclusions: The stimulation of TLR4 in human AVICs upregulates NT3 expression and promotes cell proliferation and collagen deposition. The NT3-Trk cascade plays a critical role in the TLR4-mediated elevation of fibrogenic activity in human AVICs. Upregulated NT3 production by endogenous TLR4 activators may contribute to aortic valve fibrosis associated with CAVD progression.

Preventative medicine: The next revolution in the treatment of aortic stenosis

Calcific aortic valve disease (CAVD) is a valvular disease frequently in the elderly individuals that can lead to the valve dysfunction. Osteoblastic differentiation of human aortic valve interstitial cells (HAVICs) induced by inflammation play a crucial role in CAVD pathophysiological processes. To date, no effective drugs for CAVD have been established, and new agents are urgently needed. Piericidin glycosides, obtained from a marine-derived Streptomyces strain, were revealed to have regulatory effects on mitochondria in previous studies. Here, we discovered that 13-hydroxypiericidin A 10-O-α-D-glucose (1→6)-β-D-glucoside (S18), a specific piericidin diglycoside, suppresses lipopolysaccharide- (LPS) induced inflammatory responses of HAVICs by alleviating mitochondrial stress in an interleukin (IL)-37-dependent manner. Knockdown of IL-37 by siRNA not only exaggerated LPS-induced HAVIC inflammation and mitochondrial stress but also abrogated the anti-inflammatory effect of S18 on HAVICs. Moreover, S18 alleviated aortic valve lesions in IL-37 transgenic mice of CAVD model. Microscale thermophoresis (MST) and docking analysis of five piericidin analogues suggested that diglycosides, but not monoglycosides, exert obvious IL-37-binding activity. These results indicate that S18 directly binds to IL-37 to alleviate inflammatory responses in HAVICs and aortic valve lesions in mice. Piericidin diglycoside S18 is a potential therapeutic agent to prevent the development of CAVD.

Analysis of Osteopontin Levels for the Identification of Asymptomatic Patients With Calcific Aortic Valve Disease

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