Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles.

Calcified aortic valve disease in its final stage leads to aortic valve stenosis, limiting cardiac function. To date, surgical intervention is the only option for treating calcific aortic valve stenosis. This study combined controlled drug delivery by nanoparticles (NPs) and active targeting by antibody conjugation. The chelating agent diethylenetriaminepentaacetic acid (DTPA) was covalently bound to human serum albumin (HSA)-based NP, and the NP surface was modified using conjugating antibodies (anti-elastin or isotype IgG control). Calcification was induced ex vivo in porcine aortic valves by preincubation in an osteogenic medium containing 2.5 mM sodium phosphate for five days. Valve calcifications mainly consisted of basic calcium phosphate crystals. Calcifications were effectively resolved by adding 1-5 mg DTPA/mL medium. Incubation with pure DTPA, however, was associated with a loss of cellular viability. Reversal of calcifications was also achieved with DTPA-coupled anti-elastin-targeted NPs containing 1 mg DTPA equivalent. The addition of these NPs to the conditioned media resulted in significant regression of the valve calcifications compared to that in the IgG-NP control without affecting cellular viability. These results represent a step further toward the development of targeted nanoparticular formulations to dissolve aortic valve calcifications.

Salidroside ameliorates pathological cardiac hypertrophy via TLR4-TAK1-dependent signaling.

Salidroside, a prominent active ingredient in traditional Chinese medicines, is garnering increased attention because of its unique pharmacological effects against ischemic heart disease via MAPK signaling, which plays a critical role in regulating the evolution of ventricular hypertrophy. However, the function of Salidroside on myocardial hypertrophy has not yet been elucidated. C57BL/6 mice were subjected to transverse aortic constriction (TAC), and treated with Salidroside (100 mg kg-1  day-1 ) by oral gavage for 3 weeks starting 1 week after surgery. Four weeks after TAC surgery, the mice were subjected to echocardiography and then sacrificed to harvest the hearts for analysis. For in vitro study, neonatal rat cardiomyocytes were used to validate the protective effects of Salidroside in response to Angiotensin II (Ang II, 1 µM) stimulation. Here, we proved that Salidroside dramatically inhibited hypertrophic reactions generated by pressure overload and isoproterenol (ISO) injection. Salidroside prevented the activation of the TAK1-JNK/p38 axis. Salidroside pretreatment of TAK1-inhibited cardiomyocytes shows no additional attenuation of Ang II-induced cardiomyocytes hypertrophy and signaling pathway activation. The overexpression of constitutively active TAK1 removed the protective effects of Salidroside on myocardial hypertrophy. TAC-induced increase of TLR4 protein expression was reduced considerably in the Salidroside treated mice. Transient transfection of small interfering RNA targeting TLR4 (siTLR4) in cardiomyocytes did not further decrease the activation of the TAK1/JNK-p38 axis. In conclusion, Salidroside functioned as a TLR4 inhibitor and displayed anti-hypertrophic action via the TAK1/JNK-p38 pathway.

ALP Inhibitors Inhibit Inflammatory Responses and Osteoblast Differentiation in hVIC via AKT-ERK Pathways.

Objective: The aim of this study was to explore the calcification process of aortic valve interstitial cells and its potential association with osteogenic differentiation and alkaline phosphatase (ALP) activity. Methods: The study patients were divided into 3 groups: the control group, the osteogenic induction medium (OM) group and the OM+ALP inhibitor group. Cell calcification was measured by alizarin red S staining and alizarin red S dye released by extracellular matrix (ECM) was quantified by spectrophotometry. Immunohistochemical staining was performed on valve tissues of patients harboring calcified and non-calcified aortic valve disease. Expression of bone morphogenetic protein (BMP), runt related transcription factor 2 (RUNX2), osteocalcin and osteopontin (OPN), was evaluated using immunohistochemistry¸and expression of osteogenic specific markers (BMP, RUNX2 and OPN) was detected using Wesern blot analysis. RNA sequencing was analyzed to further study the exact mechanism of ALP inhibitors in terms of inhibiting the osteogenic differentiation of valvular interstitial cells (VIC). The mRNA levels of tumor necrosis factor alpha (TNF-α), Toll-like receptor 4 (TLR4) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3), were detected using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In addition, Western blot analysis was performed to evaluate the expression of phosphorylated extracellular regulated protein kinases (ERK), nuclear factor κ B inhibitor α (IκBα) and protein kinase B (AKT) in protein. Results: Alizarin red staining was positive in the OM and OM+ALP inhibitor groups, and calcified nodules were formed in VIC, which showed a significant difference compared with the control group (P < .05). The semi-quantitative level of calcification in the OM group was higher than in the control group (P < .05), and the semi-quantitative level of calcification in the OM+ALP inhibitor group was lower than in the OM group (P < .05). ALP staining intensity, ALP activity and messenger RNA (mRNA) levels of BMP, RUNX2, osteocalcin, OPN, ERK, IκBα, AKT, TNF-α, Toll-like receptor 4 (TLR4) and NLRP3 inflammasome (NLRP3) in the OM group were higher than in the control group (P < .05). ALP staining intensity, ALP activity and mRNA expressions of BMP, RUNX2, osteocalcin, OPN, phosphorylated ERK, IκBα, AKT, TNF-α and NLRP3 in the OM+ALP inhibitor group were lower than in the OM group (P < .05). Compared with the control group, 723 genes were upregulated and 248 genes were downregulated in the OM group. Compared with the OM group, 352 genes were upregulated and 586 genes were downregulated in the OM+ALP inhibitor group. Conclusion: We suggest that ALP inhibitors have potential in terms of inhibiting the inflammatory response and osteoblast differentiation of human VIC (hVIC) via the TLR4, AKT, ERK and NLRP3 pathways.

[Berberine inhibits osteogenic differentiation of aortic valve interstitial cells induced by osteogenic induction medium].

This research focused on the effect and mechanism of berberine on osteogenic differentiation of valve interstitial cells(VICs) induced by osteogenic induction medium, in order to provide new insights into the clinical treatment of calcified aortic valve disease. The expression of osteogenic and fibrotic makers in three cases of calcified valve tissues and one case of normal control was assayed by Western blot. After the porcine aortic VICs were isolated, the effects of different concentrations of berberine on their viability were examined by MTT assay for determining the optimal concentration range. VICs were cultured in osteogenic induction medium and treated with different concentrations of berberine. Western blot and q-PCR were conducted to detect the effects of berberine on the expression of osteogenic and fibrotic makers in VICs. The effects of berberine on osteogenic differentiation of VICs in the early and late stages were separately measured by ALP staining and alizarin red S staining. The effects of berberine on the phosphorylation of ERK1/2 at different time points were assayed by Western blot. And PD98059, an inhibitor of ERK1/2, was added for verification. The results suggested that related osteogenic and fibrotic makers were significantly up-regulated in calcified valve tissues as compared with those in the normal control. The up-regulated fibrosis and osteogenic makers of VICs under osteogenic conditions were reversed by berberine and the ALP activity and calcium deposition in VICs were also reduced obviously. The level of ERK1/2 phosphorylation was decreased. Similarly, the osteogenic and fibrotic makers of VICs induced by osteogenic induction medium were lowered by PD98059. This study has confirmed that berberine is able to inhibit the differentiation of VICs into myofibroblasts or osteoblast-like cells, which may be associated with the inhibition of ERK1/2 signaling pathway.

Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification.

AIMS: Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. METHODS AND RESULTS: Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis. CONCLUSION: The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability.

Basic fibroblast growth factor inhibits aortic valvular interstitial cells calcification via Notch1 pathway.

Calcific aortic valve disease (CAVD) is an active pathological process mediated by abnormal activation and transdifferentiation of valvular interstitial cells (VICs). The present study aims to investigate the function and underlying mechanism of the basic fibroblast growth factor (BFGF) on osteogenic differentiation of VICs. Porcine VICs cultured with osteogenic induction medium are supplemented with or without BFGF. Morphology of VICs is identified by fluorescein isothiocyanate-labeled phalloidin, the cell viability is assessed by the cell counting kit-8 method, and protein and mRNA expression level of osteogenic differentiation markers, including Runx2, osteopontin, and Sp7, are verified by western blot analysis and quantitative real-time PCR, respectively. RNA sequencing is used to identify changes in gene profiles. Alizarin Red S staining is used to measure calcium deposition. The results demonstrate that the content of calcium deposition and the expression level of osteogenic markers are downregulated by supplementing BFGF. Notch1 signaling pathway is extracted as a candidate target after bioinformatics analysis by RNA sequencing. The transfection of si-Notch1 abolishes the calcification inhibitory effect of BFGF. Taken together, our findings shed the light on the mechanism and potential therapeutics of BFGF for CAVD.

A Novel Two-Dimensional Echocardiography Method to Objectively Quantify Aortic Valve Calcium and Predict Aortic Stenosis Severity.

Aortic valve calcium (AVC) is a strong predictor of aortic stenosis (AS) severity and is typically calculated by multidetector computed tomography (MDCT). We propose a novel method using pixel density quantification software to objectively quantify AVC by two-dimensional (2D) transthoracic echocardiography (TTE) and distinguish severe from non-severe AS. A total of 90 patients (mean age 76 ± 10 years, 75% male, mean AV gradient 32 ± 11 mmHg, peak AV velocity 3.6 ± 0.6 m/s, AV area (AVA) 1.0 ± 0.3 cm2, dimensionless index (DI) 0.27 ± 0.08) with suspected severe aortic stenosis undergoing 2D echocardiography were retrospectively evaluated. Parasternal short axis aortic valve views were used to calculate a gain-independent ratio between the average pixel density of the entire aortic valve in short axis at end diastole and the average pixel density of the aortic annulus in short axis (2D-AVC ratio). The 2D-AVC ratio was compared to echocardiographic hemodynamic parameters associated with AS, MDCT AVC quantification, and expert reader interpretation of AS severity based on echocardiographic AVC interpretation. The 2D-AVC ratio exhibited strong correlations with mean AV gradient (r = 0.72, p < 0.001), peak AV velocity (r = 0.74, p < 0.001), AVC quantified by MDCT (r = 0.71, p <0.001) and excellent accuracy in distinguishing severe from non-severe AS (area under the curve = 0.93). Conversely, expert reader interpretation of AS severity based on echocardiographic AVC was not significantly related to AV mean gradient (t = 0.23, p = 0.64), AVA (t = 2.94, p = 0.11), peak velocity (t = 0.59, p = 0.46), or DI (t = 0.02, p = 0.89). In conclusion, these data suggest that the 2D-AVC ratio may be a complementary method for AS severity adjudication that is readily quantifiable at time of TTE.

Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification.

The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.

Ubiquinol supplementation in elderly patients undergoing aortic valve replacement: biochemical and clinical aspects.

Epidemiological data show a rise in the mean age of patients affected by heart disease undergoing cardiac surgery. Senescent myocardium reduces the tolerance to ischemic stress and there are indications about age-associated deficit in post-operative cardiac performance. Coenzyme Q10 (CoQ10), and more specifically its reduced form ubiquinol (QH), improve several conditions related to bioenergetic deficit or increased exposure to oxidative stress. This trial (Eudra-CT 2009-015826-13) evaluated the clinical and biochemical effects of ubiquinol in 50 elderly patients affected by severe aortic stenosis undergoing aortic valve replacement and randomized to either placebo or 400 mg/day ubiquinol from 7 days before to 5 days after surgery. Plasma and cardiac tissue CoQ10 levels and oxidative status, circulating troponin I, CK-MB (primary endpoints), IL-6 and S100B were assessed. Moreover, main cardiac adverse effects, NYHA class, contractility and myocardial hypertrophy (secondary endpoints) were evaluated during a 6-month follow-up visit. Ubiquinol treatment counteracted the post-operative plasma CoQ10 decline (p<0.0001) and oxidation (p=0.038) and curbed the post-operative increase in troponin I (QH, 1.90 [1.47-2.48] ng/dL; placebo, 4.03 [2.45-6.63] ng/dL; p=0.007) related to cardiac surgery. Moreover, ubiquinol prevented the adverse outcomes that might have been associated with defective left ventricular ejection fraction recovery in elderly patients.

FADS1 (Fatty Acid Desaturase 1) Genotype Associates With Aortic Valve FADS mRNA Expression, Fatty Acid Content and Calcification.

BACKGROUND: Aortic stenosis (AS) contributes to cardiovascular mortality and morbidity but disease mechanisms remain largely unknown. Recent evidence associates a single nucleotide polymorphism rs174547 within the FADS1 gene, encoding FADS1 (fatty acid desaturase 1), with risk of several cardiovascular outcomes, including AS. FADS1 encodes a rate-limiting enzyme for ω-3 and ω-6 fatty acid metabolism. The aim of this study was to decipher the local transcriptomic and lipidomic consequences of rs174547 in tricuspid aortic valves from patients with AS. METHODS: Expression quantitative trait loci study was performed using data from Illumina Human610-Quad BeadChip, Infinium Global Screening Arrays, and Affymetrix Human Transcriptome 2.0 arrays in calcified and noncalcified aortic valve tissue from 58 patients with AS (mean age, 74.2; SD, 5.9). Fatty acid content was assessed in aortic valves from 25 patients with AS using gas chromatography. Δ5 and Δ6 desaturase activity was assessed by the product-to-precursor ratio. RESULTS: The minor C-allele of rs174547, corresponding to the protective genotype for AS, was associated with higher FADS2 mRNA levels in calcified valve tissue, whereas FADS1 mRNA and other transcripts in proximity of the single nucleotide polymorphism were unaltered. In contrast, the FADS1 Δ5-desaturase activity and the FADS2 Δ6-desaturase activity were decreased. Finally, docosahexaenoic acid was decreased in calcified tissue compared with non-calcified tissue and C-allele carriers exhibited increased docosahexaenoic acid levels. Overall desaturase activity measured with ω-3 fatty acids was higher in C-allele carriers. CONCLUSIONS: The association between the FADS1 genotype and AS may implicate effects on valvular fatty acids.

Fibrotic aortic valve disease after radiotherapy: an immunohistochemical study in breast cancer and lymphoma patients.

BACKGROUND: Radiation-associated aortic valve (AV) stenosis is frequently seen as a late sequela after thoracic radiotherapy (RT). Although the clinical relationship between thoracic radiotherapy and valvular dysfunction has been established, the process leading to accelerated aortic valve stenosis remains unclear. The aim of this study was to determine whether increased inflammatory cell infiltration, fibrosis, and calcification is present in aortic valves after radiotherapy at the time of aortic valve replacement. METHODS: Stenotic aortic valve specimens from 43 patients were obtained after surgical aortic valve replacement. A total 28 patients had previously undergone radiotherapy for breast cancer or malignant lymphoma. A total 15 patients were included as control. The valve leaflets were assessed by (immuno)histochemistry for inflammatory cell composition (CD3, CD20, CD68, and CD163) and extracellular matrix changes (collagen and calcification). RESULTS: Aortic valve cell density after radiotherapy for lymphoma was markedly decreased when compared with other groups. Irradiated aortic valve show similar (low) degrees of late T and B lymphocyte infiltration as control valves, whereas macrophage marker CD68 was decreased after radiotherapy for breast cancer. Collagen content was increased following radiotherapy. Aortic valves of patients with lymphoma contained significantly less calcified tissue when compared with the other groups. CONCLUSION: High-dose radiation at a young age (patients with lymphoma) results in cell loss and premature fibrotic aortic valve stenosis as opposed to the degenerative calcific stenosis observed in patients with breast cancer. Our findings suggest a possible dose-dependent effect of radiotherapy on aortic valve fibrosis. The active presence of inflammatory cells may be limited to the acute phase after radiotherapy.

Nobiletin exhibits potent inhibition on tumor necrosis factor alpha-induced calcification of human aortic valve interstitial cells via targeting ABCG2 and AKR1B1.

Inflammation is considered to be one of the initial critical factors in the occurrence of calcific heart valve disease. This study was to prove Nobiletin (NBT) inhibits inflammation-caused calcification of human valve interstitial cells (hVICs) and to elucidate the involved molecular mechanisms. Tumor necrosis factor-alpha (TNF-α)-induced hVICs were treated with or without NBT. Cell growth and calcification of hVICs were assessed. RNA sequencing was utilized to investigate the gene expression changes. Molecular target prediction and docking assay were further performed. NBT interfered with hVIC growth under TNF-α condition in a dose-dependent manner also presented a gradual decrease of positive Alizarin Red S staining, down-regulation of BMP2, and RUNX2 gene expression. Based on the global gene expression cluster, control and TNF-α plus NBT group showed a high similarity versus TNF-α only group. After Venn interaction of differential expression genes (DEGs), 2,236 common DEGs were identified to display different biological functions and signaling pathways. ABCG2 and AKR1B1 were further selected as prediction targets of NBT involved in RELA, TNF, BMP2, RUNX2, etc. interactions in mediating hVIC calcification. The results show that NBT is a natural product to prevent the occurrence of heart valve calcification.

Impact of high-fat diet and vitamin D3 supplementation on aortic stenosis establishment in waved-2 epidermal growth factor receptor mutant mice.

OBJECTIVE: The use of animal models of aortic stenosis (AS) remains essential to further elucidate its pathophysiology and to evaluate new therapeutic strategies. The waved-2 mouse AS model has been proposed; data have indicated that while aortic regurgitation (AR) is effectively induced, development of AS is rare. We aimed to evaluate the effect of high-fat diet (HFD) and vitamin D3 supplementation in this model. METHODS: HFD and subcutaneous vitamin D3 injections were initiated at the age of 6 weeks until the age of 6 (n = 16, 6-month treatment group) and 9 (n = 11, 9-month treatment group) months. Twelve waved-2 mice without supplementation were used as control. Echocardiography was performed at 3, 6 and 9 months. Blood serum analysis (calcium, 1,25(OH)2D3 and cholesterol), histology and immunohistochemistry (CD-31, CD-68 and osteopontin) were evaluated at the end of the experiment (6 or 9 months). RESULTS: Total cholesterol and 1,25(OH)2D3 were significantly increased relative to the control group. HFD and vitamin D3 supplementation did result in improvements to the model, since AS was only detected in 6 (15.3%) mice (2 in the 3 groups) and AR was developed in the remaining animals. Echocardiographic parameters, fibrosis, thickness, inflammation and valvular calcification, were not significantly different between the 6-month treatment and control groups. Similar results were also observed in the 9-month treatment group. CONCLUSION: These results suggest that HFD and vitamin D3 supplementation have no effect in the waved-2 mouse model. This model essentially mimics AR and rarely AS. Further studies are needed to find a reliable animal model of AS.

Warfarin, but not rivaroxaban, promotes the calcification of the aortic valve in ApoE-/- mice.

INTRODUCTION: Vitamin K antagonists, such as warfarin, are known to promote arterial calcification through blockade of gamma-carboxylation of Matrix-Gla-Protein. It is currently unknown whether other oral anticoagulants such as direct inhibitors of Factor Xa can have protective effects on the progression of aortic valve calcification. AIMS: To compare the effect of warfarin and rivaroxaban on the progression of aortic valve calcification in atherosclerotic mice. RESULTS: 42 ApoE-/- mice fed with Western-type Diet (WTD) were randomized to treatment with warfarin (n = 14), rivaroxaban (n = 14) or control (n = 14) for 8 weeks. Histological analyses were performed to quantify the calcification of aortic valve leaflets and the development of atherosclerosis. The analyses showed a significant increase in valve calcification in mice treated with warfarin as compared to WTD alone (P = .025) or rivaroxaban (P = .005), whereas no significant differences were found between rivaroxaban and WTD (P = .35). Quantification of atherosclerosis and intimal calcification was performed on the innominate artery of the mice and no differences were found between the 3 treatments as far as atherogenesis and calcium deposition is concerned. In vitro experiments performed using bovine interstitial valve cells (VIC) showed that treatment with rivaroxaban did not prevent the osteogenic conversion of the cells but reduce the over-expression of COX-2 induced by inflammatory mediators. CONCLUSION: We showed that warfarin, but not rivaroxaban, could induce calcific valve degeneration in a mouse model of atherosclerosis. Both the treatments did not significantly affect the progression of atherosclerosis. Overall, these data suggest a safer profile of rivaroxaban on the risk of cardiovascular disease progression.

Beneficial Effects of Galectin-3 Blockade in Vascular and Aortic Valve Alterations in an Experimental Pressure Overload Model.

Galectin-3 (Gal-3) is involved in cardiovascular fibrosis and aortic valve (AV) calcification. We hypothesized that Gal-3 pharmacological inhibition with modified citrus pectin (MCP) could reduce aortic and AV remodeling in normotensive rats with pressure overload (PO). Six weeks after aortic constriction, vascular Gal-3 expression was up-regulated in male Wistar rats. Gal-3 overexpression was accompanied by an increase in the aortic media layer thickness, enhanced total collagen, and augmented expression of fibrotic mediators. Further, vascular inflammatory markers as well as inflammatory cells content were greater in aorta from PO rats. MCP treatment (100 mg/kg/day) prevented the increase in Gal-3, media thickness, fibrosis, and inflammation in the aorta of PO rats. Gal-3 levels were higher in AVs from PO rats. This paralleled enhanced AV fibrosis, inflammation, as well as greater expression of calcification markers. MCP treatment prevented the increase in Gal-3 as well as fibrosis, inflammation, and calcification in AVs. Overall, Gal-3 is overexpressed in aorta and AVs from PO rats. Gal-3 pharmacological inhibition blocks aortic and AV remodeling in experimental PO. Gal-3 could be a new therapeutic approach to delay the progression and the development of aortic remodeling and AV calcification in PO.

Role for Galectin-3 in Calcific Aortic Valve Stenosis.

BACKGROUND: Aortic stenosis (AS) is a chronic inflammatory disease, and calcification plays an important role in the progression of the disease. Galectin-3 (Gal-3) is a proinflammatory molecule involved in vascular osteogenesis in atherosclerosis. Therefore, we hypothesized that Gal-3 could mediate valve calcification in AS. METHODS AND RESULTS: Blood samples and aortic valves (AVs) from 77 patients undergoing AV replacement were analyzed. As controls, noncalcified human AVs were obtained at autopsy (n=11). Gal-3 was spontaneously expressed in valvular interstitial cells (VICs) from AVs and increased in AS as compared to control AVs. Positive correlations were found between circulating and valvular Gal-3 levels. Valvular Gal-3 colocalized with the VICs markers, alpha-smooth muscle actin and vimentin, and with the osteogenic markers, osteopontin, bone morphogenetic protein 2, runt-related transcription factor 2, and SRY (sex-determining region Y)-box 9. Gal-3 also colocalized with the inflammatory markers cd68, cd80 and tumor necrosis factor alpha. In vitro, in VICs isolated from AVs, Gal-3 induced expression of inflammatory, fibrotic, and osteogenic markers through the extracellular signal-regulated kinase 1 and 2 pathway. Gal-3 expression was blocked in VICs undergoing osteoblastic differentiation using its pharmacological inhibitor, modified citrus pectin, or the clustered regularly interspaced short palindromic repeats/Cas9 knockout system. Gal-3 blockade and knockdown decreased the expression of inflammatory, fibrotic, and osteogenic markers in differentiated VICs. CONCLUSIONS: Gal-3, which is overexpressed in AVs from AS patients, appears to play a central role in calcification in AS. Gal-3 could be a new therapeutic approach to delay the progression of AV calcification in AS.

Omega-3-fatty acid adds to the protective effect of flax lignan concentrate in pressure overload-induced myocardial hypertrophy in rats via modulation of oxidative stress and apoptosis.

Objective of the present investigation was to study the effect of the flax lignan concentrate (FLC) and Omega-3-fatty acid (O-3-FA) on myocardial apoptosis, left ventricular (LV) contractile dysfunction and electrocardiographic abnormalities in pressure overload-induced cardiac hypertrophy. The rats were divided into five groups such as sham, aortic stenosis (AS), AS+FLC, AS+O-3-FA and AS+FLC+O-3-FA. Cardiac hypertrophy was produced in rats by abdominal aortic constriction. The rats were treated with FLC (400mg/kg, p.o.), O-3-FA (400mg/kg, p.o.) and FLC+O-3-FA orally per day for four weeks. The LV function, myocardial apoptosis, and oxidative stress were quantified. FLC+O-3-FA treatment significantly reduced hemodynamic changes, improved LV contractile dysfunction, reduced cardiomyocyte apoptosis and cellular oxidative stress. Moreover, it significantly up-regulated the VEGF expression and decreased TNF-alpha level in serum. The histological analysis also revealed that FLC+O-3-FA treatment markedly preserved the cardiac structure and inhibited interstitial fibrosis. In conclusion, FLC+O-3-FA treatment improved LV dysfunction, inhibited cardiomyocyte apoptosis, improved myocardial angiogenesis, conserved activities of membrane-bound phosphatase enzymes and suppressed inflammation through reduced oxidative stress in an additive manner than FLC alone and O-3-FA alone treatment in pressure overload-induced cardiac hypertrophy.

Biophysical analysis of dystrophic and osteogenic models of valvular calcification.

Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder characterized by the formation of calcific nodules (CN) on the valve. In vitro assays studying the formation of these nodules were developed and have led to many significant mechanistic findings; however, the biophysical properties of CNs have not been clearly defined. A thorough analysis of dystrophic and osteogenic nodules utilizing scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and atomic force microscopy (AFM) was conducted to describe calcific nodule properties and provide a link between calcific nodule morphogenesis in vitro and in vivo. Unique nodule properties were observed for dystrophic and osteogenic nodules, highlighting the distinct mechanisms occurring in valvular calcification.

Extracellular pyrophosphate is reduced in aortic interstitial valve cells acquiring a calcifying profile: implications for aortic valve calcification.

OBJECTIVES: Pyrophosphate (PPi) is a potent inhibitor of ectopic mineralization but its role during aortic valve calcification is not known. METHODS: Anti-calcific effect of PPi was investigated by using an in vitro model of serum-driven calcification of collagen sponges and decellularized porcine aortic valve leaflets. Bovine interstitial valve cells (VIC), seeded either within the collagen matrices or in transwell chambers, were used to test cellular ability to inhibit serum-induced calcification. PPi metabolism was investigated in clonal VIC harboring different calcifying potential. RESULTS: In a cell-free system, high serum levels induced a dose-dependent calcification of type I collagen matrices which was prevented by PPi and ATP supplementation. Blockade of serum-driven calcification by PPi and ATP was also observed when using decellularized porcine aortic valve leaflets. A similar anti-calcific effect was also seen for bovine VIC, either statically seeded into the collagen matrices or co-cultured by using a transwell system. However, when we performed co-culture experiments by using clonal VIC harboring different calcifying potential, we observed that the subset of cells acquiring a pro-calcific profile lost the ability to protect the collagen from serum-driven calcification. Pro-calcific differentiation of the clonal VIC was accompanied by increase in ALP along with significant reduction in NPP activity and ATP/PPi extracellular accumulation. These changes were not observed in the clonal subtype with lower propensity towards calcification. CONCLUSIONS: We showed that PPi and ATP are potent inhibitors of serum-driven calcification of collagen matrix and that their extracellular accumulation is reduced in calcifying VIC.

Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5.

Pressure overload-induced TGF-ß signaling activates cardiac fibroblasts (CFB) and leads to increased extracellular matrix (ECM) protein synthesis including fibrosis. Excessive ECM accumulation may in turn affect cardiac function contributing to development of heart failure. The aim of this study was to examine the effects of SM16, an orally active small molecular inhibitor of ALK5, on pressure overload-induced cardiac fibrosis. One week after aortic banding (AB), C57Bl/6J mice were randomized to standard chow or chow with SM16. Sham operated animals served as controls. Following 4 weeks AB, mice were characterized by echocardiography and cardiovascular magnetic resonance before sacrifice. SM16 abolished phosphorylation of SMAD2 induced by AB in vivo and by TGF-ß in CFB in vitro. Interestingly, Masson Trichrome and Picrosirius Red stained myocardial left ventricular tissue revealed reduced development of fibrosis and collagen cross-linking following AB in the SM16 treated group, which was confirmed by reduced hydroxyproline incorporation. Furthermore, treatment with SM16 attenuated mRNA expression following induction of AB in vivo and stimulation with TGF-ß in CFB in vitro of Col1a2, the cross-linking enzyme LOX, and the pro-fibrotic glycoproteins SPARC and osteopontin. Reduced ECM synthesis by CFB and a reduction in myocardial stiffness due to attenuated development of fibrosis and collagen cross-linking might have contributed to the improved diastolic function and cardiac output seen in vivo, in combination with reduced lung weight and ANP expression by treatment with SM16. Despite these beneficial effects on cardiac function and development of heart failure, mice treated with SM16 exhibited increased mortality, increased LV dilatation and inflammatory heart valve lesions that may limit the use of SM16 and possibly also other small molecular inhibitors of ALK5, as future therapeutic drugs.