Calcific Aortic Valve Disease: Molecular Mechanisms And Therapeutic Approaches

Calcification occurs in atherosclerotic vascular lesions and in the aortic valve. Calcific aortic valve disease (CAVD) is a slow, progressive disorder that ranges from mild valve thickening without obstruction of blood flow, termed aortic sclerosis, to severe calcification with impaired leaflet motion, termed aortic stenosis. In the past, this process was thought to be ‘degenerative’ because of time-dependent wear and tear of the leaflets, with passive calcium deposition. The presence of osteoblasts in atherosclerotic vascular lesions and in CAVD implies that calcification is an active, regulated process akin to atherosclerosis, with lipoprotein deposition and chronic inflammation. If calcification is active, via pro-osteogenic pathways, one might expect that development and progression of calcification could be inhibited. The overlap in the clinical factors associated with calcific valve disease and atherosclerosis provides further support for a shared disease mechanism. In our recent research we used an in vitro porcine valve interstitial cell model to study spontaneous calcification and potential promoters and inhibitors. Using this model, we found that denosumab, a human monoclonal antibody targeting the receptor activator of nuclear factor-κB ligand may, at a working concentration of 50 μg/mL, inhibit induced calcium deposition to basal levels

Inhibitors and Promotors of Calcific Aortic Stenosis

Dissertation embargoed until 2020-11-1

Stem cells and their “niche” in the human heart: toward cell therapies of cardiac disease

Replacement of dead cardiomyocytes by scar tissue, unable to mediate normal cardiac contraction, is the most dramatic change following heart infarction. Current dogma investigates potential endogenous “self-repairing” capacity of the myocardium as a means to minimize such degenerative process. Spontaneous regeneration of the myocardium could be exploited to treat severe and frequently fatal conditions. Post ischaemic intrinsic cardiac healing capability could arise because: i) Cardiomyocytes may proliferate and regenerate damaged tissue, or; i) Endogenous cardiac stem cells (CSCs) may divide and differentiate to exhibit cardiac repairing potential. Although CSCs existence has been documented, their identity, intra-cardiac localization and exact potential remain elusive. Cardiac pericytes, which ensheath blood vessels and express cardiac repair capabilities, could interact with c-kit+ CSCs nested in vascular niches during cellular response to injury. I tested the hypothesis that pericytes and endogenous cardiac progenitor’s stem cells might increase their interaction within vascular niches under ischaemic conditions. Focussing on the potential of cell therapies of cardiac disease, I investigated the spatial relationship between pericytes and endogenous cardiac progenitors within stem cells' niches localised in different regions of the human foetal, adult healthy and ischaemic heart. Immunostaining of foetal human cardiac tissues showed that c-kit+ cells expression and their association with pericytes decrease with heart development. Clear decrease is already evident by 19th week of the gestation. Pericytes and c-kit+ cell populations isolated from foetal hearts and expanded in culture reveal that pericytes’ cells express higher levels of the mesodermal cardiac progenitor factor KDR: 3751± 61(SD) vs 398± 19.9 (SD); (P< 0.05) and for the marker towards cardiac lineage Islet1: 1146±155 (SD) vs 728± 124 (SD); (P< 0.05) while c-kit+ cells express higher levels than pericytes of the stemness marker SSEA3:1655± 40.6 (SD) vs 747± 27 (SD); (P< 0.05), known to progressively decrease with cell differentiation. Absence of staining for CD31 marker in cultured cardiac pericytes and c-kit+ cells is replicated by results of endothelial differentiation assessment, which shows that cardiac pericytes and c-kit+ cells do not form CD31+ networks. The cardiac marker α-actin was present in both cell populations. In healthy adult heart, pericytes marker CD146 localise within the vasculature. Following ischaemia this pericyte marker becomes also evident outside the vasculature. In healthy adult atrium, c-kit expression is low and coexpression with other markers inconspicuous. Ischaemia leads to increased c-kit expression in the microvasculature. Furthermore, following ischaemia c-kit, endothelium and pericyte markers colocalize within the same atrial cells. Colocalization studies in ischaemic hearts revealed low levels of co-occurrence (M1/M2) between c-kit and vascular markers in vessels <50μm but a high degree of correlation (PCC). Ischaemia leads to increased c-kit expression, particularly in blood vessels <50um diameter. Blood vessels >50μm diameter show mostly, staining for endothelial (vWF) and pericyte (CD146) markers. Acute ischaemia of the left ventricle affected the detection of cardiac stem cells markers in the infarcted area. The absence of coexpression of markers during acute ischaemia of the left ventricle suggests that post-ischaemia markers coexpresion is time dependent. Conclusion: Foetal heart pericytes and c-kit+/CD117 cells express early cardiac transcription factors and show trans-differentiation potential, which decreases in healthy adult hearts. The preservation and activity of cardiac stem cells’ niches within the atrium vasculature, appears re-activated in post-ischaemic hearts. Better understanding of cardiac c-kit+ and pericyte cells’ interactions during-human embryonic development and during ischaemia may identify alternative novel therapeutic strategy against coronary artery disease

Denosumab could be a potential Inhibitor of Valvular Interstitial Cells Calcification in vitro

OBJECTIVE: Denosumab is a fully human monoclonal antibody and novel antiresorptive agent that works by binding receptor activator of nuclear factor kappa-β ligand (RANKL) and inhibiting the signaling cascade that causes osteoclast maturation, activity, and survival. We aimed to elucidate the effect of Denosumab in the process of spontaneous and induced calcification in an in vitro porcine valvular interstitial cells (VICs) model. MATERIALS AND METHODS: VICs were extracted from fresh porcine hearts by serial collagenase digestion. Spontaneous calcification of VICs was increased in vitro by adding Na(3)PO(4) (3 mM, pH 7.4) and different concentrations (0.1, 1 and 10 ng/ml) of transforming growth factor beta (TGFß). The degree of calcification before and after treatment with Denosumab was estimated by Alizarin Red staining for calcium deposition, and Sirius Red staining for collagen. Colorimetric techniques were used to determine calcium and collagen deposition quantitatively. For statistical analysis we used SPSS and Microsoft Office Excel 2013. RESULTS: Porcine aortic VICs in vitro were induced to calcify by the addition of either 3 mM Na(3)PO(4), showing a 5.2 fold increase by 14 days (P<0.001), or 3 mM Na(3)PO(4) + 10 ng/ml of TGFβ, showing a 7 fold increase by Day 14 (P<0.001). Denosumab inhibited induced calcification by 3 mM Na(3)PO(4) and 3 mM Na(3)PO(4) with the addition of TGFß at either 0.1, 1 or 10 ng/ml to basal levels only at a concentration of 50 μg/ml (P<0.001). CONCLUSION: This study has proved that Denosumab could be a potential inhibitor of the calcification of VICs in vitro. A fuller understanding of the actions of Denosumab may identify a novel therapeutic strategy for clinical intervention against aortic valve calcification and aortic stenosis

Using Na3PO4 to Enhance In vitro Animal Models of Aortic Valve Calcification

BACKGROUND/OBJECTIVES: The pathogenesis of calcific aortic valvular disease (CAVD) involves an active inflammatory process of valvular interstitial cells (VICs) characterized by the activation of specific osteogenic signaling pathways and apoptosis. This process can be studied by analyzing certain molecular markers and gene expression pathways of spontaneous calcification. The purpose of our study is to investigate the role of sodium phosphate (Na3PO4) as a calcification promoter, with the aim of improving in vitro animal models for testing potential calcification inhibitors. MATERIALS AND METHODS: VICs were extracted from 6 healthy 6-month-old fresh porcine hearts by serial collagenase digestion. Quantitative polymerase chain reaction (qPCR) was used to quantify trans-differentiation of genes of interest during spontaneous calcification of VICs. Spontaneous calcification of VICs was increased by adding Na(3)PO(4) (3 mM, pH 7.4). The degree of calcification was estimated by Alizarin Red staining for calcium deposition, and Sirius Red staining for collagen. Colorimetric techniques were used to determine calcium and collagen deposition quantitatively. Additionally, the enzymatic activity of alkaline phosphatase (ALP) was measured by a kinetic assay. For statistical analysis we used SPSS and Microsoft Office Excel 2013. RESULTS: Porcine VICs calcify spontaneously with demonstrable calcium and collagen deposition. In this study we observed an increase of calcium and collagen deposition from day 0 to day 14 (calcium: 376%; P<0.001, collagen: 3553%; P<0.001). qPCR analysis of mRNA by day 14 showed the following results: α-actin, a marker of myoblast phenotype, was increased to 1.6-fold; P<0.001. Runx2, an osteoblast marker, rose to 1.3 fold; P<0.05, TGF-β, a promoter of osteogenesis, increased to 3.2-fold; P<0.001, and RhoA, a regulator of nodular formation in myoblasts, increased to 4.5-fold; P<0.001, compared to their levels at day 0. RANKL mRNA and calponin did not change significantly. Treatment of porcine VICs with Na3PO4 (3 mM, pH 7.4) led to a marked increase in calcium deposition by day 14 (522%; P<0.001), and a significant increase in ALP activity by day 7 (228%; P<0.05). There were no significant changes in ALP activity between the groups by day 14. CONCLUSION: This study has demonstrated the upregulation of some specific molecules during spontaneous calcification of aortic VICs with an active increase of calcium, collagen and ALP activity. In this in vitro model it was possible to increase spontaneous VICs calcification with Na(3)PO(4) (3 mM, pH 7.4) to a level in which inhibitors of calcification could be tested to identify a novel potential therapeutic strategy against calcific aortic stenosis

Risk of COVID-19 after natural infection or vaccinationResearch in context

Background: While vaccines have established utility against COVID-19, phase 3 efficacy studies have generally not comprehensively evaluated protection provided by previous infection or hybrid immunity (previous infection plus vaccination). Individual patient data from US government-supported harmonized vaccine trials provide an unprecedented sample population to address this issue. We characterized the protective efficacy of previous SARS-CoV-2 infection and hybrid immunity against COVID-19 early in the pandemic over three-to six-month follow-up and compared with vaccine-associated protection. Methods: In this post-hoc cross-protocol analysis of the Moderna, AstraZeneca, Janssen, and Novavax COVID-19 vaccine clinical trials, we allocated participants into four groups based on previous-infection status at enrolment and treatment: no previous infection/placebo; previous infection/placebo; no previous infection/vaccine; and previous infection/vaccine. The main outcome was RT-PCR-confirmed COVID-19 >7–15 days (per original protocols) after final study injection. We calculated crude and adjusted efficacy measures. Findings: Previous infection/placebo participants had a 92% decreased risk of future COVID-19 compared to no previous infection/placebo participants (overall hazard ratio [HR] ratio: 0.08; 95% CI: 0.05–0.13). Among single-dose Janssen participants, hybrid immunity conferred greater protection than vaccine alone (HR: 0.03; 95% CI: 0.01–0.10). Too few infections were observed to draw statistical inferences comparing hybrid immunity to vaccine alone for other trials. Vaccination, previous infection, and hybrid immunity all provided near-complete protection against severe disease. Interpretation: Previous infection, any hybrid immunity, and two-dose vaccination all provided substantial protection against symptomatic and severe COVID-19 through the early Delta period. Thus, as a surrogate for natural infection, vaccination remains the safest approach to protection. Funding: National Institutes of Health

Risk of COVID-19 after natural infection or vaccinationResearch in context

Summary: Background: While vaccines have established utility against COVID-19, phase 3 efficacy studies have generally not comprehensively evaluated protection provided by previous infection or hybrid immunity (previous infection plus vaccination). Individual patient data from US government-supported harmonized vaccine trials provide an unprecedented sample population to address this issue. We characterized the protective efficacy of previous SARS-CoV-2 infection and hybrid immunity against COVID-19 early in the pandemic over three-to six-month follow-up and compared with vaccine-associated protection. Methods: In this post-hoc cross-protocol analysis of the Moderna, AstraZeneca, Janssen, and Novavax COVID-19 vaccine clinical trials, we allocated participants into four groups based on previous-infection status at enrolment and treatment: no previous infection/placebo; previous infection/placebo; no previous infection/vaccine; and previous infection/vaccine. The main outcome was RT-PCR-confirmed COVID-19 >7–15 days (per original protocols) after final study injection. We calculated crude and adjusted efficacy measures. Findings: Previous infection/placebo participants had a 92% decreased risk of future COVID-19 compared to no previous infection/placebo participants (overall hazard ratio [HR] ratio: 0.08; 95% CI: 0.05–0.13). Among single-dose Janssen participants, hybrid immunity conferred greater protection than vaccine alone (HR: 0.03; 95% CI: 0.01–0.10). Too few infections were observed to draw statistical inferences comparing hybrid immunity to vaccine alone for other trials. Vaccination, previous infection, and hybrid immunity all provided near-complete protection against severe disease. Interpretation: Previous infection, any hybrid immunity, and two-dose vaccination all provided substantial protection against symptomatic and severe COVID-19 through the early Delta period. Thus, as a surrogate for natural infection, vaccination remains the safest approach to protection. Funding: National Institutes of Health