Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis.
ABSTRACT: Diabetes contributes directly to the development of cardiovascular aortic valve disease. There is currently no drug therapy available for a dysfunctional valve and this urges the need for additional research to identify distinctive mechanisms of cardiovascular aortic valve disease evolution. The aim of this study was to evaluate changes of valvular aortic lesions induced in a hyperlipemic ApoE-/- mouse model by early type 1 diabetes onset (at 4 and 7?days after streptozotocin induction). The haemodynamic valve parameters were evaluated by echography and blood samples and aortic valves were collected. Plasma parameters were measured, and inflammatory, remodelling and osteogenic markers were evaluated in the aortic valves. Next, correlations between all parameters were determined. The results showed early aortic valve dysfunction detected by echography after 1?week of diabetes; lesions were found in the aortic root. Moreover, increased expression of cell adhesion molecules, extracellular matrix remodelling and osteogenic markers were detected in hyperlipemic ApoE-/- diabetic mice. Significant correlations were found between tissue valve biomarkers and plasmatic and haemodynamic parameters. Our study may help to understand the mechanisms of aortic valve disease in the diabetic milieu in order to discover and validate new biomarkers of cardiovascular aortic valve disease in diabetes and reveal new possible targets for nanobiotherapies.
Project description:Aortic valve stenosis (AVS), a consequence of increased fibrosis and calcification of the aortic valve leaflets, causes progressive narrowing of the aortic valve. Proteoglycans, structural components of the aortic valve, accumulate in regions with fibrosis and moderate calcification. Particularly, proteoglycan 4 (PRG4) has been identified in fibrotic parts of aortic valves. However, the role of PRG4 in the context of AVS and aortic valve calcification has not yet been determined. Here, transcriptomics, histology, and immunohistochemistry were performed in human aortic valves from patients undergoing aortic valve replacement. Human valve interstitial cells (VICs) were used for calcification experiments and RNA expression analysis. PRG4 was significantly upregulated in thickened and calcified regions of aortic valves compared with healthy regions. In addition, mRNA levels of PRG4 positively associated with mRNA for proteins involved in cardiovascular calcification. Treatment of VICs with recombinant human PRG4 enhanced phosphate-induced calcification and increased the mRNA expression of bone morphogenetic protein 2 and the runt-related transcription factor 2. In summary, PRG4 was upregulated in the development of AVS and promoted VIC osteogenic differentiation and calcification. These results suggest that an altered valve leaflet proteoglycan composition may play a role in the progression of AVS.
Project description: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-?B phosphorylation induced by lipopolysaccharide was markedly reduced by inhibition or silencing of Notch1 and enhanced by Jagged1. Inhibition of ERK1/2 or nuclear factor-?B 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-?B activation. These pathways could be potential therapeutic targets for prevention of the progression of calcific aortic valve disease.
Project description:Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.
Project description:Given that the bioactive lipid sphingosine 1-phosphate is involved in cardiovascular pathophysiology, and since lipid accumulation and inflammation are hallmarks of calcific aortic stenosis, the role of sphingosine 1-phosphate on the pro-inflammatory/pro-osteogenic pathways in human interstitial cells from aortic and pulmonary valves was investigated. Real-time PCR showed sphingosine 1-phosphate receptor expression in aortic valve interstitial cells. Exposure of cells to sphingosine 1-phosphate induced pro-inflammatory responses characterized by interleukin-6, interleukin-8, and cyclooxygenase-2 up-regulations, as observed by ELISA and Western blot. Strikingly, cell treatment with sphingosine 1-phosphate plus lipopolysaccharide resulted in the synergistic induction of cyclooxygenase-2, and intercellular adhesion molecule 1, as well as the secretion of prostaglandin E2, the soluble form of the intercellular adhesion molecule 1, and the pro-angiogenic factor vascular endothelial growth factor-A. Remarkably, the synergistic effect was significantly higher in aortic valve interstitial cells from stenotic than control valves, and was drastically lower in cells from pulmonary valves, which rarely undergo stenosis. siRNA and pharmacological analysis revealed the involvement of sphingosine 1-phosphate receptors 1/3 and Toll-like receptor-4, and downstream signaling through p38/MAPK, protein kinase C, and NF-?B. As regards pro-osteogenic pathways, sphingosine 1-phosphate induced calcium deposition and the expression of the calcification markers bone morphogenetic protein-2 and alkaline phosphatase, and enhanced the effect of lipopolysaccharide, an effect that was partially blocked by inhibition of sphingosine 1-phosphate receptors 3/2 signaling. In conclusion, the interplay between sphingosine 1-phosphate receptors and Toll-like receptor 4 signaling leads to a cooperative up-regulation of inflammatory, angiogenic, and osteogenic pathways in aortic valve interstitial cells that seems relevant to the pathogenesis of aortic stenosis and may allow the inception of new therapeutic approaches.
Project description:Studies of human diseased aortic valves have demonstrated increased expression of genetic markers of valve progenitors and osteogenic differentiation associated with pathogenesis. Three potential mouse models of valve disease were examined for cellular pathology, morphology, and induction of valvulogenic, chondrogenic, and osteogenic markers. Osteogenesis imperfecta murine (Oim) mice, with a mutation in Col1a2, have distal leaflet thickening and increased proteoglycan composition characteristic of myxomatous valve disease. Periostin null mice also exhibit dysregulation of the ECM with thickening in the aortic midvalve region, but do not have an overall increase in valve leaflet surface area. Klotho null mice are a model for premature aging and exhibit calcific nodules in the aortic valve hinge-region, but do not exhibit leaflet thickening, ECM disorganization, or inflammation. Oim/oim mice have increased expression of valve progenitor markers Twist1, Col2a1, Mmp13, Sox9 and Hapln1, in addition to increased Col10a1 and Asporin expression, consistent with increased proteoglycan composition. Periostin null aortic valves exhibit relatively normal gene expression with slightly increased expression of Mmp13 and Hapln1. In contrast, Klotho null aortic valves have increased expression of Runx2, consistent with the calcified phenotype, in addition to increased expression of Sox9, Col10a1, and osteopontin. Together these studies demonstrate that oim/oim mice exhibit histological and molecular characteristics of myxomatous valve disease and Klotho null mice are a new model for calcific aortic valve disease.
Project description:Aortic valve sclerosis (AVSc) is a hallmark of several cardiovascular conditions ranging from chronic heart failure and myocardial infarction to calcific aortic valve stenosis (AVS). AVSc, present in 25-30% of patients over 65 years of age, is characterized by thickening of the leaflets with marginal effects on the mechanical proprieties of the valve making its presentation asymptomatic. Despite its clinical prevalence, few studies have investigated the pathogenesis of this disease using human AVSc specimens. Here, we investigate in vitro and ex vivo BMP4-mediated transdifferentiation of human valve interstitial cells (VICs) towards an osteogenic-like phenotype in AVSc.Human specimens from 60 patients were collected at the time of aortic valve replacement (AVS) or through the heart transplant programme (Controls and AVSc). We show that non-calcified leaflets from AVSc patients can be induced to express markers of osteogenic transdifferentiation and biomineralization through the combinatory effect of BMP4 and mechanical stimulation. We show that BMP4 antagonist Noggin attenuates VIC activation and biomineralization. Additionally, patient-derived VICs were induced to transdifferentiate using either cell culture or a Tissue Engineering (TE) Aortic Valve model. We determine that while BMP4 alone is not sufficient to induce osteogenic transdifferentiation of AVSc-derived cells, the combinatory effect of BMP4 and mechanical stretch induces VIC activation towards a phenotype typical of late calcified stage of the disease.This work demonstrates, for the first time using AVSc specimens, that human sclerotic aortic valves can be induced to express marker of osteogenic-like phenotype typical of advanced severe aortic stenosis.
Project description:Arterial endothelial cells maintain vascular homeostasis and vessel tone in part through the secretion of nitric oxide (NO). In this study, we determined how aortic valve endothelial cells (VEC) regulate aortic valve interstitial cell (VIC) phenotype and matrix calcification through NO. Using an anchored in vitro collagen hydrogel culture system, we demonstrate that three-dimensionally cultured porcine VIC do not calcify in osteogenic medium unless under mechanical stress. Co-culture with porcine VEC, however, significantly attenuated VIC calcification through inhibition of myofibroblastic activation, osteogenic differentiation, and calcium deposition. Incubation with the NO donor DETA-NO inhibited VIC osteogenic differentiation and matrix calcification, whereas incubation with the NO blocker l-NAME augmented calcification even in 3D VIC-VEC co-culture. Aortic VEC, but not VIC, expressed endothelial NO synthase (eNOS) in both porcine and human valves, which was reduced in osteogenic medium. eNOS expression was reduced in calcified human aortic valves in a side-specific manner. Porcine leaflets exposed to the soluble guanylyl cyclase inhibitor ODQ increased osteocalcin and ?-smooth muscle actin expression. Finally, side-specific shear stress applied to porcine aortic valve leaflet endothelial surfaces increased cGMP production in VEC. Valve endothelial-derived NO is a natural inhibitor of the early phases of valve calcification and therefore may be an important regulator of valve homeostasis and pathology.
Project description:Calcific aortic valve disease (CAVD) is the most common heart valve disorder, yet its mechanism remains poorly understood. Valve interstitial cells (VICs) are the prevalent cells in aortic valve and their osteogenic differentiation may be responsible for calcific nodule formation in CAVD pathogenesis. Emerging evidence shows microRNA (miRNA, or miR) can function as important regulators of many pathological processes, including osteogenic differentiation. Here, we aimed to explore the function of miR-449c-5p in CAVD pathogenesis. In this study, we demonstrated the role of miR-449c-5p in VICs osteogenesis. MiRNA microarray assay and qRT-PCR results revealed miR-449c-5p was significantly down-regulated in calcified aortic valves compared with non-calcified valves. MiR-449c-5p overexpression inhibited VICs osteogenic differentiation in vitro, whereas down-regulation of miR-449c-5p enhanced the process. Target prediction analysis and dual-luciferase reporter assay confirmed Smad4 was a direct target of miR-449c-5p. Furthermore, knockdown of Smad4 inhibited VICs osteogenic differentiation, similar to the effect observed in up-regulation miR-449c-5p. In addition, animal experiments proved indirectly miR-449c-5p could alleviate aortic valve calcification. Our data suggested miR-449c-5p could function as a new inhibitory regulator of VICs osteogenic differentiation, which may act by targeting Smad4. MiR-449c-5p may be a potential therapeutic target for CAVD.
Project description:OBJECTIVE:The activation of valve interstitial cells (VICs) toward an osteogenic phenotype characterizes aortic valve sclerosis, the early asymptomatic phase of calcific aortic valve disease. Osteopontin is a phosphorylated acidic glycoprotein that accumulates within the aortic leaflets and labels VIC activation even in noncalcified asymptomatic patients. Despite this, osteopontin protects VICs against in vitro calcification. Here, we hypothesize that the specific interaction of osteopontin with CD44v6, and the related intracellular pathway, prevents calcium deposition in human-derived VICs from patients with aortic valve sclerosis. APPROACH AND RESULTS:On informed consent, 23 patients and 4 controls were enrolled through the cardiac surgery and heart transplant programs. Human aortic valves and VICs were tested for osteogenic transdifferentiation, ex vivo and in vitro. Osteopontin-CD44 interaction was analyzed using proximity ligation assay and the signaling pathways investigated. A murine model based on angiotensin II infusion was used to mimic early pathological remodeling of the aortic valves. We report osteopontin-CD44 functional interaction as a hallmark of early stages of calcific aortic valve disease. We demonstrated that osteopontin-CD44 interaction mediates calcium deposition via phospho-Akt in VICs from patients with noncalcified aortic valve sclerosis. Finally, microdissection analysis of murine valves shows increased cusp thickness in angiotensin II-treated mice versus saline infused along with colocalization of osteopontin and CD44 as seen in human lesions. CONCLUSIONS:Here, we unveil a specific protein-protein association and intracellular signaling mechanisms of osteopontin. Understanding the molecular mechanisms of early VIC activation and calcium deposition in asymptomatic stage of calcific aortic valve disease could open new prospective for diagnosis and therapeutic intervention.
Project description:BACKGROUND:There is an ever-growing number of patients requiring aortic valve replacement (AVR). Limited data is available on the long-term outcomes and structural integrity of bioprosthetic valves in younger patients undergoing surgical AVR. METHODS:The INSPIRIS RESILIA Durability Registry (INDURE) is a prospective, open-label, multicentre, international registry with a follow-up of 5?years to assess clinical outcomes of patients younger than 60?years who undergo surgical AVR using the INSPIRIS RESILIA aortic valve. INDURE will be conducted across 20-22 sites in Europe and Canada and intends to enrol minimum of 400 patients. Patients will be included if they are scheduled to undergo AVR with or without concomitant root replacement and/or coronary bypass surgery. The primary objectives are to 1) determine VARC-2 defined time-related valve safety at one-year (depicted as freedom from events) and 2) determine freedom from stage 3 structural valve degeneration (SVD) presenting as morphological abnormalities and severe haemodynamic valve degeneration at 5?years. Secondary objectives include the assessment of the haemodynamic performance of the valve, all stages of SVD, potential valve-in-valve procedures, clinical outcomes (in terms of New York Heart Association [NYHA] function class and freedom from valve-related rehospitalisation) and change in patient quality-of-life. DISCUSSION:INDURE is a prospective, multicentre registry in Europe and Canada, which will provide much needed data on the long-term performance of bioprosthetic valves in general and the INSPIRIS RESILIA valve in particular. The data may help to gather a deeper understanding of the longevity of bioprosthetic valves and may expand the use of bioprosthetic valves in patients under the age of 60?years. TRIAL REGISTRATION:ClinicalTrials.gov identifier: NCT03666741 (registration received September, 12th, 2018).