Human Valvular Interstitial Cells Research Articles

Substrate Chemistry and Morphology Influence the Valvular Interstitial Cells Mechanobiology

Calcific Aortic Valve Disease (CAVD) is the most common form of valve disease in the Western world and represents a major healthcare burden. The primary driver for valvular calcification is the differentiation of valvular interstitial cells (VICs) into a diseased phenotype, the osteoblastic-like cells. Another feature of this disease is the significant change in the organization, composition and mechanical properties of the extracellular matrix (ECM) that beside being the result of the dysfunction of the valve cells, seems to contribute to the progression of the pathology altering cellular molecular signaling. Here, we studied the mechanical, morphological and molecular properties of VICs grown on substrated with different surface chemistry and/or mechanical properties, which mimic the valve ECM structure, to highlight the matrix effect on VICs behavior and the role played in calcified tissue formation. Using atomic force microscopy (AFM) and immunofluorescence microscopy we assessed the biomechanics, morphology and the pathway involved in the mechanical stress and in onset of pathology of porcine and human VICs grown on polyacrylamide gels of different stiffness and on variable-geometry carpets of vertically aligned carbon nanotubes. Our results will allow to identify a suitable material for engineering aortic valve artificial scaffolds.

Abstract 15192: Leptin Effects on Osteoblast Differentiation of Human Valvular Interstitial Cells: New Insight Into Calcific Aortic Valve Disease

Introduction: Calcific aortic valve disease (CAVD) affects 2% to 6% of the population over 65 years and results from dysregulated processes such as calcification, supported in part by the osteoblast differentiation of valvular interstitial cells (VIC), the most prevalent cell type in the human aortic valves. Leptin has recently been linked to aortic valve calcification in ApoE-/- mice. Hypothesis: Our hypothesis is that leptin could play a role in the calcifying processes implicated in CAVD via direct effects on human VIC. Methods: Patients who underwent aortic valve replacement for severe CAVD (n=43) or with coronary artery disease (CAD) but without CAVD (n=129) were included in this study. Presence of leptin was analyzed in human explanted calcified aortic valves and blood samples. Leptin receptors expression was analyzed in aortic valves and VIC isolated from aortic valves. Leptin effects on osteoblast differentiation of VIC in presence or not of Akt and ERK inhibitors were investigated by alizarin red staining, alkaline phosphatase (ALP) activity, and RT-qPCR analysis for osteopontin, ALP, bone morphogenetic protein BMP-2, and RUNX2. Results: Patients with CAVD have significant higher serum leptin concentration than CAD patients (p=0.002). The presence of leptin was observed by immunochemistry in human calcified aortic valves, with higher concentrations in calcified vs non-calcified zones (p=0.01). Both short and long leptin receptor isoforms were expressed in VIC. Chronic leptin stimulation of VIC enhanced ALP, BMP-2 and RUNX2 expression and decreased osteopontin expression. This treatment led to a higher, dose dependent, ALP activity and calcium deposition in VIC. Inhibiting Akt or ERK during leptin stimulation led to a reduced calcification by bringing the expression of calcification genes to the control levels. Conclusions: Together, these novel findings depict the potential role of leptin in the process of CAVD by triggering calcification processes in human VIC.

Mesenchymal Stem Cells and their Potential for Microengineering the Chondrocyte Niche

Mesenchymal Stem Cells and their Potential for Microengineering the Chondrocyte Niche

Activated human valvular interstitial cells sustain interleukin-17 production to recruit neutrophils in infective endocarditis.

The mechanisms that underlie valvular inflammation in streptococcus-induced infective endocarditis (IE) remain unclear. We previously demonstrated that streptococcal glucosyltransferases (GTFs) can activate human heart valvular interstitial cells (VIC) to secrete interleukin-6 (IL-6), a cytokine involved in T helper 17 (Th17) cell differentiation. Here, we tested the hypothesis that activated VIC can enhance neutrophil infiltration through sustained IL-17 production, leading to valvular damage. To monitor cytokine and chemokine production, leukocyte recruitment, and the induction or expansion of CD4(+) CD45RA(-) CD25(-) CCR6(+) Th17 cells, primary human VIC were cultured in vitro and activated by GTFs. Serum cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA), and neutrophils and Th17 cells were detected by immunohistochemistry in infected valves from patients with IE. The expression of IL-21, IL-23, IL-17, and retinoic acid receptor-related orphan receptor C (Rorc) was upregulated in GTF-activated VIC, which may enhance the proliferation of memory Th17 cells in an IL-6-dependent manner. Many chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), were upregulated in GTF-activated VIC, which might recruit neutrophils and CD4(+) T cells. Moreover, CXCL1 production in VIC was induced in a dose-dependent manner by IL-17 to enhance neutrophil chemotaxis. CXCL1-expressing VIC and infiltrating neutrophils could be detected in infected valves, and serum concentrations of IL-17, IL-21, and IL-23 were increased in patients with IE compared to healthy donors. Furthermore, elevated serum IL-21 levels have been significantly associated with severe valvular damage, including rupture of chordae tendineae, in IE patients. Our findings suggest that VIC are activated by bacterial modulins to recruit neutrophils and that such activities might be further enhanced by the production of Th17-associated cytokines. Together, these factors can amplify the release of neutrophilic contents in situ, which might lead to severe valvular damage.

0312: Characterization of human valvular interstitial cells isolated from normal and fibrocalcified aortic valves

Purpose Aortic Valve Stenosis (AVS) affects 2% to 6% of population over 65 years in industrialized countries. This atherosclerosis-like pathology involves Valve Interstitial Cell (VIC) proliferation and commitment to osteoblast- like cells. This prevalent cell type of aortic valve presents five identifiable phenotypes: embryonic progenitor endothelial/mesenchymal cells, progenitor, quiescent, activated and osteoblastic VICs. To study the pathophysiology of AVS, their in vitro cultures are frequently used. Our purpose is to characterize VICs isolated from normal and fibrocalcified human aortic valves and analyze their in vitro behavior. Methods We collected 5 normal and 5 fibrocalcified human aortic valves. VICs were isolated by collagenase digestion. Characterization is assessed at different passages (2 to 5) by immunofluorescence. Analyzed markers consist of progenitor cell markers (SSEA4, ABCG2, CD90, NG2 and OsteoBlast CaDHerin (OB-CDH)), fibroblast markers (vimentin and HSP47) and smooth muscle cell (SMC) marker (α-actin). By blue trypan and MTS, we compared the viability and proliferation of VICs in standard and starvation medium at 48 hours. Results Independently of their origin, VICs express all progenitor cell markers. Fibroblasts markers are expressed twice more by pathological VICs and four times more for SMC marker. In standard medium, VICs viability is similar (96,7±2,4% vs 96,4±2,3% ; normal vs pathological ± SEM). Pathological VICs proliferate more than normal VICs (2,2±0,7 vs 1,6±0,4 ; OD/OD control). In starvation medium, viability is significantly reduced for pathological VICs (89,6±7,9% vs 76,5±5,3%) but still proliferate in opposition with normal VICs (1,7±0,6 vs 1,2±0,3). Conclusion All VICs phenotypes are found in vitro with no culture selection but in different ratios according to their origin. These new data in VICs isolated from normal or pathological human aortic valves allow us to approve their use in vitro .

Electrospun PGS:PCL microfibers align human valvular interstitial cells and provide tunable scaffold anisotropy.

Tissue engineered heart valves (TEHV) can be useful in the repair of congenital or acquired valvular diseases due to their potential for growth and remodeling. The development of biomimetic scaffolds is a major challenge in heart valve tissue engineering. One of the most important structural characteristics of mature heart valve leaflets is their intrinsic anisotropy, which is derived from the microstructure of aligned collagen fibers in the extracellular matrix (ECM). In the present study, a directional electrospinning technique is used to fabricate fibrous poly(glycerol sebacate):poly(caprolactone) (PGS:PCL) scaffolds containing aligned fibers, which resemble native heart valve leaflet ECM networks. In addition, the anisotropic mechanical characteristics of fabricated scaffolds are tuned by changing the ratio of PGS:PCL to mimic the native heart valve's mechanical properties. Primary human valvular interstitial cells (VICs) attach and align along the anisotropic axes of all PGS:PCL scaffolds with various mechanical properties. The cells are also biochemically active in producing heart-valve-associated collagen, vimentin, and smooth muscle actin as determined by gene expression. The fibrous PGS:PCL scaffolds seeded with human VICs mimick the structure and mechanical properties of native valve leaflet tissues and would potentially be suitable for the replacement of heart valves in diverse patient populations.

Abstract 303: CD44 Activation Protects Human Sclerotic-derived Valvular Interstitial Cells from Calcification

Introduction. The activation of Valvular Interstitial Cells (VIC) towards an osteoblast-like phenotype is a cellular hallmark of pathological progression towards Aortic Stenosis (AS). In recent years several clinical trials have failed to halt or revert the progression of this prevalent disease. The ability to prevent end-stage AS requires the understanding of the molecular events associated with the early phase of valve degeneration, a condition known as Aortic Sclerosis (ASc). In the last few years the transmembrane receptor CD44 has been studied as a putative molecule for cardiovascular drug therapy. We reported that the functional interaction between CD44 and one of its ligand, Osteopontin (OPN), protects vascular smooth muscle cells from calcification. More recently, we demonstrated that sclerotic tissues show increased expression of Bone Morphogenetic Protein 4 (BMP4) and BMP4 directly stimulates osteoblast-like transdifferentiation and calcification of VICs. Therefore we hypothesized a direct role of CD44 activation in protecting human Aortic Sclerosis-derived VICs from calcification. Methods. Human VICs from Control, ASc, and AS (n=5 each group) were isolated. Histological, cellular and molecular analysis, and in situ Proximity Ligation Assay were used to investigate the role of CD44 and OPN in VIC calcification. BMP4 treatments were used to promote VIC activation. Osteoblast-like transdifferentiation was analyzed using Alkaline Phosphatase (ALP) expression. Results. CD44 and OPN, as well as their functional binding, were increased in sclerotic and stenotic tissues compared to healthy controls in vitro and ex vivo. CD44-OPN binding prevented in vitro calcification induced by inorganic phosphate on human ASc-derived VICs. A neutralizing antibody against CD44, under BMP4 treatments, promoted calcium deposition along with increased expression of OPN and ALP. Conclusion. Our results generate an important insight into the molecular mechanism of VIC calcification. We proved that CD44-OPN direct interaction inhibits calcification of Aortic Sclerosis-derived VICs, suggesting that CD44 activation could have a protective role against VIC osteoblast-like transdifferentiation and calcification in the early stage of the disease.

Valvular heart disease in a patient taking 3,4‐methylenedioxymethamphetamine (MDMA, ‘Ecstasy’)

3, 4-methylenedioxymethamphetamine, (MDMA, ‘Ecstasy’), an amphetamine-derived drug, is a psychoactive stimulant used as a recreational drug. In vitro experiments have suggested that ecstasy can lead to fenfluramine-like proliferation of cardiac valves through activation of 5-hydroxytryptamine 2B (5-HT2B) receptors and can induce mitogenic responses in human valvular interstitial cells [1]. We report the first observation of valvular heart disease (VHD) with pathological confirmation after long term use of ecstasy. A 33-year-old male patient was admitted for shortness of breath and chest pain. The only risk factor reported was active smoking. He reported long lasting exposure to ecstasy (several pills week−1 since the age of 17 years). At examination a systolic and diastolic mitral murmur was diagnosed. Mean blood pressure was 106/70 mmHg. ECG showed sinus rhythm (91 beats min−1) with a right bundle branch block. Echocardiography revealed a slight left ventricular dilation (endiastolic diameter of 55 mm) with an ejection fraction of 55% and typical features of mitral valve disease. There was a left atrial enlargement (area of 42 cm2) thick mitral valve with decreased opening of the mitral valve leaflets. Mean pressure gradient across the mitral valve was of 14.7 mmHg with mitral valve areas of 1.56 cm2. These moderate to severe mitral stenosis features were associated with severe mitral regurgitation (regurgitant orifice area = 0.49 cm2) with a central blood jet. Other valves and heart chambers were normal. This patient had pulmonary hypertension (40 mm Hg) without right heart failure. Coronary angiogram showed normal coronary arteries with a decreased cardiac index and post capillary pulmonary hypertension. Surgery was decided. Macroscopic analysis made the surgeon hypothesize about a possible cardiac tumour. Mitral valve leaflets and the chordae tendinae were markedly thickened and fibrous. The chordae tendinae were retracted (Figure 1). After resection, the surgeon decided to perform mitral valve replacement with a mechanical Saint Jude prosthesis despite severe damage of the mitral annulus. At histology, the endocardium on both sides was fibrous up to 2 mm thick. It was stratified with layers of collagen bundles and spindle cells. Density of spindle cells was heterogeneous, but more frequently low. In this abundant extracellular matrix, rare foci contained abundant elastic fibres. No inflammation or myxoid matrix accumulations were observed, ruling out rheumatic and Barlow diseases (Figure 1B, C). Common disease (Osler endocarditis, carcinoid syndrome, rheumatic fever, autoimmune disease …) or intake of other drugs (ergot derivatives, appetite-suppressant drugs including benfluorex) known to affect cardiac valves were not found. One month later, the patient came back with chest pain, fatigue and loss of weight. Trans-thoracic echocardiography showed a para-prosthetic leak with prosthesis dehiscence. The patient underwent re-operation with fixation of his prosthesis. At follow-up there were no complications. Figure 1 Anatomical and histological findings. (A) a short leaflet mitral valve (left) and a large leaflet mitral valve with a part of the left ventricle which was also removed (right). (B) Gross pathology of the mitral valve: on this section the valve is markedly ... This is the first case of VHD with pathological confirmation related to long term exposure to ecstasy. Except for an in vitro study [1], few data are available [1]. Among 29 subjects using ecstasy, eight had abnormal mitral valve echocardiography vs. 0 in controls [2]. The fact that the lesions in our patient were very similar to those described with appetite-suppressant drugs [3] and the ability of ecstasy to activate 5-HT2B receptors, like fenfluramine [1, 4], suggests its role in the pathophysiology of this VHD. VHD is unusual in young subjects. The present work allows the addition of ecstasy to the list of substances able to induce VHD [5]. The data also underline the important (and probably underestimated) harm associated with the now large use of ecstasy in young subjects. Information must be given about this risk.

Assembly and Testing of Stem Cell-Seeded Layered Collagen Constructs for Heart Valve Tissue Engineering

Tissue engineering holds great promise for treatment of valvular diseases. Despite excellent progress in the field, current approaches do not fully take into account each patient's valve anatomical uniqueness, the presence of a middle spongiosa cushion that allows shearing of external fibrous layers (fibrosa and ventricularis), and the need for autologous valvular interstitial cells. In this study we propose a novel approach to heart valve tissue engineering based on bioreactor conditioning of mesenchymal stem cell-seeded, valve-shaped constructs assembled from layered collagenous scaffolds. Fibrous scaffolds were prepared by decellularization of porcine pericardium and spongiosa scaffolds by decellularization and elastase treatment of porcine pulmonary arteries. To create anatomically correct constructs, we created silicone molds from native porcine aortic valves, dried two identical fibrous scaffolds onto the molds, and stabilized them with penta-galloyl-glucose a reversible collagen-binding polyphenol that reduces biodegradation. The layers were fused with a protein/aldehyde scaffold bio-adhesive and neutralized to reduce cytotoxicity. Spongiosa scaffolds, seeded with human bone marrow-derived stem cells, were inserted within the valve-shaped layered scaffolds and sutured inside the original aortic root. The final product was mounted in a heart valve bioreactor and cycled in cell culture conditions. Most cells were alive after 8 days, elongated significantly, and stained positive for vimentin, similar to native human valvular interstitial cells, indicating feasibility of our approach.

Comments on “The relation between Helicobacter pylori infection and cardiac syndrome X: A preliminary study", Assadi M, et al.

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3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") induces fenfluramine-like proliferative actions on human cardiac valvular interstitial cells in vitro.

Recent findings have implicated the 5-hydroxytryptamine 2B (5-HT2B) serotonin receptor in mediating the heart valve fibroplasia [valvular heart disease (VHD)] and primary pulmonary hypertension observed in patients taking the now-banned appetite suppressant fenfluramine (Pondimin, Redux). Via large-scale, random screening of a portion of the receptorome, we have discovered that the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") and its N-demethylated metabolite 3,4-methylenedioxyamphetamine (MDA) each preferentially bind to and activate human recombinant 5-HT2B receptors. We also demonstrate that MDMA and MDA, like fenfluramine and its N-deethylated metabolite norfenfluramine, elicit prolonged mitogenic responses in human valvular interstitial cells via activation of 5-HT2B receptors. We also report that pergolide and dihydroergotamine, two drugs recently demonstrated to induce VHD in humans, potently activate 5-HT2B receptors, thus validating this assay system for its ability to predict medications that might induce VHD. Our discovery that MDMA and a major metabolite, MDA, induce prolonged mitogenic responses in vitro similar to those induced by fenfluramine and norfenfluramine in vivo (i.e., valvular interstitial cell fibroplasia) predict that long-term MDMA use could lead to the development of fenfluramine-like VHD. Because of the widespread abuse of MDMA, these findings have major public health implications. These findings also underscore the necessity of screening current and future drugs at h5-HT2B receptors for agonist actions before their use in humans.