The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D-printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12 to 22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over 10-fold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 minutes, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0, and 73.3±5.2% for 22, 17, and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6, and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment.
The aortic heart valve undergoes geometric and mechanical changes over time. The cusps of a normal, healthy valve thicken and become less extensible over time. In the disease calcific aortic stenosis (CAS), calcified nodules progressively stiffen the cusps. The local mechanical changes in the cusps, due to either normal aging or pathological processes, affect overall function of the valve. In this paper, we propose a computational model for the aging aortic valve that connects local changes to overall valve function. We extend a previous model for the healthy valve to describe aging. To model normal/uncomplicated aging, leaflet thickness and extensibility are varied versus age according to experimental data. To model calcification, initial sites are defined and a simple growth law is assumed. The nodules then grow over time, so that the area of calcification increases from one model to the next model representing greater age. Overall valve function is recorded for each individual model to yield a single simulation of valve function over time. This simulation is the first theoretical tool to describe the temporal behavior of aortic valve calcification. The ability to better understand and predict disease progression will aid in design and timing of patient treatments for CAS.
Approximately 5 million people are affected with aortic valve disease (AoVD) in the United States. The most common treatment is aortic valve (AoV) replacement surgery, however, replacement valves are susceptible to failure, necessitating additional surgeries. The molecular mechanisms underlying disease progression and late AoV calcification are not well understood. Recent studies suggest that genes involved in bone and cartilage development play an active role in osteogenic-like calcification in human calcific AoVD (CAVD). In an effort to define the molecular pathways involved in AoVD progression and calcification, expression of markers of valve mesenchymal progenitors, chondrogenic precursors, and osteogenic differentiation was compared in pediatric non-calcified and adult calcified AoV specimens. Valvular interstitial cell (VIC) activation, extracellular matrix (ECM) disorganization, and markers of valve mesenchymal and skeletal chondrogenic progenitor cells were observed in both pediatric and adult AoVD. However, activated BMP signaling, increased expression of cartilage and bone-type collagens, and increased expression of the osteogenic marker Runx2 are observed in adult diseased AoVs and are not observed in the majority of pediatric diseased valves, representing a marked distinction in the molecular profile between pediatric and adult diseased AoVs. The combined evidence suggests that an actively regulated osteochondrogenic disease process underlies the pathological changes affecting AoVD progression, ultimately resulting in stenotic AoVD. Both pediatric and adult diseased AoVs express protein markers of valve mesenchymal and chondrogenic progenitor cells while adult diseased AoVs also express proteins involved in osteogenic calcification. These findings provide specific molecular indicators of AoVD progression, which may lead to identification of early disease markers and the development of potential therapeutics.
Aortic valve disease; Valvular interstitial cells; Calcification; Extracellular matrix
Dystrophic calcific nodule formation in vitro involves differentiation of aortic valve interstitial cells (AVICs) into a myofibroblast phenotype. Interestingly, inhibition of the kinase MEK1/2 prevents calcific nodule formation despite leading to myofibroblast activation of AVICs, indicating the presence of an additional mechanotransductive component required for calcific nodule morphogenesis. In this study, we assess the role of TGF-β1-induced cadherin-11 expression in calcific nodule formation.
Methods and Results
As shown previously, porcine AVICs treated with TGF-β1 prior to cyclic strain exhibit increased myofibroblast activation and significant calcific nodule formation. In addition to an increase in contractile myofibroblast markers, TGF-β1 treated AVICs exhibit significantly increased expression of cadherin-11. This expression is inhibited by the addition of U0126, a specific MEK1/2 inhibitor. The role of increased cadherin-11 is revealed through a wound assay, which demonstrates increased intercellular tension in TGF-β1 treated AVICs possessing cadherin-11. Furthermore, when siRNA is used to knockdown cadherin-11, calcific nodule formation is abrogated, indicating that robust cell-cell connections are necessary in generating tension for calcific nodule morphogenesis. Finally, we demonstrate enrichment of cadherin-11 in human calcified leaflets.
These results indicate the necessity of cadherin-11 for dystrophic calcific nodule formation, which proceeds through an Erk1/2 dependent pathway.
Calcific aortic valve disease (CAVD) results in impaired function through the inability of valves to fully open and close, but the causes of this pathology are unknown. Stiffening of the aorta is associated with CAVD and results in exposing the aortic valves to greater mechanical strain. Transforming growth factor β1 (TGF-β1) is enriched in diseased valves and has been shown to combine with strain to synergistically alter aortic valve interstitial cell (AVIC) phenotypes. Therefore, we investigated the role of strain and TGF-β1 on the calcification of AVICs. Following TGF-β1 pretreatment, strain induced intact monolayers to aggregate and calcify. Using a wound assay, we confirmed that TGF-β1 increases tension in the monolayer in parallel with α-smooth muscle actin (αSMA) expression. Continual exposure to strain accelerates aggregates to calcify into mature nodules that contain a necrotic core surrounded by an apoptotic ring. This phenotype appears to be mediated by strain inhibition of AVIC migration after the initial formation of aggregates. To better interpret the extent to which externally applied strain physically impacts this process, we modified the classical Lamé solution, derived using principles from linear elasticity, to reveal strain magnification as a novel feature occurring in a mechanical environment that supports nodule formation. These results indicate that strain can impact multiple points of nodule formation: by modifying tension in the monolayer, remodeling cell contacts, migration, apoptosis, and mineralization. Therefore, strain induced nodule formation provides new directions for developing strategies to address CAVD.
Calcific Aortic Valve Disease; Mechanobiology; TGF-β1; αSMA; Mechanical Strain; Apoptosis; Dystrophic Calcification; Aortic Valve Interstitial Cell; Myofibroblast
Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of Sox9 expression has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism.
Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, Triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, versus Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-Mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies we have further investigated the effectiveness of MABP in experiments that examined: 1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; 2) Furthermore, recognizing the importance of alkaline phosphatase in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of alkaline phosphatase activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced alkaline phosphatase activity, and in vivo have no significant calcification in long term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.
The regulation of valvular endothelial phenotypes by the hemodynamic environments of the human aortic valve is poorly understood. The nodular lesions of calcific aortic stenosis (CAS) develop predominantly beneath the aortic surface of the valve leaflets in the valvular fibrosa layer. However, the mechanisms of this regional localization remain poorly characterized. In this study, we combine numerical simulation with in vitro experimentation to investigate the hypothesis that the previously documented differences between valve endothelial phenotypes are linked to distinct hemodynamic environments characteristic of these individual anatomical locations. A finite-element model of the aortic valve was created, describing the dynamic motion of the valve cusps and blood in the valve throughout the cardiac cycle. A fluid mesh with high resolution on the fluid boundary was used to allow accurate computation of the wall shear stresses. This model was used to compute two distinct shear stress waveforms, one for the ventricular surface and one for the aortic surface. These waveforms were then applied experimentally to cultured human endothelial cells and the expression of several pathophysiological relevant genes was assessed. Compared to endothelial cells subjected to shear stress waveforms representative of the aortic face, the endothelial cells subjected to the ventricular waveform showed significantly increased expression of the “atheroprotective” transcription factor Kruppel-like factor 2 (KLF2) and the matricellular protein Nephroblastoma overexpressed (NOV), and suppressed expression of chemokine Monocyte-chemotactic protein-1 (MCP-1). Our observations suggest that the difference in shear stress waveforms between the two sides of the aortic valve leaflet may contribute to the documented differential side-specific gene expression, and may be relevant for the development and progression of CAS and the potential role of endothelial mechanotransduction in this disease.
Aortic valve; Calcific aortic stenosis; Endothelial mechanotransduction; Shear stress; Cell mechanics and mechanotransduction; Valvular disease
While the prevalence of calcified aortic valve disease continues to rise and no pharmacological treatments exist, little is known regarding the pathogenesis of the disease. Proteoglycans and the glycosaminoglycan hyaluronan are involved in calcification in arteriosclerosis and their characterization in calcified aortic valves may lend insight into the pathogenesis of the disease.
14 calcified aortic valves removed during valve replacement surgery were immunohistochemically stained for the proteoglycans (PGs) decorin, biglycan, and versican, as well as the glycosaminoglycan hyaluronan. Staining intensity was evaluated in the following regions of interest: center of calcified nodule, edge of nodule, tissue directly surrounding nodule; center and tissue surrounding small “prenodules”; and fibrosa layer of normal regions of the leaflet distanced from the nodule.
Decorin, biglycan, and versican, as well as hyaluronan, were abundantly present immediately surrounding the calcified nodules, but minimally within the nodule itself. Expression of decorin and biglycan in and surrounding prenodules was greater than in the edge and center regions of mature nodules. The levels of expression of the PGs and hyaluronan were highly correlated with one another in the different regions of the valve.
The three PGs and hyaluronan demonstrated distinctive localization relative to nodules within calcified aortic valves, where they likely mediate lipid retention, cell proliferation, and extracellular matrix remodeling, and motivate further study. Comparisons between expression of these components in mature nodules and prenodules suggest distinct roles for these components in nodule progression, especially in the tissues surrounding the nodules.
proteoglycans; hyaluronan; calcification; aortic valve; immunohistochemistry
Acquired aortic valve disease and valvular calcification is highly prevalent in adult populations worldwide and is associated with significant cardiovascular morbidity and mortality. At present, there are no medical therapies that will prevent or regress aortic valve calcification or stenosis and surgical or transcatheter aortic valve replacement remain the only effective therapies for treating this disease. In the setting of valve injury as a result of exposure to biochemical mediators or hemodynamic forces, normal homeostatic processes are disrupted resulting in extracellular matrix degradation, aberrant matrix deposition and fibrosis, inflammatory cell infiltration, lipid accumulation, and neoangiogenesis of the valve tissue and, ultimately, calcification of the valve. Calcification of the aortic valve is now understood to be an active process that involves the coordinated actions of resident valve endothelial and interstitial cells, circulating inflammatory and immune cells, and bone marrow-derived cells. These cells may undergo a phenotype transition to become osteoblast-like cells and elaborate bone matrix, endothelial-to-mesenchymal transition, and form matrix vesicles that serve as a nidus for microcalcifications. Each of these mechanisms has been shown to contribute to aortic valve calcification suggesting that strategies that target these cellular events may lead to novel therapeutic interventions to halt the progression or reverse aortic valve calcification.
Calcification; Aortic Valve
Irradiation of the chest or chest wall has been shown to caause calcific aortic stenosis. However, the mechanisms are unknown. Aortic valve interstitial cells (AVICs) have been implicated in the pathogenesis of aortic stenosis; they have been shown to change from the phenotype of a myofibroblast to an osteoblast-like cell. We therefore hypothesized that irradiation of human AVICs induces an osteogenic phenotype. In isolated human AVICs, our purpose was to determine the effect of irradiation on the production of osteogenic factors: (a) bone morphogenetic protein-2 (BMP-2) (b) osteopontin (OPN) (c) alkaline phosphatase (ALP), and (d) the transcription factor Runx2.
Human AVICs were isolated from normal aortic valves obtained from explanted hearts of patients undergoing cardiac transplantation (n=4) and grown in culture. The cells were grown to confluence, irradiated with 10 Gy using a cesium-137 irradiator and then lysed 24 hours following irradiation. Cell lysates were analyzed via immunoblot and densitometry for BMP-2, OPN, ALP and Runx2. Statistics were by ANOVA. P < 0.05 was significant.
Irradiation induced an osteogenic phenotype in human AVICs. Irradiation induced a 2-fold increase in BMP-2, a 7-fold increase in OPN, a 3-fold increase in ALP, and a 2-fold increase in Runx2.
Radiation induces an osteogenic phenotype in human AVICs. The irradiated cells had significantly increased expression of the osteogenic factors BMP-2, OPN, ALP and Runx2. These data offer mechanistic insight into the pathogenesis of radiation-induced valvular heart disease.
aortic stenosis; osteogenesis; radiation
Calcific aortic stenosis (CAS) is a pathological condition of the aortic valve characterized by dystrophic calcification of the valve leaflets. Despite the high prevalence and mortality associated with CAS, little is known about its pathogenetic mechanisms. Characterized by progressive dystrophic calcification of the valve leaflets, the early stages of aortic valve degeneration are similar to the active inflammatory process of atherosclerosis including endothelial disruption, inflammatory cell infiltration, lipid deposition, neo-vascularization and calcification. In the vascular system, the endothelium is an important regulator of physiological and pathological conditions; however, the contribution of endothelial dysfunction to valvular degeneration at the cellular and molecular level has received little attention. Endothelial cell (EC) activation and neo-vascularization of the cusps characterizes all stages of aortic valvular degeneration from aortic sclerosis to aortic stenosis. Here we reported the role of osteopontin (OPN) in the regulation of EC activation in vitro and in excised tissue from CAS patients and controls. OPN is an important pro-angiogenic factor in several pathologies. High levels of OPN have been demonstrated in both tissue and plasma of patients with aortic valve sclerosis and stenosis. The characterization of valvular ECs as a cellular target for OPN will help us uncover the pathogenesis of aortic valve degeneration and stenosis, opening new perspectives for the prevention and therapy of this prevalent disease.
During heart valve remodeling and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-positive stress fibers, the hallmark of activated myofibroblasts, are also observed in VICs cultured on stiff substrates especially in the presence of transforming growth factor-beta1 (TGF-β1), yet the detailed relationship between stiffness and VIC phenotype has not been explored. The goal of this study was to characterize VIC activation as a function of substrate stiffness over a wide range of stiffness levels including that of diseased valves (stiff), normal valves (compliant), and hydrogels for heart valve tissue engineering (very soft). VICs obtained from porcine aortic valves were cultured on stiff tissue culture plastic to activate them, then cultured on collagen-coated polyacrylamide substrates of predefined stiffness in a high-throughput culture system to assess the persistence of activation. Quantitative metrics extracted from regression analysis demonstrate that relative to a compliant substrate, stiff substrates result in higher cell numbers, more pronounced expression of αSMA-positive stress fibers, and larger spread area which is in qualitative agreement with previous studies. Our data also indicate that VICs require a much lower substrate stiffness level to “deactivate” them than previously thought. The high sensitivity of VICs to substrate stiffness demonstrates the importance of the mechanical properties of materials used for valve repair or for engineering valve tissue.
valvular interstitial cells; stiffness; phenotype; polyacrylamide; TGF-β1
Calcific aortic stenosis, characterized by excessive fibrosis and deposition of bone-like calcified tissue, affects roughly 2–3% of the US population over 65. Recent studies have suggested that statins have a positive effect on the progression of aoritic stenosis, likely due to their ability to affect the resident cell population, known as valvular interstitial cells (VICs). VICs are fibroblastic cells that can differentiate to form activated myofibroblasts, displaying increased alpha smooth muscle actin (αSMA) expression, contractility, and collagen production.
Methods and Results
In culture, VICs spontaneously form multicellular aggregates that subsequently develop into calcified nodules, providing an in vitro model for aortic stenosis. Using real-time microscopic tracking, we observed that confluent VIC monolayers spontaneously contract into rounded nodules, suggesting that myofibroblastic contractility is a critical step in the process of nodule formation. Over-expression of αSMA increased VIC calcific nodule formation and contractility, while knock-down of αSMA with siRNAs reduced these phenotypes, suggesting that the expression and contractile properties of αSMA are essential to the formation of nodules. Statin treatment of VICs reduced αSMA expression, inhibited contractility, and decreased nodule formation. When statins were used to treat preformed nodules, no decrease in the number of calcified nodules was observed, suggesting that statins may play more of a preventative role in aortic stenosis than a cure.
Our studies provide evidence of a causal relationship between VIC myofibroblastic activity and initial VIC calcific nodule formation. Furthermore we demonstrate that pravastatin inhibition of calcific nodule formation is related to inhibition of myofibroblastic activity.
valvular interstitial cells; myofibroblast; calcific nodule; statin; contractility
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.
Aortic valve disease; valvular interstitial cells; calcification; extracellular matrix; Mouse models of human disease
Aortic valve disease is estimated to affect 2% of the United States population. There is increasing evidence that aortic valve (AoV) disease has a basis in development, as congenital valve malformations are prevalent in patients undergoing valve replacement surgery. In fact, a number of genetic mutations have been linked to valve malformations and disease. In the initial stages of AoV pathogenesis, the valvular interstitial cells become activated, undergo cell proliferation, and participate in extracellular matrix remodeling. Many of these cell properties are shared with mesenchymal progenitor cells of the normally developing valves and bones. Historically, valve calcification was thought to be a passive process reflecting end stage disease. However, recent evidence describes the increased expression of transcription factors in diseased AoV that are common to valvulogenic and osteogenic processes. These studies lend support to the idea that a developmental gene program is re-activated in aortic valve disease and may contribute to the molecular mechanisms underlying valve calcification in disease.
valve development; aortic valve disease; transcriptional regulation
The lack of therapies that inhibit valvular calcification and the conflicting outcomes of clinical studies regarding the impact of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors on valve disease highlight the need for controlled investigations to characterize the interactions between HMG-CoA reductase inhibitors and valve tissue. Thus, we applied multiple in vitro disease stimuli to valvular interstitial cell (VIC) cultures and examined the impact of simvastatin treatment on VIC function.
Methods and Results
VICs were cultured on three different substrates that supported various levels of nodule formation. TGF-β1 was also applied as a disease stimulus to VICs on 2-D surfaces or encapsulated in 3-D collagen gels and combined with different temporal applications of simvastatin. Simvastatin inhibited calcific nodule formation in a dose-dependent manner on all materials, although the level of statin efficacy was highly substrate-dependent. Simvastatin treatment significantly altered nodule morphology, resulting in dramatic nodule dissipation over time, also in a substrate-dependent manner. These effects were mimicked in 3-D cultures, wherein simvastatin reversed TGF-β1-induced contraction. Decreases in nodule formation were not achieved via the HMG-CoA reductase pathway, but were correlated with decreases in ROCK activity.
These studies represent a significant contribution to understanding how simvastatin may impact heart valve calcification.
heart valves; valvular interstitial cells; simvastatin; calcification; extracellular matrix
Calcific aortic stenosis is the third leading cause of adult heart disease and the most common form of acquired valvular disease in developed countries. However, the molecular pathways leading to calcification are poorly understood. We reported two families in which heterozygous mutations in NOTCH1 caused bicuspid aortic valve and severe aortic valve calcification. NOTCH1 is part of a highly conserved signaling pathway involved in cell fate decisions, cell differentiation, and cardiac valve formation. In this study, we examined the mechanism by which NOTCH1represses aortic valve calcification. Heterozygous Notch1-null (Notch1+/−) mice had greater than fivefold more aortic valve calcification than age- and sex-matched wildtype littermates. Inhibition of Notch signaling in cultured sheep aortic valve interstitial cells (AVICs) also increased calcification more than fivefold and resulted in gene expression typical of osteoblasts. We found that Notch1 normally represses the gene encoding bone morphogenic protein 2 (Bmp2) in murine aortic valves in vivo and in aortic valve cells in vitro. siRNA-mediated knockdown of Bmp2 blocked the calcification induced by Notch inhibition in AVICs. These findings suggest that Notch1 signaling in aortic valve cells represses osteoblast-like calcification pathways mediated by Bmp2.
Aortic Valve Calcification; Notch1; Bmp2; Gene regulation; Aortic valve interstitial cells
The pathogenesis of valvar calcification, which complicates the course of cardiac valve disease and also affects tissue valve prostheses, is incompletely understood. The present work explores the possible role of the vitamin K-dependent, calcium-binding amino acid, γ-carboxyglutamic acid (Gla) in valve mineralization. Gla is normally found in the vitamin K-dependent clotting factor proteins, and is also present in unique calcium binding proteins in bone, kidney, and lung. Unique Gla-containing proteins have also been isolated from pathologic calcifications including calcium containing renal stones and calcified atherosclerotic plaque. Calcified valves including specimens with calcific aortic stenosis, calcified porcine xenograft valves, and a calcified aortic homograft valve were analyzed for Gla content, complete amino acid analysis, and tissue calcium and phosphorus levels. Normal porcine valves contained protein-bound Gla (2.0-10.6 Gla/104 amino acids): no Gla was present in normal valve leaflets. Furthermore, Gla levels paralleled tissue calcium content in the calcified valves. In addition, complete amino acid analysis indicated a decline in valvar collagen content plus increased acidic proteins in conjunction with valvar calcification and the presence of Gla-containing proteins. These results suggest that calcific valvar disease may result in part from vitamin K-dependent processes.
Phenotypically, the aortic valve interstitial cell (AVIC) is a dynamic myofibroblast, appearing contractile and activated in times of development, disease, and remodeling. The precise mechanism of phenotypic modulation is unclear, but it is speculated that both biomechanical and biochemical factors are influential. Therefore, we hypothesized that isolated and combined treatments of cyclic tension and TGF-β1 would alter the phenotype and subsequent collagen biosynthesis of AVICs in situ.
Methods and Results
Porcine aortic valve leaflets received 7 and 14 day treatments of 15% cyclic stretch (Tension), 0.5 ng/ml TGF-β1 (TGF), 15% cyclic stretch and 0.5 ng/ml TGF-β1 (Tension+TGF), or neither mechanical nor cytokine stimuli (Null). Tissues were homogenized and assayed for AVIC phenotype (smooth muscle α-actin (SMA)) and collagen biosynthesis (via heat shock protein 47 (Hsp47) which was further verified with type I collagen C-terminal propeptide (CICP)). At both 7 and 14 days, SMA, Hsp47, and CICP quantities were significantly greater (p<0.001) in the Tension+TGF group compared to all other groups. Additionally, Tension alone appeared to maintain SMA and Hsp47 levels that were measured at day 0, while TGF alone elicited an increase in SMA and Hsp47 compared to day 0 levels. Null treatment revealed diminished proteins at both time points.
Elevated TGF-β1 levels, in the presence of cyclic mechanical tension, resulted in synergistic increases in the contractile and biosynthetic proteins in AVICs. Since cyclic mechanical stimuli can never be relieved in vivo, the presence of TGF-β1 (possibly from infiltrating macrophages) may result in overly biosynthetic AVICs, leading to altered ECM architecture, compromised valve function, and ultimately degenerative valvular disease.
Aortic valve interstitial cells; degenerative valve disease; TGF-β1; heart valve remodeling; myofibroblasts
Pulmonary autograft valve replacement has been simulated by implanting the pulmonary valve into the aortic position of the same cadaver heart from both human and porcine sources. The forces acting on the pulmonary valve leaflets have been calculated on the basis of a triaxial ellipsoid mathematical model. These forces on the pulmonary autograft valve were shown to be essentially similar to those previously reported for aortic valve leaflets. Biomechanical measurements have been made on the simulated autograft valves and on the isolated pulmonary valve cusps. The tensile strengths of the pulmonary valve cusps in both circumferential and radial directions were roughly three times greater than those of aortic valve cusps. This indicated the ability of the pulmonary valves to accept, ab initio, aortic valve closing pressures. Pressure-induced changes in dimension, calculated on the basis of diameters of the simulated pulmonary autograft root, also indicated that the distensibility of the autograft valve was limited. It reached a maximum at 30 mm Hg (4 kPa) without any suggestion of further distension to the point of distortion and incompetence. The combination of the calculated forces acting on the valve and the biomechanical measurements have shown that pulmonary valves used as autograft aortic valve replacements are able to tolerate aortic pressures from the time of implantation. These experimental results from simulated autografts support the clinical use of this valve over the past 13 years.
Aortic valve (AV) disease is often characterized by the formation of calcific nodules within AV leaflets that alter functional biomechanics. In vitro, formation of these nodules is associated with osteogenic differentiation and/or increased contraction and apoptosis of AV interstitial cells (AVICs), leading to growth of calcium phosphate crystal structures. In several other cell types, increased intracellular Ca2+ has been shown to be an important part in activation of osteogenic differentiability. However, elevated intracellular Ca2+ is known to mediate cell contraction, and has also been shown to lead to apoptosis in many cell types. Therefore, a rise in intracellular Ca2+ may precede cellular changes that lead to calcification, and fibroblasts similar to AVICs have been shown to exhibit increases in intracellular Ca2+ in response to mechanical strain. In this study, we hypothesized that strain induces intracellular Ca2+ accumulation through stretch-activated calcium channels. We were also interested in assessing possible correlations between intracellular Ca2+ increases and apoptosis in AVICs. To test our hypothesis, cultured porcine AVICs were used to assess correlates between strain, intracellular Ca2+, and apoptosis. Ca2+ sensitive fluorescent dyes were utilized to measure real-time intracellular Ca2+ changes in strained AVICs. Ca2+ changes were then correlated with AVIC apoptosis using flow cytometric Annexin V apoptosis assays. These data indicate that strain-dependent accumulation of intracellular Ca2+ is correlated with apoptosis in AVICs. We believe that these findings indicate early mechanotransductive events that may initiate AV calcification pathways.
Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. Although exact causes and mechanisms of AV calcification are unclear, previous studies suggest that mechanical forces play a role. Since calcium deposits occur almost exclusively on the aortic surfaces of AV leaflets, it has been hypothesized that adverse patterns of fluid shear stress on the aortic surface of AV leaflets promote calcification. The current study characterizes AV leaflet aortic surface fluid shear stresses using Laser Doppler velocimetry and an in vitro pulsatile flow loop. The valve model used was a native porcine valve mounted on a suturing ring and preserved using 0.15% glutaraldehyde solution. This valve model was inserted in a mounting chamber with sinus geometries, which is made of clear acrylic to provide optical access for measurements. To understand the effects of hemodynamics on fluid shear stress, shear stress was measured across a range of conditions: varying stroke volumes at the same heart rate and varying heart rates at the same stroke volume. Systolic shear stress magnitude was found to be much higher than diastolic shear stress magnitude due to the stronger flow in the sinuses during systole, reaching up to 20 dyn/cm2 at mid-systole. Upon increasing stroke volume, fluid shear stresses increased due to stronger sinus fluid motion. Upon increasing heart rate, fluid shear stresses decreased due to reduced systolic duration that restricted the formation of strong sinus flow. Significant changes in the shear stress waveform were observed at 90 beats/ min, most likely due to altered leaflet dynamics at this higher heart rate. Overall, this study represents the most well-resolved shear stress measurements to date across a range of conditions on the aortic side of the AV. The data presented can be used for further investigation to understand AV biological response to shear stresses.
Native aortic valve; Aortic valve leaflet; Fluid shear stress; Laser Doppler velocimetry; Aortic surface; Heart valve fluid mechanics
Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.
In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1−/− mouse aortic tissue.
This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention.
The mitral valve was replaced by a pig aortic valve in 33 patients at Groote Schuur Hospital. Eleven of the failed heterograft aortic valves were examined at intervals of from 2 to 32 months after insertion. Fourteen control pig aortic valves were also examined. Electron microscopy was performed on two of the failed heterograft valves and three control pig valves. Failure of the heterograft was due to stretching and deformation of the cusps with resultant valvular incompetence. Stretching of the cusp was a result of reduction in the amount of its collagen content. The elastic tissue appeared little altered. A microscopic layer of fibrin thrombus was present on the surface of 8 of the 11 valves. Only 2 of the 11 valves showed invasion of the graft by immunologically competent cells. No valve showed any sign of infection or calcification. The denatured collagen of the heterograft has a low antigenicity and also, infortunately, a limited durability.