On the role of predicted in vivo mitral valve interstitial cell deformation on its biosynthetic behavior.

Ischemic mitral regurgitation (IMR), a frequent complication of myocardial infarction, is characterized by regurgitation of blood from the left ventricle back into the left atrium. Physical interventions via surgery or less-invasive techniques are the only available therapies for IMR, with valve repair via undersized ring annuloplasty (URA) generally preferred over valve replacement. However, recurrence of IMR after URA occurs frequently and is attributed to continued remodeling of the MV and infarct region of the left ventricle. The mitral valve interstitial cells (MVICs) that maintain the tissue integrity of the MV leaflets are highly mechanosensitive, and altered loading post-URA is thought to lead to aberrant MVIC-directed tissue remodeling. Although studies have investigated aspects of mechanically directed VIC activation and remodeling potential, there remains a substantial disconnect between organ-level biomechanics and cell-level phenomena. Herein, we utilized an extant multiscale computational model of the MV that linked MVIC to organ-level MV biomechanical behaviors to simulate changes in MVIC deformation following URA. A planar biaxial bioreactor system was then used to cyclically stretch explanted MV leaflet tissue, emulating the in vivo changes in loading following URA. This simulation-directed experimental investigation revealed that post-URA deformations resulted in decreased MVIC activation and collagen mass fraction. These results are consistent with the hypothesis that URA failures post-IMR are due, in part, to reduced MVIC-mediated maintenance of the MV leaflet tissue resulting from a reduction in physical stimuli required for leaflet tissue homeostasis. Such information can inform the development of novel URA strategies with improved durability.

Mitral valve leaflet response to ischaemic mitral regurgitation: from gene expression to tissue remodelling.

Ischaemic mitral regurgitation (IMR), a frequent complication following myocardial infarction (MI), leads to higher mortality and poor clinical prognosis if untreated. Accumulating evidence suggests that mitral valve (MV) leaflets actively remodel post MI, and this remodelling increases both the severity of IMR and the occurrence of MV repair failures. However, the mechanisms of extracellular matrix maintenance and modulation by MV interstitial cells (MVICs) and their impact on MV leaflet tissue integrity and repair failure remain largely unknown. Herein, we sought to elucidate the multiscale behaviour of IMR-induced MV remodelling using an established ovine model. Leaflet tissue at eight weeks post MI exhibited significant permanent plastic radial deformation, eliminating mechanical anisotropy, accompanied by altered leaflet composition. Interestingly, no changes in effective collagen fibre modulus were observed, with MVICs slightly rounder, at eight weeks post MI. RNA sequencing indicated that YAP-induced genes were elevated at four weeks post MI, indicating elevated mechanotransduction. Genes related to extracellular matrix organization were downregulated at four weeks post MI when IMR occurred. Transcriptomic changes returned to baseline by eight weeks post MI. This multiscale study suggests that IMR induces plastic deformation of the MV with no functional damage to the collagen fibres, providing crucial information for computational simulations of the MV in IMR.

On the simulation of mitral valve function in health, disease, and treatment.

The mitral valve (MV) is the heart valve that regulates blood ?ow between the left atrium and left ventricle (LV). In situations where the MV fails to fully cover the left atrioventricular ori?ce during systole, the resulting regurgitation causes pulmonary congestion, leading to heart failure and/or stroke. The causes of MV insuf?ciency can be either primary (e.g. myxomatous degeneration) where the valvular tissue is organically diseased, or secondary (typically inducded by ischemic cardiomyopathy) termed ischemic mitral regurgitation (IMR), is brought on by adverse LV remodeling. IMR is present in up to 40% of patients and more than doubles the probability of cardiovascular morbidity after 3.5 years. There is now agreement that adjunctive procedures are required to treat IMR caused by lea?et tethering. However, there is no consensus regarding the best procedure. Multicenter registries and randomized trials would be necessary to prove which procedure is superior. Given the number of proposed procedures and the complexity and duration of such studies, it is highly unlikely that IMR procedure optimization will be achieved by prospective clinical trials. There is thus an urgent need for cell and tissue physiologically based quantitative assessments of MV function to better design surgical solutions and associated therapies. Novel computational approaches directed towards optimized surgical repair procedures can substantially reduce the need for such trial-and-error approaches. We present the details of our MV modeling techniques, with an emphasis on what is known and investigated at various length scales.

Porphyrin-Based SOD Mimic MnTnBu OE -2-PyP5+ Inhibits Mechanisms of Aortic Valve Remodeling in Human and Murine Models of Aortic Valve Sclerosis.

Background Aortic valve sclerosis ( AVS c), the early asymptomatic presentation of calcific aortic valve (AV) disease, affects 25% to 30% of patients aged >65 years. In vitro and ex vivo experiments with antioxidant strategies and antagonists of osteogenic differentiation revealed that AVS c is reversible. In this study, we characterized the underlying changes in the extracellular matrix architecture and valve interstitial cell activation in AVSc and tested in vitro and in vivo the activity of a clinically approved SOD (superoxide dismutase) mimic and redox-active drug MnTnBu OE -2-PyP5+ ( BMX -001). Methods and Results After receiving informed consent, samples from patients with AVS c, AV stenosis, and controls were collected. Uniaxial mechanical stimulation and in vitro studies on human valve interstitial cells were performed. An angiotensin II chronic infusion model was used to impose AV thickening and remodeling. We characterized extracellular matrix structures by small-angle light scattering, scanning electron microscopy, histology, and mass spectrometry. Diseased human valves showed altered collagen fiber alignment and ultrastructural changes in AVS c, accumulation of oxidized cross-linking products in AV stenosis, and reversible expression of extracellular matrix regulators ex vivo. We demonstrated that MnTnBu OE -2-PyP5+ inhibits human valve interstitial cell activation and extracellular matrix remodeling in a murine model (C57 BL /6J) of AVS c by electron microscopy and histology. Conclusions AVS c is associated with architectural remodeling despite marginal effects on the mechanical properties in both human and mice. MnTnBu OE -2-PyP5+ controls AV thickening in a murine model of AVS c. Because this compound has been approved recently for clinical use, this work could shift the focus for the treatment of calcific AV disease, moving from AV stenosis to an earlier presentation ( AVS c) that could be more responsive to medical therapies.

Serotonin receptor 2B signaling with interstitial cell activation and leaflet remodeling in degenerative mitral regurgitation.

AIMS: Mitral valve interstitial cells (MVIC) play an important role in the pathogenesis of degenerative mitral regurgitation (MR) due to mitral valve prolapse (MVP). Numerous clinical studies have observed serotonin (5HT) dysregulation in cardiac valvulopathies; however, the impact of 5HT-mediated signaling on MVIC activation and leaflet remodeling in MVP have been investigated to a limited extent. Here we test the hypothesis that 5HT receptors (5HTRs) signaling contributes to MVP pathophysiology. METHODS AND RESULTS: Diseased human MV leaflets were obtained during cardiac surgery for MVP; normal MV leaflets were obtained from heart transplants. MV RNA was used for microarray analysis of MVP patients versus control, highlighting genes that indicate the involvement of 5HTR pathways and extracellular matrix remodeling in MVP. Human MV leaflets were also studied in vitro and ex vivo with biomechanical testing to assess remodeling in the presence of a 5HTR2B antagonist (LY272015). MVP leaflets from Cavalier King Charles Spaniels were used as a naturally acquired in vivo model of MVP. These canine MVP leaflets (N=5/group) showed 5HTR2B upregulation. This study also utilized CB57.1ML/6 mice in order to determine the effect of Angiotensin II infusion on MV remodeling. Histological analysis showed that MV thickening due to chronic Angiotensin II remodeling is mitigated by a 5HTR2B antagonist (LY272015) but not by 5HTR2A inhibitors. CONCLUSION: In humans, MVP is associated with an upregulation in 5HTR2B expression and increased 5HT receptor signaling in the leaflets. Antagonism of 5HTR2B mitigates MVIC activation in vitro and MV remodeling in vivo. These observations support the view that 5HTR signaling is involved not only in previously reported 5HT-related valvulopathies, but it is also involved in the pathological remodeling of MVP.

The Three-Dimensional Microenvironment of the Mitral Valve: Insights into the Effects of Physiological Loads.

INTRODUCTION: In the mitral valve (MV), numerous pathological factors, especially those resulting from changes in external loading, have been shown to affect MV structure and composition. Such changes are driven by the MV interstitial cell (MVIC) population via protein synthesis and enzymatic degradation of extracellular matrix (ECM) components. METHODS: While cell phenotype, ECM composition and regulation, and tissue level changes in MVIC shape under stress have been studied, a detailed understanding of the three-dimensional (3D) microstructural mechanisms are lacking. As a first step in addressing this challenge, we applied focused ion beam scanning electron microscopy (FIB-SEM) to reveal novel details of the MV microenvironment in 3D. RESULTS: We demonstrated that collagen is organized into large fibers consisting of an average of 605 ± 113 fibrils, with a mean diameter of 61.2 ± 9.8 nm. In contrast, elastin was organized into two distinct structural subtypes: (1) sheet-like lamellar elastin, and (2) circumferentially oriented elastin struts, based on both the aspect ratio and transmural tilt. MVICs were observed to have a large cytoplasmic volume, as evidenced by the large mean surface area to volume ratio 3.68 ± 0.35, which increased under physiological loading conditions to 4.98 ± 1.17. CONCLUSIONS: Our findings suggest that each MVIC mechanically interacted only with the nearest 3-4 collagen fibers. This key observation suggests that in developing multiscale MV models, each MVIC can be considered a mechanically integral part of the local fiber ensemble and is unlikely to be influenced by more distant structures.

Regulation of valve interstitial cell homeostasis by mechanical deformation: implications for heart valve disease and surgical repair.

Mechanical stress is one of the major aetiological factors underlying soft-tissue remodelling, especially for the mitral valve (MV). It has been hypothesized that altered MV tissue stress states lead to deviations from cellular homeostasis, resulting in subsequent cellular activation and extracellular matrix (ECM) remodelling. However, a quantitative link between alterations in the organ-level in vivo state and in vitro-based mechanobiology studies has yet to be made. We thus developed an integrated experimental-computational approach to elucidate MV tissue and interstitial cell responses to varying tissue strain levels. Comprehensive results at different length scales revealed that normal responses are observed only within a defined range of tissue deformations, whereas deformations outside of this range lead to hypo- and hyper-synthetic responses, evidenced by changes in α-smooth muscle actin, type I collagen, and other ECM and cell adhesion molecule regulation. We identified MV interstitial cell deformation as a key player in leaflet tissue homeostatic regulation and, as such, used it as the metric that makes the critical link between in vitro responses to simulated equivalent in vivo behaviour. Results indicated that cell responses have a delimited range of in vivo deformations that maintain a homeostatic response, suggesting that deviations from this range may lead to deleterious tissue remodelling and failure.

Heart Valve Biomechanics and Underlying Mechanobiology.

Heart valves control unidirectional blood flow within the heart during the cardiac cycle. They have a remarkable ability to withstand the demanding mechanical environment of the heart, achieving lifetime durability by processes involving the ongoing remodeling of the extracellular matrix. The focus of this review is on heart valve functional physiology, with insights into the link between disease-induced alterations in valve geometry, tissue stress, and the subsequent cell mechanobiological responses and tissue remodeling. We begin with an overview of the fundamentals of heart valve physiology and the characteristics and functions of valve interstitial cells (VICs). We then provide an overview of current experimental and computational approaches that connect VIC mechanobiological response to organ- and tissue-level deformations and improve our understanding of the underlying functional physiology of heart valves. We conclude with a summary of future trends and offer an outlook for the future of heart valve mechanobiology, specifically, multiscale modeling approaches, and the potential directions and possible challenges of research development. © 2016 American Physiological Society. Compr Physiol 6:1743-1780, 2016.

A meso-scale layer-specific structural constitutive model of the mitral heart valve leaflets.

Fundamental to developing a deeper understanding of pathophysiological remodeling in mitral valve (MV) disease is the development of an accurate tissue-level constitutive model. In the present work, we developed a novel meso-scale (i.e. at the level of the fiber, 10-100 µm in length scale) structural constitutive model (MSSCM) for MV leaflet tissues. Due to its four-layer structure, we focused on the contributions from the distinct collagen and elastin fiber networks within each tissue layer. Requisite collagen and elastin fibrous structural information for each layer were quantified using second harmonic generation microscopy and conventional histology. A comprehensive mechanical dataset was also used to guide model formulation and parameter estimation. Furthermore, novel to tissue-level structural constitutive modeling approaches, we allowed the collagen fiber recruitment function to vary with orientation. Results indicated that the MSSCM predicted a surprisingly consistent mean effective collagen fiber modulus of 162.72 MPa, and demonstrated excellent predictive capability for extra-physiological loading regimes. There were also anterior-posterior leaflet-specific differences, such as tighter collagen and elastin fiber orientation distributions (ODF) in the anterior leaflet, and a thicker and stiffer atrialis in the posterior leaflet. While a degree of angular variance was observed, the tight valvular tissue ODF also left little room for any physically meaningful angular variance in fiber mechanical responses. Finally, a novel fibril-level (0.1-1 µm) validation approach was used to compare the predicted collagen fiber/fibril mechanical behavior with extant MV small angle X-ray scattering data. Results demonstrated excellent agreement, indicating that the MSSCM fully captures the tissue-level function. Future utilization of the MSSCM in computational models of the MV will aid in producing highly accurate simulations in non-physiological loading states that can occur in repair situations, as well as guide the form of simplified models for real-time simulation tools.

Quantification and simulation of layer-specific mitral valve interstitial cells deformation under physiological loading.

Within each of the four layers of mitral valve (MV) leaflet tissues there resides a heterogeneous population of interstitial cells that maintain the structural integrity of the MV tissue via protein biosynthesis and enzymatic degradation. There is increasing evidence that tissue stress-induced MV interstitial cell (MVIC) deformations can have deleterious effects on their biosynthetic states that are potentially related to the reduction of tissue-level maintenance and to subsequent organ-level failure. To better understand the interrelationships between tissue-level loading and cellular responses, we developed the following integrated experimental-computational approach. Since in vivo cellular deformations are not directly measurable, we quantified the in-situ layer-specific MVIC deformations for each of the four layers under a controlled biaxial tension loading device coupled to multi-photon microscopy. Next, we explored the interrelationship between the MVIC stiffness and deformation to layer-specific tissue mechanical and structural properties using a macro-micro finite element computational model. Experimental results indicated that the MVICs in the fibrosa and ventricularis layers deformed significantly more than those in the atrialis and spongiosa layers, reaching a nucleus aspect ratio of 3.3 under an estimated maximum physiological tension of 150N/m. The simulated MVIC moduli for the four layers were found to be all within a narrow range of 4.71-5.35kPa, suggesting that MVIC deformation is primarily controlled by each tissue layer's respective structure and mechanical behavior rather than the intrinsic MVIC stiffness. This novel result further suggests that while the MVICs may be phenotypically and biomechanically similar throughout the leaflet, they experience layer-specific mechanical stimulatory inputs due to distinct extracellular matrix architecture and mechanical behaviors of the four MV leaflet tissue layers. This also suggests that MVICs may behave in a layer-specific manner in response to mechanical stimuli in both normal and surgically modified MVs.

Anisotropic poly(ethylene glycol)/polycaprolactone hydrogel-fiber composites for heart valve tissue engineering.

The recapitulation of the material properties and structure of the native aortic valve leaflet, specifically its anisotropy and laminate structure, is a major design goal for scaffolds for heart valve tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for this purpose as they are biocompatible, can be modified for their mechanical and biofunctional properties, and can be laminated. This study investigated augmenting PEG hydrogels with polycaprolactone (PCL) as an analog to the fibrosa to improve strength and introduce anisotropic mechanical behavior. However, due to its hydrophobicity, PCL must be modified prior to embedding within PEG hydrogels. In this study, PCL was electrospun (ePCL) and modified in three different ways, by protein adsorption (pPCL), alkali digestion (hPCL), and acrylation (aPCL). Modified PCL of all types maintained the anisotropic elastic moduli and yield strain of unmodified anisotropic ePCL. Composites of PEG and PCL (PPCs) maintained anisotropic elastic moduli, but aPCL and pPCL had isotropic yield strains. Overall, PPCs of all modifications had elastic moduli of 3.79±0.90 MPa and 0.46±0.21 MPa in the parallel and perpendicular directions, respectively. Valvular interstitial cells seeded atop anisotropic aPCL displayed an actin distribution aligned in the direction of the underlying fibers. The resulting scaffold combines the biocompatibility and tunable fabrication of PEG with the strength and anisotropy of ePCL to form a foundation for future engineered valve scaffolds.

The tensile and viscoelastic properties of aortic valve leaflets treated with a hyaluronidase gradient.

Purpose: When diseased, aortic valves are typically replaced with bioprosthetic heart valves (BPHVs), porcine valves or bovine pericardium that are fixed in glutaraldehyde. These replacements fail within 10-15 years due to calcification and fatigue, and their failure coincides with a loss of glycosaminoglycans (GAGs). This study investigates this relationship between GAG concentration and the tensile and viscoelastic properties of aortic valve leaflets. Methods: Aortic valve leaflets were dissected from porcine hearts and digested in hyaluronidase in concentrations ranging from 0-5 U/mL for 0-24 hours, yielding a spectrum of GAG concentrations that was measured using the uronic acid assay and confirmed by Alcian Blue staining. Digested leaflets with varying GAG concentrations were then tested in tension in the circumferential and radial directions with varying strain rate, as well as in stress relaxation. Results: The GAG concentration of the leaflets was successfully reduced using hyaluronidase, although water content was not affected. Elastic modulus, the maximum stress, and hysteresis significantly increased with decreasing GAG concentration. Extensibility and the radius of transition curvature did not change with GAG concentration. The stress relaxation behavior and strain-rate independent nature of the leaflet did not change with GAG concentration. Conclusions: These results suggest that GAGs in the spongiosa lubricate tissue motion and reduce stresses experienced by the leaflet. This study forms the basis for predictive models of BPHV mechanics based on GAG concentration, and guides the rational design of future heart valve replacements.