Radiofrequency ablation alters the microstructural organization of healthy and enzymatically digested porcine mitral valves.

BACKGROUND: Myxomatous mitral valve degeneration is a common cause of mitral regurgitation and is often associated with mitral valve prolapse. With no known targets to pharmacologically treat mitral valve prolapse, surgery is often the only treatment option. Recently, radiofrequency ablation has been proposed as a percutaneous alternative to surgical resection for the reduction of mitral valve leaflet area. OBJECTIVE: Using an in vitro model of porcine mitral valve anterior leaflet enlargement following enzymatic digestion, we sought to investigate mechanisms by which radiofrequency ablation alters the geometry, microstructural organization, and mechanical properties of healthy and digested leaflets. METHODS: Paired measurements before and after ablation revealed the impact of radiofrequency ablation on leaflet properties. Multiphoton imaging was used to characterize changes in the structure and organization of the valvular extracellular matrix; planar biaxial mechanical testing and constitutive modeling were used to estimate mechanical properties of healthy and digested leaflets. RESULTS: Enzymatic digestion increased leaflet area and thickness to a similar extent as clinical mitral valve disease. Radiofrequency ablation altered extracellular matrix alignment and reduced the area of digested leaflets to that of control. Additionally, enzymatic digestion resulted in fiber alignment and reorientation toward the radial direction, causing increased forces during ablation and a structural stiffening which was improved by radiofrequency ablation. CONCLUSION: Radiofrequency ablation induces radial extracellular matrix alignment and effectively reduces the area of enlarged mitral valve leaflets. Hence, this technique may be a therapeutic approach for myxomatous mitral valve disease and is thus an avenue for future study.

Multi-view digital image correlation systems for in vitro testing of arteries from mice to humans.

Background: Digital image correlation (DIC) methods are increasingly used for non-contact optical assessment of geometry and deformation in soft tissue biomechanics, thus providing the full-field strain estimates needed for robust inverse material characterization. Despite the well-known flexibility and ease of use of DIC, issues related to spatial resolution and depth-of-field remain challenging in studies of quasi-cylindrical biological samples such as arteries. Objective: After demonstrating that standard surrounding multi-view DIC systems are inappropriate for such usage, we submit that both the optical setup and the data analysis need to be specifically designed with respect to the size of the arterial sample of interest. Accordingly, we propose novel and optimized DIC systems for two distinct ranges of arterial diameters: less than 2.5 mm (murine arteries) and greater than 10 mm (human arteries). Methods: We designed, set up, and validated a four-camera panoramic-DIC system for testing murine arteries and a multi-biprism DIC system for testing human arteries. Both systems enable dynamic 360-deg measurements with refraction correction over the entire surface of submerged samples in their native geometries. Results: Illustrative results for 3D shape and full-surface deformation fields were obtained for a mouse infrarenal aorta and a latex cylinder of size similar to the human infrarenal aorta. Conclusion: Results demonstrated the feasibility and accuracy of both proposed methods in providing quantitative information on the regional behavior of arterial samples tested in vitro under physiologically relevant loading.

Immuno-driven and Mechano-mediated Neotissue Formation in Tissue Engineered Vascular Grafts.

In vivo development of a neovessel from an implanted biodegradable polymeric scaffold depends on a delicate balance between polymer degradation and native matrix deposition. Studies in mice suggest that this balance is dictated by immuno-driven and mechanotransduction-mediated processes, with neotissue increasingly balancing the hemodynamically induced loads as the polymer degrades. Computational models of neovessel development can help delineate relative time-dependent contributions of the immunobiological and mechanobiological processes that determine graft success or failure. In this paper, we compare computational results informed by long-term studies of neovessel development in immuno-compromised and immuno-competent mice. Simulations suggest that an early exuberant inflammatory response can limit subsequent mechano-sensing by synthetic intramural cells and thereby attenuate the desired long-term mechano-mediated production of matrix. Simulations also highlight key inflammatory differences in the two mouse models, which allow grafts in the immuno-compromised mouse to better match the biomechanical properties of the native vessel. Finally, the predicted inflammatory time courses revealed critical periods of graft remodeling. We submit that computational modeling can help uncover mechanisms of observed neovessel development and improve the design of the scaffold or its clinical use.

Differential cell-matrix mechanoadaptations and inflammation drive regional propensities to aortic fibrosis, aneurysm or dissection in hypertension.

The embryonic lineage of intramural cells, microstructural organization of the extracellular matrix, local luminal and wall geometry, and haemodynamic loads vary along the length of the aorta. Yet, it remains unclear why certain diseases manifest differentially along the aorta. Toward this end, myriad animal models provide insight into diverse disease conditions-including fibrosis, aneurysm and dissection-but inherent differences across models impede general interpretations. We examined region-specific cellular, matrix, and biomechanical changes in a single experimental model of hypertension and atherosclerosis, which commonly coexist. Our findings suggest that (i) intramural cells within the ascending aorta are unable to maintain the intrinsic material stiffness of the wall, which ultimately drives aneurysmal dilatation, (ii) a mechanical stress-initiated, inflammation-driven remodelling within the descending aorta results in excessive fibrosis, and (iii) a transient loss of adventitial collagen within the suprarenal aorta contributes to dissection propensity. Smooth muscle contractility helps to control wall stress in the infrarenal aorta, which maintains mechanical properties near homeostatic levels despite elevated blood pressure. This early mechanoadaptation of the infrarenal aorta does not preclude subsequent acceleration of neointimal formation, however. Because region-specific conditions may be interdependent, as, for example, diffuse central arterial stiffening can increase cyclic haemodynamic loads on an aneurysm that is developing proximally, there is a clear need for more systematic assessments of aortic disease progression, not simply a singular focus on a particular region or condition.

Comparison of 10 murine models reveals a distinct biomechanical phenotype in thoracic aortic aneurysms.

Thoracic aortic aneurysms are life-threatening lesions that afflict young and old individuals alike. They frequently associate with genetic mutations and are characterized by reduced elastic fibre integrity, dysfunctional smooth muscle cells, improperly remodelled collagen and pooled mucoid material. There is a pressing need to understand better the compromised structural integrity of the aorta that results from these genetic mutations and renders the wall vulnerable to dilatation, dissection or rupture. In this paper, we compare the biaxial mechanical properties of the ascending aorta from 10 murine models: wild-type controls, acute elastase-treated, and eight models with genetic mutations affecting extracellular matrix proteins, transmembrane receptors, cytoskeletal proteins, or intracellular signalling molecules. Collectively, our data for these diverse mouse models suggest that reduced mechanical functionality, as indicated by a decreased elastic energy storage capability or reduced distensibility, does not predispose to aneurysms. Rather, despite normal or lower than normal circumferential and axial wall stresses, it appears that intramural cells in the ascending aorta of mice prone to aneurysms are unable to maintain or restore the intrinsic circumferential material stiffness, which may render the wall biomechanically vulnerable to continued dilatation and possible rupture. This finding is consistent with an underlying dysfunctional mechanosensing or mechanoregulation of the extracellular matrix, which normally endows the wall with both appropriate compliance and sufficient strength.

A hidden structural vulnerability in the thrombospondin-2 deficient aorta increases the propensity to intramural delamination.

Mice lacking thrombospondin-2 (TSP2) represent an animal model of impaired collagen fibrillogenesis. Collagen constitutes ~1/3 of the wall of the normal murine descending thoracic aorta (DTA) and is thought to confer mechanical strength at high pressures. Microstructural analysis of the DTA from TSP2-null mice revealed irregular and disorganized collagen fibrils in the adventitia and at the interface between the media and adventitia. Yet, biaxial mechanical tests performed under physiologic loading conditions showed that most mechanical metrics, including stress and stiffness, were not different between mutant and control DTAs at 20- and 40-weeks of age, thus suggesting that the absence of TSP2 is well compensated under normal conditions. A detailed bilayered analysis of the wall mechanics predicted, however, that the adventitia of TSP2-null DTAs fails to engage at high pressures, which could render the media vulnerable to mechanical damage. Failure tests confirmed that the pressure at which the DTA ruptures is significantly lower in 20-week-old TSP2-null mice compared to age-matched controls (640±37 vs. 1120±45mmHg). Moreover, half of the 20-week-old and all 40-week-old mutant DTAs failed by delamination, not rupture. This delamination occurred at the interface between the media and the adventitia, with separation planes often observed at ~45 degrees with respect to the circumferential/axial directions. Combined with the observed microstructural anomalies, our theoretical-experimental biomechanical results suggest that TSP2-null DTAs are more susceptible to material failure when exposed to high pressures and this vulnerability may result from a reduced resistance to shear loading at the medial/adventitial border.

Loss of Elastic Fiber Integrity Compromises Common Carotid Artery Function: Implications for Vascular Aging.

Competent elastic fibers endow central arteries with the compliance and resilience that are fundamental to their primary mechanical function in vertebrates. That is, by enabling elastic energy to be stored in the arterial wall during systole and then to be used to work on the blood during diastole, elastic fibers decrease ventricular workload and augment blood flow in pulsatile systems. Indeed, because elastic fibers are formed during development and stretched during somatic growth, their continual tendency to recoil contributes to the undulation of the stiffer collagen fibers, which facilitates further the overall compliance of the wall under physiologic pressures while allowing the collagen to limit over-distension during acute increases in blood pressure. In this paper, we use consistent methods of measurement and quantification to compare the biaxial material stiffness, structural stiffness, and energy storage capacity of murine common carotid arteries having graded degrees of elastic fiber integrity - normal, elastin-deficient, fibrillin-1 deficient, fibulin-5 null, and elastase-treated. The finding that the intrinsic material stiffness tends to be maintained nearly constant suggests that intramural cells seek to maintain a favorable micromechanical environment in which to function. Nevertheless, a loss of elastic energy storage capability due to the loss of elastic fiber integrity severely compromises the primary function of these central arteries.

Decreased elastic energy storage, not increased material stiffness, characterizes central artery dysfunction in fibulin-5 deficiency independent of sex.

Central artery stiffness has emerged over the past 15 years as a clinically significant indicator of cardiovascular function and initiator of disease. Loss of elastic fiber integrity is one of the primary contributors to increased arterial stiffening in aging, hypertension, and related conditions. Elastic fibers consist of an elastin core and multiple glycoproteins; hence defects in any of these constituents can adversely affect arterial wall mechanics. In this paper, we focus on mechanical consequences of the loss of fibulin-5, an elastin-associated glycoprotein involved in elastogenesis. Specifically, we compared the biaxial mechanical properties of five central arteries-the ascending thoracic aorta, descending thoracic aorta, suprarenal abdominal aorta, infrarenal abdominal aorta, and common carotid artery-from male and female wild-type and fibulin-5 deficient mice. Results revealed that, independent of sex, all five regions in the fibulin-5 deficient mice manifested a marked increase in structural stiffness but also a marked decrease in elastic energy storage and typically an increase in energy dissipation, with all differences being most dramatic in the ascending and abdominal aortas. Given that the primary function of large arteries is to store elastic energy during systole and to use this energy during diastole to work on the blood, fibulin-5 deficiency results in a widespread diminishment of central artery function that can have significant effects on hemodynamics and cardiac function.

Consistent biomechanical phenotyping of common carotid arteries from seven genetic, pharmacological, and surgical mouse models.

The continuing lack of longitudinal histopathological and biomechanical data for human arteries in health and disease highlights the importance of studying the many genetic, pharmacological, and surgical models that are available in mice. As a result, there has been a significant increase in the number of reports on the biomechanics of murine arteries over the past decade, particularly for the common carotid artery. Whereas most of these studies have focused on wild-type controls or comparing controls vs. a single model of altered hemodynamics or vascular disease, there is a pressing need to compare results across many different models to understand more broadly the effects of genetic mutations, pharmacological treatments, or surgical alterations on the evolving hemodynamics and the microstructure and biomechanical properties of these vessels. This paper represents a first step toward this goal, that is, a biomechanical phenotyping of common carotid arteries from control mice and seven different mouse models that represent alterations in elastic fiber integrity, collagen remodeling, and smooth muscle cell functionality.

Biomechanical phenotyping of central arteries in health and disease: advantages of and methods for murine models.

The stiffness and structural integrity of the arterial wall depends primarily on the organization of the extracellular matrix and the cells that fashion and maintain this matrix. Fundamental to the latter is a delicate balance in the continuous production and removal of structural constituents and the mechanical state in which such turnover occurs. Perturbations in this balance due to genetic mutations, altered hemodynamics, or pathological processes result in diverse vascular phenotypes, many of which have yet to be well characterized biomechanically. In this paper, we emphasize the particular need to understand regional variations in the biaxial biomechanical properties of central arteries in health and disease and, in addition, the need for standardization in the associated biaxial testing and quantification. As an example of possible experimental methods, we summarize testing protocols that have evolved in our laboratory over the past 8 years. Moreover, we note advantages of a four fiber family stress-stretch relation for quantifying passive biaxial behaviors, the use of stored energy as a convenient scalar metric of the associated material stiffness, and the utility of appropriate linearizations of the nonlinear, anisotropic relations both for purposes of comparison across laboratories and to inform computational fluid-solid-interaction models. We conclude that, notwithstanding prior advances, there remain many opportunities to advance our understanding of arterial mechanics and mechanobiology, particularly via the diverse genetic, pharmacological, and surgical models that are, or soon will be, available in the mouse.

Disparate Changes in the Mechanical Properties of Murine Carotid Arteries and Aorta in Response to Chronic Infusion of Angiotensin-II.

Chronic infusion of angiotensin-II has proved useful for generating dissecting aortic aneurysms in atheroprone mice. These lesions preferentially form in the suprarenal abdominal aorta and sometimes in the ascending aorta, but reasons for such localization remain unknown. This study focused on why these lesions do not form in other large (central) arteries. Toward this end, we quantified and compared the geometry, composition, and biaxial material behavior (using a nonlinear constitutive relation) of common carotid arteries from three groups of mice: non-treated controls as well as mice receiving a subcutaneous infusion of angiotensin-II for 28 days that either did or did not lead to the development of a dissecting aortic aneurysm. Consistent with the mild hypertension induced by the angiotensin-II, the carotid wall thickened as expected and remodeled modestly. There was no evidence, however, of a marked loss of elastic fibers or smooth muscle cells, each of which appear to be initiating events for the development of aneurysms, and there was no evidence of intramural discontinuities that might give rise to dissections.