Tissue properties and collagen remodeling in heart valve tissue engineering

Valvular heart disease is a major health problem worldwide causing morbidity and mortality. Heart valve replacement is frequently applied to avoid serious cardiac, pulmonary, or systemic problems. However, the current replacements do not consist of living tissue and, consequently, cannot grow, repair, or remodel in response to changing functional demands. Heart valve tissue engineering (HVTE) seeks to overcome the shortcomings of the existing valve replacements by creating living autologous heart valves. One of the main challenges of HVTE is to control tissue formation, collagen remodeling and consequent tissue mechanical properties during the in vitro culture phase. Additionally, it is important to define benchmarks based on the target native heart valve tissues to compare with the tissue structure and mechanical properties of tissue-engineered (TE) heart valves. The aim of this thesis is to define benchmarks, understand and optimize tissue development and resulting tissue mechanical properties of TE heart valves, with special emphasis on collagen remodeling. In order to provide insights into the evolution and maturation of the extracellular matrix and mechanical properties and to provide benchmarks for TE heart valves, matrix composition, maturation and mechanical properties of native human aortic and pulmonary heart valves were studied. It was observed that the matrix composition and the mechanical properties change with age and that a significant part of the mechanical behaviour of the human native heart valve leaflets is defined by the composition and maturation of the matrix. Tissue (mechanical) properties of TE heart valves should be optimized towards the provided benchmarks during the in vitro culture phase. To this end, possible indicators of in vitro tissue outcome were determined to enable prediction of the properties of the autologous tissues cultured for individual patients. It was found that a-Smooth muscle actin (aSMA) might be such an indicator. In addition, interspecies differences in tissue (mechanical) properties were evaluated to determine whether ovine TE heart valves are representative of human TE heart valves as the ovine model is the prescribed animal model to evaluate heart valve replacements. This study suggested that the culture process of ovine tissue can be controlled, whereas the mechanical properties, and hence functionality, of tissues cultured with human cells are more difficult to predict, indicating once more the importance of early markers to predict tissue outcome. As a further step towards clinical application and to circumvent the use of animal-derived medium components in the culture protocol, fetal bovine serum was replaced by human platelet lysate for the culture of autologous TE heart valve constructs. Although tissue composition and maturation were similar, mechanical properties were much lower for the tissues cultured in platelet lysate, most likely due to an increased production of matrix-degrading enzymes leading to an altered collagen architecture. Thus, collagen architecture, rather than collagen content alone, is dominant in defining the tissue mechanical properties. To stimulate tissue formation and maturation towards the right collagen architecture for in vivo mechanical functionality, mechanical conditioning of the engineered tissue is commonly pursued. Previous studies indicated that intermittent conditioning, in which cyclic and static strain are alternated, is favoured to obtain mature tissues in a short time period. To unravel the underlying mechanism of intermittent conditioning, the effects of cyclic strain and static strain after cyclic strain were examined at gene expression level. This study indicated that a period of static strain is required for collagen synthesis and remodeling, while continuous cyclic strain shifts this balance towards collagen remodeling and maturation. These results imply that the mechanical conditioning protocol should change over time from intermittent conditioning to continuous cyclic strain to improve collagen maturation after its synthesis and, therewith, the mechanical properties of TE heart valves. In summary, the results from this thesis suggest that in addition to collagen content and maturation, collagen organization is particularly important in defining the tissue mechanical properties. Thus, optimization of culture protocols should focus on obtaining the proper collagen architecture for creating mechanically functioning TE heart valves. Autologous culture of TE heart valves using human platelet lysate is not preferred, since it prevents the formation of a load-bearing organized collagen network. Mechanical conditioning protocols should start with intermittent conditioning, followed by continuous cyclic strain to enhance collagen maturation after its synthesis. Considering the interpatient variability in tissue outcome of tissues cultured with similar protocols, it must be noted that further refinement, or even personalization, of culture protocols might be necessary. To this end, markers of tissue outcome, such as aSMA, are necessary to predict and adapt culture protocols and, therewith, individual tissue outcome at an early stage during culture. Although these suggestions require additional (in vivo) study, the results of this thesis provide substantial insight on how to improve in vitro HVTE strategies to control tissue properties and collagen remodeling for optimization of TE heart valves towards their native counterparts

Sheep-specific immunohistochemical panel for the evaluation of regenerative and inflammatory processes in tissue-engineered heart valves

\u3cp\u3eThe creation of living heart valve replacements via tissue engineering is actively being pursued by many research groups. Numerous strategies have been described, aimed either at culturing autologous living valves in a bioreactor (in vitro) or inducing endogenous regeneration by the host via resorbable scaffolds (in situ). Whereas a lot of effort is being invested in the optimization of heart valve scaffold parameters and culturing conditions, the pathophysiological in vivo remodeling processes to which tissue-engineered heart valves are subjected upon implantation have been largely under-investigated. This is partly due to the unavailability of suitable immunohistochemical tools specific to sheep, which serves as the gold standard animal model in translational research on heart valve replacements. Therefore, the goal of this study was to comprise and validate a comprehensive sheep-specific panel of antibodies for the immunohistochemical analysis of tissue-engineered heart valve explants. For the selection of our panel we took inspiration from previous histopathological studies describing the morphology, extracellular matrix composition and cellular composition of native human heart valves throughout development and adult stages. Moreover, we included a range of immunological markers, which are particularly relevant to assess the host inflammatory response evoked by the implanted heart valve. The markers specifically identifying extracellular matrix components and cell phenotypes were tested on formalin-fixed paraffin-embedded sections of native sheep aortic valves. Markers for inflammation and apoptosis were tested on ovine spleen and kidney tissues. Taken together, this panel of antibodies could serve as a tool to study the spatiotemporal expression of proteins in remodeling tissue-engineered heart valves after implantation in a sheep model, thereby contributing to our understanding of the in vivo processes which ultimately determine long-term success or failure of tissue-engineered heart valves.\u3c/p\u3

Decreased mechanical properties of heart valve tissue constructs cultured in platelet lysate as compared to fetal bovine serum

In autologous heart valve tissue engineering, there is an ongoing search for alternatives of fetal bovine serum (FBS). Human platelet-lysate (PL) might be a promising substitute. In the present article, we aimed to examine the tissue formation, functionality, and mechanical properties of engineered three-dimensional tissue constructs cultured in PL as a substitute for FBS. Our results show that tissue constructs that were cultured in PL and FBS produce similar amounts of collagen, glycosoaminoglycans, and collagen crosslinks, and that the cellular phenotype remains unchanged. Nevertheless, mechanical testing showed that the ultimate tensile strength in PL constructs was on average approximately three times lower as compared to FBS (0.25 vs. 0.74¿MPa, respectively,

F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries and veins

Vascular endothelial-cadherin- and integrin-based cell adhesions are crucial for endothelial barrier function. Formation and disassembly of these adhesions controls endothelial remodeling during vascular repair, angiogenesis, and inflammation. In vitro studies indicate that vascular cytokines control adhesion through regulation of the actin cytoskeleton, but it remains unknown whether such regulation occurs in human vessels. We aimed to investigate regulation of the actin cytoskeleton and cell adhesions within the endothelium of human arteries and veins. We used an ex vivo protocol for immunofluorescence in human vessels, allowing detailed en face microscopy of endothelial monolayers. We compared arteries and veins of the umbilical cord and mesenteric, epigastric, and breast tissues and find that the presence of central F-actin fibers distinguishes the endothelial phenotype of adult arteries from veins. F-actin in endothelium of adult veins as well as in umbilical vasculature predominantly localizes cortically at the cell boundaries. By contrast, prominent endothelial F-actin fibers in adult arteries anchor mostly to focal adhesions containing integrin-binding proteins paxillin and focal adhesion kinase and follow the orientation of the extracellular matrix protein fibronectin. Other arterial F-actin fibers end in vascular endothelial-cadherin-based endothelial focal adherens junctions. In vitro adhesion experiments on compliant substrates demonstrate that formation of focal adhesions is strongly induced by extracellular matrix rigidity, irrespective of arterial or venous origin of endothelial cells. Our data show that F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries from veins. We conclude that the biomechanical properties of the vascular extracellular matrix determine this endothelial characteristi

Age-dependent changes of stress and strain in the human heart valve and their relation with collagen remodeling

\u3cp\u3eIn order to create tissue-engineered heart valves with long-term functionality, it is essential to fully understand collagen remodeling during neo-tissue formation. Collagen remodeling is thought to maintain mechanical tissue homeostasis. Yet, the driving factor of collagen remodeling remains unidentified. In this study, we determined the collagen architecture and the geometric and mechanical properties of human native semilunar heart valves of fetal to adult age using confocal microscopy, micro-indentation and inverse finite element analysis. The outcomes were used to predict age-dependent changes in stress and stretch in the heart valves via finite element modeling. The results indicated that the circumferential stresses are different between the aortic and pulmonary valve, and, moreover, that the stress increases considerably over time in the aortic valve. Strikingly, relatively small differences were found in stretch with time and between the aortic and pulmonary valve, particularly in the circumferential direction, which is the main determinant of the collagen fiber stretch. Therefore, we suggest that collagen remodeling in the human heart valve maintains a stretch-driven homeostasis. Next to these novel insights, the unique human data set created in this study provides valuable input for the development of numerical models of collagen remodeling and optimization of tissue engineering. Statement of significance Annually, over 280,000 heart valve replacements are performed worldwide. Tissue engineering has the potential to provide valvular disease patients with living valve substitutes that can last a lifetime. Valve functionality is mainly determined by the collagen architecture. Hence, understanding collagen remodeling is crucial for creating tissue-engineered valves with long-term functionality. In this study, we determined the structural and material properties of human native heart valves of fetal to adult age to gain insight into the mechanical stimuli responsible for collagen remodeling. The age-dependent evolutionary changes in mechanical state of the native valve suggest that collagen remodeling in heart valves is a stretch-driven process.\u3c/p\u3

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils

Age-dependent changes in geometry, tissue composition and mechanical properties of fetal to adult cryopreserved human heart valves

\u3cp\u3eThere is limited information about age-specific structural and functional properties of human heart valves, while this information is key to the development and evaluation of living valve replacements for pediatric and adolescent patients. Here, we present an extended data set of structure-function properties of cryopreserved human pulmonary and aortic heart valves, providing age-specific information for living valve replacements. Tissue composition, morphology, mechanical properties, and maturation of leaflets from 16 pairs of structurally unaffected aortic and pulmonary valves of human donors (fetal-53 years) were analyzed. Interestingly, no major differences were observed between the aortic and pulmonary valves. Valve annulus and leaflet dimensions increase throughout life. The typical three-layered leaflet structure is present before birth, but becomes more distinct with age. After birth, cell numbers decrease rapidly, while remaining cells obtain a quiescent phenotype and reside in the ventricularis and spongiosa. With age and maturation-but more pronounced in aortic valves-the matrix shows an increasing amount of collagen and collagen cross-links and a reduction in glycosaminoglycans. These matrix changes correlate with increasing leaflet stiffness with age. Our data provide a new and comprehensive overview of the changes of structure-function properties of fetal to adult human semilunar heart valves that can be used to evaluate and optimize future therapies, such as tissue engineering of heart valves. Changing hemodynamic conditions with age can explain initial changes in matrix composition and consequent mechanical properties, but cannot explain the ongoing changes in valve dimensions and matrix composition at older age.\u3c/p\u3

The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions

Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissu