Cells and Materials for Cardiac Repair and Regeneration.

After more than 20 years following the introduction of regenerative medicine to address the problem of cardiac diseases, still questions arise as to the best cell types and materials to use to obtain effective clinical translation. Now that it is definitively clear that the heart does not have a consistent reservoir of stem cells that could give rise to new myocytes, and that there are cells that could contribute, at most, with their pro-angiogenic or immunomodulatory potential, there is fierce debate on what will emerge as the winning strategy. In this regard, new developments in somatic cells' reprogramming, material science and cell biophysics may be of help, not only for protecting the heart from the deleterious consequences of aging, ischemia and metabolic disorders, but also to boost an endogenous regeneration potential that seems to be lost in the adulthood of the human heart.

Mechanosensor YAP cooperates with TGF-ß1 signaling to promote myofibroblast activation and matrix stiffening in a 3D model of human cardiac fibrosis.

Cardiac fibrosis is characterized by a maladaptive remodeling of the myocardium, which is controlled by various inflammatory pathways and cytokines. This remodeling is accompanied by a significant stiffening of the matrix, which may contribute to further activate collagen synthesis and scar formation. Evidence suggests that TGF-ß1 signaling, the main pro-fibrotic pathway in cardiac fibrosis, might cooperates with the Hippo transcriptional pathway by activating YAP. To directly test the cooperation of mechanical cues and paracrine signaling in cardiac fibrosis, we developed a 3D model of cardiac extracellular matrix remodeling by generating tissue blocks with Gelatin Methacrylate, a bioink with tunable stiffness, and human cardiosphere-derived stromal cells. Using this strategy, we assessed the cooperation of TGF-ß1 and YAP transcriptional factor to matrix compaction. Using mechanical compression tests, Masson's trichrome staining, immunofluorescence, and RT-qPCR, we demonstrate that pharmacological inhibition of YAP complex reverts almost completely the pro-compaction phenotype and the matrix-remodeling activity of cells treated with TGF-ß1. Our data show a direct connection between the classical pro-fibrotic signaling driven by TGF-ß1 and the mechanically activated pathways under the control of YAP in cardiac remodeling. Treatment with the elective drug targeting YAP is sufficient to override this cooperation with potential benefits for anti-fibrotic therapeutic applications. STATEMENT OF SIGNIFICANCE: Heart failure is a pathology in continuous growth worldwide, characterized by a progressive fibrosis, which decreases the pumping efficiency of the heart. Experimental evidences suggest that fibroblasts, normally responsible for the turnover of the cardiac matrix, are involved in myocardial fibrosis by differentiating into 'myofibroblasts'. These cells remodel extensively the cardiac extracellular matrix and deposit abundant collagen with a consequent increase in stiffness. In the present contribution, we propose a new 3D model of cell-mediated cardiac extracellular matrix stiffening to investigate the mechano-chemical mechanisms underlying the onset of the pathology. We also consolidate a pharmacological treatment able to prevent the pathological activation of fibroblasts with potential benefits for anti-fibrotic treatment of the failing heart.

Mechanical Strain Induces Transcriptomic Reprogramming of Saphenous Vein Progenitors.

Intimal hyperplasia is the leading cause of graft failure in aortocoronary bypass grafts performed using human saphenous vein (SV). The long-term consequences of the altered pulsatile stress on the cells that populate the vein wall remains elusive, particularly the effects on saphenous vein progenitors (SVPs), cells resident in the vein adventitia with a relatively wide differentiation capacity. In the present study, we performed global transcriptomic profiling of SVPs undergoing uniaxial cyclic strain in vitro. This type of mechanical stimulation is indeed involved in the pathology of the SV. Results showed a consistent stretch-dependent gene regulation in cyclically strained SVPs vs. controls, especially at 72 h. We also observed a robust mechanically related overexpression of Adhesion Molecule with Ig Like Domain 2 (AMIGO2), a cell surface type I transmembrane protein involved in cell adhesion. The overexpression of AMIGO2 in stretched SVPs was associated with the activation of the transforming growth factor ß pathway and modulation of intercellular signaling, cell-cell, and cell-matrix interactions. Moreover, the increased number of cells expressing AMIGO2 detected in porcine SV adventitia using an in vivo arterialization model confirms the upregulation of AMIGO2 protein by the arterial-like environment. These results show that mechanical stress promotes SVPs' molecular phenotypic switching and increases their responsiveness to extracellular environment alterations, thus prompting the targeting of new molecular effectors to improve the outcome of bypass graft procedure.

Reduction of Cardiac Fibrosis by Interference With YAP-Dependent Transactivation.

BACKGROUND: Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation. METHODS: We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. RESULTS: Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-ß1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. CONCLUSIONS: Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

From dissection of fibrotic pathways to assessment of drug interactions to reduce cardiac fibrosis and heart failure.

Cardiac fibrosis is characterized by extracellular matrix deposition in the cardiac interstitium, and this contributes to cardiac contractile dysfunction and progression of heart failure. The main players involved in this process are the cardiac fibroblasts, which, in the presence of pro-inflammatory/pro-fibrotic stimuli, undergo a complete transformation acquiring a more proliferative, a pro-inflammatory and a secretory phenotype. This review discusses the cellular effectors and molecular pathways implicated in the pathogenesis of cardiac fibrosis and suggests potential strategies to monitor the effects of specific drugs designed to slow down the progression of this disease by specifically targeting the fibroblasts.

Vascular dysfunction and pathology: focus on mechanical forces.

The role of mechanical forces is emerging as a new player in the pathophysiologic programming of the cardiovascular system. The ability of the cells to 'sense' mechanical forces does not relate only to perception of movement or flow, as intended traditionally, but also to the biophysical properties of the extracellular matrix, the geometry of the tissues, and the force distribution inside them. This is also supported by the finding that cells can actively translate mechanical cues into discrete gene expression and epigenetic programming. In the present review, we will contextualize these new concepts in the vascular pathologic programming.

Human cardiosphere-derived stromal cells exposed to SARS-CoV-2 evolve into hyper-inflammatory/pro-fibrotic phenotype and produce infective viral particles depending on the levels of ACE2 receptor expression.

AIMS: Patients with severe respiratory syndrome caused by SARS-CoV-2 undergo cardiac complications due to hyper-inflammatory conditions. Although the presence of the virus has been detected in the myocardium of infected patients, and infection of induced pluripotent cell-derived cardiomyocytes has been demonstrated, the reported expression of Angiotensin-Converting Enzyme-2 (ACE2) in cardiac stromal cells suggests that SARS-CoV-2 may determine cardiac injury by sustaining productive infection and increasing inflammation. METHODS AND RESULTS: We analysed expression of ACE2 receptor in primary human cardiac stromal cells derived from cardiospheres, using proteomics and transcriptomics before exposing them to SARS-CoV-2 in vitro. Using conventional and high sensitivity PCR methods, we measured virus release in the cellular supernatants and monitored the intracellular viral bioprocessing. We performed high-resolution imaging to show the sites of intracellular viral production and demonstrated the presence of viral particles in the cells with electron microscopy. We finally used RT-qPCR assays to detect genes linked to innate immunity and fibrotic pathways coherently regulated in cells after exposure to the virus. CONCLUSIONS: Our findings indicate that cardiac stromal cells are susceptible to SARS-CoV-2 infection and produce variable viral yields depending on the extent of cellular ACE2 receptor expression. Interestingly, these cells also evolved towards hyper-inflammatory/pro-fibrotic phenotypes independently of ACE2 levels. Thus, SARS-CoV-2 infection of myocardial stromal cells could be involved in cardiac injury and explain the high number of complications observed in severe cases of COVID-19.

Digital PCR for high sensitivity viral detection in false-negative SARS-CoV-2 patients.

Patients requiring diagnostic testing for coronavirus disease 2019 (COVID-19) are routinely assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) amplification of Sars-CoV-2 virus RNA extracted from oro/nasopharyngeal swabs. Despite the good specificity of the assays certified for SARS-CoV-2 molecular detection, and a theoretical sensitivity of few viral gene copies per reaction, a relatively high rate of false negatives continues to be reported. This is an important challenge in the management of patients on hospital admission and for correct monitoring of the infectivity after the acute phase. In the present report, we show that the use of digital PCR, a high sensitivity method to detect low amplicon numbers, allowed us to correctly detecting infection in swab material in a significant number of false negatives. We show that the implementation of digital PCR methods in the diagnostic assessment of COVID-19 could resolve, at least in part, this timely issue.

A fluorogenic peptide-based smartprobe for the detection of neutrophil extracellular traps and inflammation.

A highly sensitive optical probe for the detection of activated neutrophils and Neutrophil Extracellular Traps (NETs) is reported. It is based on a triple-quenched, super-silent tri-branched probe that generates >20 fold increase in fluorescence upon cleavage. The probe was highly specific for human neutrophil elastase, a protease that mediates a variety of inflammatory diseases, and detected NETosis and neutrophil activation in in vitro differentiated neutrophils and isolated human neutrophils.

Harnessing Mechanosensation in Next Generation Cardiovascular Tissue Engineering.

The ability of the cells to sense mechanical cues is an integral component of "social" cell behavior inside tissues with a complex architecture. Through "mechanosensation" cells are in fact able to decrypt motion, geometries and physical information of surrounding cells and extracellular matrices by activating intracellular pathways converging onto gene expression circuitries controlling cell and tissue homeostasis. Additionally, only recently cell mechanosensation has been integrated systematically as a crucial element in tissue pathophysiology. In the present review, we highlight some of the current efforts to assess the relevance of mechanical sensing into pathology modeling and manufacturing criteria for a next generation of cardiovascular tissue implants.

Coronary artery mechanics induces human saphenous vein remodelling via recruitment of adventitial myofibroblast-like cells mediated by Thrombospondin-1.

Rationale: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease. Methods: We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results in vitro and using a model of SV carotid interposition in pigs. Results: Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22α) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-ß-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-ß-dependent pathway, and validated in a saphenous vein into carotid interposition pig model. Conclusions: Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors.

Mechanotransduction in the Cardiovascular System: From Developmental Origins to Homeostasis and Pathology.

With the term 'mechanotransduction', it is intended the ability of cells to sense and respond to mechanical forces by activating intracellular signal transduction pathways and the relative phenotypic adaptation. While a known role of mechanical stimuli has been acknowledged for developmental biology processes and morphogenesis in various organs, the response of cells to mechanical cues is now also emerging as a major pathophysiology determinant. Cells of the cardiovascular system are typically exposed to a variety of mechanical stimuli ranging from compression to strain and flow (shear) stress. In addition, these cells can also translate subtle changes in biophysical characteristics of the surrounding matrix, such as the stiffness, into intracellular activation cascades with consequent evolution toward pro-inflammatory/pro-fibrotic phenotypes. Since cellular mechanotransduction has a potential readout on long-lasting modifications of the chromatin, exposure of the cells to mechanically altered environments may have similar persisting consequences to those of metabolic dysfunctions or chronic inflammation. In the present review, we highlight the roles of mechanical forces on the control of cardiovascular formation during embryogenesis, and in the development and pathogenesis of the cardiovascular system.

Activation of human aortic valve interstitial cells by local stiffness involves YAP-dependent transcriptional signaling.

Differentiation of valve interstitial cells (VICs) into pro-calcific cells is one of the central events in calcific aortic valve (AoV) disease (CAVD). While the paracrine pathways and the responsivity of VICs to mechanical compliance of the surrounding environment are well characterized, the molecular programming related to variations in local stiffness, and its link to cytoskeleton dynamics, is less consolidated. By using a simple method to produce 2D poly-acrylamide gels with stiffness controlled with atomic force microscopy (AFM), we manufactured adhesion substrates onto which human VICs from stenotic valves were plated, and subsequently investigated for cytoskeleton dynamics and activation of the mechanosensing-related transcription factor YAP. As a comparison, we employed VICs from patients undergoing valve substitution for valve insufficiency, a non-calcific AoV disease, which does not involve extensive inflammation. While the two VICs types did not differ for basic responses onto substrates with different stiffness values (e.g. adhesion and proliferation), they were subject to a different dynamics of stiffness-dependent YAP nuclear shuttling, revealing for the first time an intracellular force transduction mechanism distinctive for calcific aortic valve disease. In VICs from stenotic valves, YAP nuclear translocation occurred in concert with an increase in cytoskeleton tensioning and loading of the myofibroblast-specific protein αSMA onto the F-actin cytoskeleton. AFM force mapping performed along radial sections of human calcific valve leaflets identified, finally, areas with high and low levels of rigidity within a similar range to those controlling YAP nuclear translocation in vitro. Since VICs juxtaposed to these areas exhibited nuclear localized YAP, we conclude that subtle variations in matrix stiffness are involved in mechanosensing-dependent VICs activation and pathological differentiation in CAVD.