Early Impairment of Paracrine and Phenotypic Features in Resident Cardiac Mesenchymal Stromal Cells after Thoracic Radiotherapy.

Radiotherapy-induced cardiac toxicity and consequent diseases still represent potential severe late complications for many cancer survivors who undergo therapeutic thoracic irradiation. We aimed to assess the phenotypic and paracrine features of resident cardiac mesenchymal stromal cells (CMSCs) at early follow-up after the end of thoracic irradiation of the heart as an early sign and/or mechanism of cardiac toxicity anticipating late organ dysfunction. Resident CMSCs were isolated from a rat model of fractionated thoracic irradiation with accurate and clinically relevant heart dosimetry that developed delayed dose-dependent cardiac dysfunction after 1 year. Cells were isolated 6 and 12 weeks after the end of radiotherapy and fully characterized at the transcriptional, paracrine, and functional levels. CMSCs displayed several altered features in a dose- and time-dependent trend, with the most impaired characteristics observed in those exposed in situ to the highest radiation dose with time. In particular, altered features included impaired cell migration and 3D growth and a and significant association of transcriptomic data with GO terms related to altered cytokine and growth factor signaling. Indeed, the altered paracrine profile of CMSCs derived from the group at the highest dose at the 12-week follow-up gave significantly reduced angiogenic support to endothelial cells and polarized macrophages toward a pro-inflammatory profile. Data collected in a clinically relevant rat model of heart irradiation simulating thoracic radiotherapy suggest that early paracrine and transcriptional alterations of the cardiac stroma may represent a dose- and time-dependent biological substrate for the delayed cardiac dysfunction phenotype observed in vivo.

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.

Shockwaves delivery for aortic valve therapy-Realistic perspective for clinical translation?

Calcific aortic valve disease (CAVD) is the most frequent valvular heart disorder, and the one with the highest impact and burden in the elderly population. While the quality and standardization of the current aortic valve replacements has reached unprecedented levels with the commercialization of minimally-invasive implants and the design of procedures for valve repair, the need of supplementary therapies able to block or retard the course of the pathology before patients need the intervention is still awaited. In this contribution, we will discuss the emerging opportunity to set up devices to mechanically rupture the calcium deposits accumulating in the aortic valve and restore, at least in part, the pliability and the mechanical function of the calcified leaflets. Starting from the evidences gained by mechanical decalcification of coronary arteries in interventional cardiology procedures, a practice already in the clinical setting, we will discuss the advantages and the potential drawbacks of valve lithotripsy devices and their potential applicability in the clinical scenario.

Long COVID and the cardiovascular system-elucidating causes and cellular mechanisms in order to develop targeted diagnostic and therapeutic strategies: a joint Scientific Statement of the ESC Working Groups on Cellular Biology of the Heart and Myocardial and Pericardial Diseases.

Long COVID has become a world-wide, non-communicable epidemic, caused by long-lasting multiorgan symptoms that endure for weeks or months after SARS-CoV-2 infection has already subsided. This scientific document aims to provide insight into the possible causes and therapeutic options available for the cardiovascular manifestations of long COVID. In addition to chronic fatigue, which is a common symptom of long COVID, patients may present with chest pain, ECG abnormalities, postural orthostatic tachycardia, or newly developed supraventricular or ventricular arrhythmias. Imaging of the heart and vessels has provided evidence of chronic, post-infectious perimyocarditis with consequent left or right ventricular failure, arterial wall inflammation, or microthrombosis in certain patient populations. Better understanding of the underlying cellular and molecular mechanisms of long COVID will aid in the development of effective treatment strategies for its cardiovascular manifestations. A number of mechanisms have been proposed, including those involving direct effects on the myocardium, microthrombotic damage to vessels or endothelium, or persistent inflammation. Unfortunately, existing circulating biomarkers, coagulation, and inflammatory markers, are not highly predictive for either the presence or outcome of long COVID when measured 3 months after SARS-CoV-2 infection. Further studies are needed to understand underlying mechanisms, identify specific biomarkers, and guide future preventive strategies or treatments to address long COVID and its cardiovascular sequelae.

Cardiac fibroblasts and mechanosensation in heart development, health and disease.

The term 'mechanosensation' describes the capacity of cells to translate mechanical stimuli into the coordinated regulation of intracellular signals, cellular function, gene expression and epigenetic programming. This capacity is related not only to the sensitivity of the cells to tissue motion, but also to the decryption of tissue geometric arrangement and mechanical properties. The cardiac stroma, composed of fibroblasts, has been historically considered a mechanically passive component of the heart. However, the latest research suggests that the mechanical functions of these cells are an active and necessary component of the developmental biology programme of the heart that is involved in myocardial growth and homeostasis, and a crucial determinant of cardiac repair and disease. In this Review, we discuss the general concept of cell mechanosensation and force generation as potent regulators in heart development and pathology, and describe the integration of mechanical and biohumoral pathways predisposing the heart to fibrosis and failure. Next, we address the use of 3D culture systems to integrate tissue mechanics to mimic cardiac remodelling. Finally, we highlight the potential of mechanotherapeutic strategies, including pharmacological treatment and device-mediated left ventricular unloading, to reverse remodelling in the failing heart.

Luminal endothelialization of small caliber silk tubular graft for vascular constructs engineering.

The constantly increasing incidence of coronary artery disease worldwide makes necessary to set advanced therapies and tools such as tissue engineered vessel grafts (TEVGs) to surpass the autologous grafts [(i.e., mammary and internal thoracic arteries, saphenous vein (SV)] currently employed in coronary artery and vascular surgery. To this aim, in vitro cellularization of artificial tubular scaffolds still holds a good potential to overcome the unresolved problem of vessel conduits availability and the issues resulting from thrombosis, intima hyperplasia and matrix remodeling, occurring in autologous grafts especially with small caliber (<6 mm). The employment of silk-based tubular scaffolds has been proposed as a promising approach to engineer small caliber cellularized vascular constructs. The advantage of the silk material is the excellent manufacturability and the easiness of fiber deposition, mechanical properties, low immunogenicity and the extremely high in vivo biocompatibility. In the present work, we propose a method to optimize coverage of the luminal surface of silk electrospun tubular scaffold with endothelial cells. Our strategy is based on seeding endothelial cells (ECs) on the luminal surface of the scaffolds using a low-speed rolling. We show that this procedure allows the formation of a nearly complete EC monolayer suitable for flow-dependent studies and vascular maturation, as a step toward derivation of complete vascular constructs for transplantation and disease modeling.

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.

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.

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.

Engineering Efforts to Refine Compatibility and Duration of Aortic Valve Replacements: An Overview of Previous Expectations and New Promises.

The absence of pharmacological treatments to reduce or retard the progression of cardiac valve diseases makes replacement with artificial prostheses (mechanical or bio-prosthetic) essential. Given the increasing incidence of cardiac valve pathologies, there is always a more stringent need for valve replacements that offer enhanced performance and durability. Unfortunately, surgical valve replacement with mechanical or biological substitutes still leads to disadvantages over time. In fact, mechanical valves require a lifetime anticoagulation therapy that leads to a rise in thromboembolic complications, while biological valves are still manufactured with non-living tissue, consisting of aldehyde-treated xenograft material (e.g., bovine pericardium) whose integration into the host fails in the mid- to long-term due to unresolved issues regarding immune-compatibility. While various solutions to these shortcomings are currently under scrutiny, the possibility to implant fully biologically compatible valve replacements remains elusive, at least for large-scale deployment. In this regard, the failure in translation of most of the designed tissue engineered heart valves (TEHVs) to a viable clinical solution has played a major role. In this review, we present a comprehensive overview of the TEHVs developed until now, and critically analyze their strengths and limitations emerging from basic research and clinical trials. Starting from these aspects, we will also discuss strategies currently under investigation to produce valve replacements endowed with a true ability to self-repair, remodel and regenerate. We will discuss these new developments not only considering the scientific/technical framework inherent to the design of novel valve prostheses, but also economical and regulatory aspects, which may be crucial for the success of these novel designs.

Lithotripsy of Calcified Aortic Valve Leaflets by a Novel Ultrasound Transcatheter-Based Device.

The increasing incidence of calcific aortic valve disease necessitates the elaboration of new strategies to retard the progression of the pathology with an innovative solution. While the increasing diffusion of the transcatheter aortic valve replacements (TAVRs) allows a mini-invasive approach to aortic valve substitution as an alternative to conventional surgical replacement (SAVR) in an always larger patient population, TAVR implantation still has contraindications for young patients. In addition, it is liable to undergo calcification with the consequent necessity of re-intervention with conventional valve surgery or repeated implantation in the so-called TAVR-in-TAVR procedure. Inspired by applications for non-cardiac pathologies or for vascular decalcification before stenting (i.e., coronary lithotripsy), in the present study, we show the feasibility of human valve treatment with a mini-invasive device tailored to deliver shockwaves to the calcific leaflets. We provide evidence of efficient calcium deposit ruptures in human calcified leaflets treated ex vivo and the safety of the treatment in pigs. The use of this device could be helpful to perform shockwaves valvuloplasty as an option to retard TAVR/SAVR, or as a pretreatment to facilitate prosthesis implantation and minimize the occurrence of paravalvular leak.

Animal models and animal-free innovations for cardiovascular research: current status and routes to be explored. Consensus document of the ESC Working Group on Myocardial Function and the ESC Working Group on Cellular Biology of the Heart.

Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies, all of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task, in particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and comorbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models do not represent a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on an organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and state-of-the-art animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction, and refinement (3R) as a guiding concept.

Circadian rhythms in ischaemic heart disease: key aspects for preclinical and translational research: position paper of the ESC working group on cellular biology of the heart.

Circadian rhythms are internal regulatory processes controlled by molecular clocks present in essentially every mammalian organ that temporally regulate major physiological functions. In the cardiovascular system, the circadian clock governs heart rate, blood pressure, cardiac metabolism, contractility, and coagulation. Recent experimental and clinical studies highlight the possible importance of circadian rhythms in the pathophysiology, outcome, or treatment success of cardiovascular disease, including ischaemic heart disease. Disturbances in circadian rhythms are associated with increased cardiovascular risk and worsen outcome. Therefore, it is important to consider circadian rhythms as a key research parameter to better understand cardiac physiology/pathology, and to improve the chances of translation and efficacy of cardiac therapies, including those for ischaemic heart disease. The aim of this Position Paper by the European Society of Cardiology Working Group Cellular Biology of the Heart is to highlight key aspects of circadian rhythms to consider for improvement of preclinical and translational studies related to ischaemic heart disease and cardioprotection. Applying these considerations to future studies may increase the potential for better translation of new treatments into successful clinical outcomes.

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.

Carbon Nanotubes Substrates Alleviate Pro-Calcific Evolution in Porcine Valve Interstitial Cells.

The quest for surfaces able to interface cells and modulate their functionality has raised, in recent years, the development of biomaterials endowed with nanocues capable of mimicking the natural extracellular matrix (ECM), especially for tissue regeneration purposes. In this context, carbon nanotubes (CNTs) are optimal candidates, showing dimensions and a morphology comparable to fibril ECM constituents. Moreover, when immobilized onto surfaces, they demonstrated outstanding cytocompatibility and ease of chemical modification with ad hoc functionalities. In this study, we interface porcine aortic valve interstitial cells (pVICs) to multi-walled carbon nanotube (MWNT) carpets, investigating the impact of surface nano-morphology on cell properties. The results obtained indicate that CNTs significantly affect cell behavior in terms of cell morphology, cytoskeleton organization, and mechanical properties. We discovered that CNT carpets appear to maintain interfaced pVICs in a sort of "quiescent state", hampering cell activation into a myofibroblasts-like phenotype morphology, a cellular evolution prodromal to Calcific Aortic Valve Disease (CAVD) and characterized by valve interstitial tissue stiffening. We found that this phenomenon is linked to CNTs' ability to alter cell tensional homeostasis, interacting with cell plasma membranes, stabilizing focal adhesions and enabling a better strain distribution within cells. Our discovery contributes to shedding new light on the ECM contribution in modulating cell behavior and will open the door to new criteria for designing nanostructured scaffolds to drive cell functionality for tissue engineering applications.

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.

COVID-19-related cardiac complications from clinical evidences to basic mechanisms: opinion paper of the ESC Working Group on Cellular Biology of the Heart.

The pandemic of coronavirus disease (COVID)-19 is a global threat, causing high mortality, especially in the elderly. The main symptoms and the primary cause of death are related to interstitial pneumonia. Viral entry also into myocardial cells mainly via the angiotensin converting enzyme type 2 (ACE2) receptor and excessive production of pro-inflammatory cytokines, however, also make the heart susceptible to injury. In addition to the immediate damage caused by the acute inflammatory response, the heart may also suffer from long-term consequences of COVID-19, potentially causing a post-pandemic increase in cardiac complications. Although the main cause of cardiac damage in COVID-19 remains coagulopathy with micro- (and to a lesser extent macro-) vascular occlusion, open questions remain about other possible modalities of cardiac dysfunction, such as direct infection of myocardial cells, effects of cytokines storm, and mechanisms related to enhanced coagulopathy. In this opinion paper, we focus on these lesser appreciated possibilities and propose experimental approaches that could provide a more comprehensive understanding of the cellular and molecular bases of cardiac injury in COVID-19 patients. We first discuss approaches to characterize cardiac damage caused by possible direct viral infection of cardiac cells, followed by formulating hypotheses on how to reproduce and investigate the hyperinflammatory and pro-thrombotic conditions observed in the heart of COVID-19 patients using experimental in vitro systems. Finally, we elaborate on strategies to discover novel pathology biomarkers using omics platforms.