Elastin-like recombinamers-based hydrogel modulates the post-ischemic remodelling in a clinically-relevant model of myocardial infarction

Myocardial infarction (MI) belongs to the family of cardiovascular diseases and represents the primary cause of worldwide mortality. Despite the promising results achieved in both small and large animal studies, recent meta-analyses highlighted the marginal effect of therapeutic strategies like bone-marrow derived stem cells once they are tested in the clinical trials. Mammalian adult cardiomyocytes are able to re-enter the cell-cycle only during the first week after birth, thus avoiding the maladaptive remodelling which characterises post-ischemic phases in the adult. In this thesis a study included the identification of significant differences in the cellular membrane glycosylation from tissue samples harvested from neonatal and adult rats. Increasing evidence has shown the crucial role that components of the extracellular matrix (ECM) can play to modulate the post-ischemic remodelling. Elastin is a natural component of the cardiac ECM and can be mimicked by genetic engineering through the production of elastin-like recombinamers (ELRs). In this thesis, two ELRs named HE5 and HRGD which contain matrix metalloproteinase-responsive and cell-adhesive sequences, respectively, were used to fabricate an acellular hydrogel. This hydrogel, lacking growth factor bioactivity, was applied in this MI model by multiple intramyocardial injections at seven days post-MI and its effects evaluated after 21 days. The ischemic core in the ELRs-treated group showed reduced fibrosis and enhanced angiogenesis. Complete functional recovery in the ejection fraction 21 days after the ELRs-hydrogel injections distinguished this group from the stable impaired behaviour in untreated animals. High-throughput analyses at expression, protein and glycan level were performed, and an enhanced preservation of GATA4+ cardiomyocytes in the border zone of the infarct was observed. The combined effect of a modulated extracellular matrix in the ischemic core and preserved cardiomyocytes in the border zone are likely to be responsible for the marked functional recovery.2021-04-1

Elastin-like recombinamers-based hydrogel modulates the post-ischemic remodelling in a clinically-relevant model of myocardial infarction

Myocardial infarction (MI) belongs to the family of cardiovascular diseases and represents the primary cause of worldwide mortality. Despite the promising results achieved in both small and large animal studies, recent meta-analyses highlighted the marginal effect of therapeutic strategies like bone-marrow derived stem cells once they are tested in the clinical trials. Mammalian adult cardiomyocytes are able to re-enter the cell-cycle only during the first week after birth, thus avoiding the maladaptive remodelling which characterises post-ischemic phases in the adult. In this thesis a study included the identification of significant differences in the cellular membrane glycosylation from tissue samples harvested from neonatal and adult rats. Increasing evidence has shown the crucial role that components of the extracellular matrix (ECM) can play to modulate the post-ischemic remodelling. Elastin is a natural component of the cardiac ECM and can be mimicked by genetic engineering through the production of elastin-like recombinamers (ELRs). In this thesis, two ELRs named HE5 and HRGD which contain matrix metalloproteinase-responsive and cell-adhesive sequences, respectively, were used to fabricate an acellular hydrogel. This hydrogel, lacking growth factor bioactivity, was applied in this MI model by multiple intramyocardial injections at seven days post-MI and its effects evaluated after 21 days. The ischemic core in the ELRs-treated group showed reduced fibrosis and enhanced angiogenesis. Complete functional recovery in the ejection fraction 21 days after the ELRs-hydrogel injections distinguished this group from the stable impaired behaviour in untreated animals. High-throughput analyses at expression, protein and glycan level were performed, and an enhanced preservation of GATA4+ cardiomyocytes in the border zone of the infarct was observed. The combined effect of a modulated extracellular matrix in the ischemic core and preserved cardiomyocytes in the border zone are likely to be responsible for the marked functional recovery.2021-04-1

Therapies to prevent post-infarction remodelling: From repair to regeneration

Myocardial infarction is the first cause of worldwide mortality, with an increasing incidence also reported in developing countries. Over the past decades, preclinical research and clinical trials continually tested the efficacy of cellular and acellular-based treatments. However, none of them resulted in a drug or device currently used in combination with either percutaneous coronary intervention or coronary artery bypass graft. Inflammatory, proliferation and remodelling phases follow the ischaemic event in the myocardial tissue. Only recently, singlecell sequencing analyses provided insights into the specific cell populations which determine the final fibrotic deposition in the affected region. In this review, ischaemia, inflammation, fibrosis, angiogenesis, cellular stress and fundamental cellular and molecular components are evaluated as therapeutic targets. Given the emerging evidence of biomaterial-based systems, the increasing use of injectable hydrogels/scaffolds and epicardial patches is reported both as acellular and cellularised/functionalised treatments. Since several variables influence the outcome of any experimented treatment, we return to the pathological basis with an unbiased view towards any specific process or cellular component. Thus, by evaluating the benefits and limitations of the approaches based on these targets, the reader can weigh the rationale of each of the strategies that reached the clinical trials stage. As recent studies focused on the relevance of the extracellular matrix in modulating ischaemic remodelling and enhancing myocardial regeneration, we aim to portray current trends in the field with this review. Finally, approaches towards feasible translational studies that are as yet unexplored are also suggested

YAP/TAZ as master regulators in cancer: modulation, function and therapeutic approaches

Our understanding of the function of the transcriptional regulators YAP and TAZ (YAP/TAZ) in cancer is advancing. In this Review, we provide an update on recent progress in YAP/TAZ biology, their regulation by Hippo signaling and mechanotransduction and highlight open questions. YAP/ TAZ signaling is an addiction shared by multiple tumor types and their microenvironments, providing many malignant attributes. As such, it represents an important vulnerability that may offer a broad window of therapeutic efficacy, and here we give an overview of the current treatment strategies and pioneering clinical trials

Understanding How Heart Metabolic Derangement Shows Differential Stage Specificity for Heart Failure with Preserved and Reduced Ejection Fraction

Heart failure (HF) is a clinical condition defined by structural and functional abnormalities in the heart that gradually result in reduced cardiac output (HFrEF) and/or increased cardiac pressures at rest and under stress (HFpEF). The presence of asymptomatic individuals hampers HF identification, resulting in delays in recognizing patients until heart dysfunction is manifested, thus increasing the chance of poor prognosis. Given the recent advances in metabolomics, in this review we dissect the main alterations occurring in the metabolic pathways behind the decrease in cardiac function caused by HF. Indeed, relevant preclinical and clinical research has been conducted on the metabolite connections and differences between HFpEF and HFrEF. Despite these promising results, it is crucial to note that, in addition to identifying single markers and reliable threshold levels within the healthy population, the introduction of composite panels would strongly help in the identification of those individuals with an increased HF risk. That said, additional research in the field is required to overcome the current drawbacks and shed light on the pathophysiological changes that lead to HF. Finally, greater collaborative data sharing, as well as standardization of procedures and approaches, would enhance this research field to fulfil its potential

An optimized protocol for combined fluorescent lectin/immunohistochemistry to characterize tissue-specific glycan distribution in human or rodent tissues

: Lectin histochemical analysis of tissues combined with immunohistochemistry is a valuable tool to characterize and correlate the spatial distribution of glycans with the presence of specific cell types or antigens of interest. The current protocol describes the application of monosaccharide motif specificity of lectin binding to glycan residues to different tissue types. In addition, we describe stereological methods to provide further quantification of the analyzed tissues. For complete details on the use and execution of this protocol, please refer to Mohd Isa et al. (2018), Contessotto et al. (2020), and Samal et al. (2020)

Synthetic/ECM-inspired hybrid platform for hollow microcarriers with ROS-triggered nanoporation hallmarks

Reactive oxygen species (ROS) are key pathological signals expressed in inflammatory diseases such as cancer, ischemic conditions and atherosclerosis. An ideal drug delivery system should not only be responsive to these signals but also should not elicit an unfavourable host response. This study presents an innovative platform for drug delivery where a natural/synthetic composite system composed of collagen type I and a synthesized polythioether, ensures a dual stimuli-responsive behaviour. Collagen type I is an extracellular matrix constituent protein, responsive to matrix metalloproteinases (MMP) cleavage per se. Polythioethers are stable synthetic polymers characterized by the presence of sulphur, which undergoes a ROS-responsive swelling switch. A polythioether was synthesised, functionalized and tested for cytotoxicity. Optimal conditions to fabricate a composite natural/synthetic hollow sphere construct were optimised by a template-based method. Collagen-polythioether hollow spheres were fabricated, revealing uniform size and ROS-triggered nanoporation features. Cellular metabolic activity of H9C2 cardiomyoblasts remained unaffected upon exposure to the spheres. Our natural/synthetic hollow microspheres exhibit the potential for use as a pathological stimuli-responsive reservoir system for applications in inflammatory diseases.This material is based upon works supported by the European Union funding under the AngioMatTrain 7th Framework Programme, Grant Agreement Number 317304. This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant Number 13/RC/2073. The authors acknowledge the use of the facilities and the scientific and technical assistance of the Centre for Microscopy and Imaging at the National University of Ireland Galway (www.imagingnuigalway.ie), a facility that is co-funded by the Irish Government’s Programme for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007–2013

Distinct glycosylation in membrane proteins within neonatal versus adult myocardial tissue

Mammalian hearts have regenerative potential restricted to early neonatal stage and lost within seven days after birth. Carbohydrates exclusive to cardiac neonatal tissue may be key regulators of regenerative potential. Although cell surface and extracellular matrix glycosylation are known modulators of tissue and cellular function and development, variation in cardiac glycosylation from neonatal tissue to maturation has not been fully examined.In this study, glycosylation of the adult rat cardiac ventricle showed no variability between the two strains analysed, nor were there any differences between the glycosylation of the right or left ventricle using lectin histochemistry and microarray profiling. However, in the Sprague-Dawley strain, neonatal cardiac glycosylation in the left ventricle differed from adult tissues using mass spectrometric analysis, showing a higher expression of high mannose structures and lower expression of complex N-linked glycans in the three-day-old neonatal tissue. Man(6)GlcNAc(2) was identified as the main high mannose N-linked structure that was decreased in adult while higher expression of sialylated N-linked glycans and lower core fucosylation for complex structures were associated with ageing. The occurrence of mucin core type 2 O-linked glycans was reduced in adult and one sulfated core type 2 O-linked structure was identified in neonatal tissue. Interestingly, O-linked glycans from mature tissue contained both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), while all sialylated N-linked glycans detected contained only Neu5Ac.As glycans are associated with intracellular communication, the specific neonatal structures found may indicate a role for glycosylation in the neonatal associated regenerative capacity of the mammalian heart. New strategies targeting tissue glycosylation could be a key contributor to achieve an effective regeneration of the mammalian heart in pathological scenarios such as myocardial infarction. (C) 2019 The Author(s). Published by Elsevier B.V

Broadly Applicable Hydrogel Fabrication Procedures Guided by YAP/TAZ-Activity Reveal Stiffness, Adhesiveness, and Nuclear Projected Area as Checkpoints for Mechanosensing

Mechanical signals are pivotal ingredients in how cells perceive and respond to their microenvironments, and to synthetic biomaterials that mimic them. In spite of increasing interest in mechanobiology, probing the effects of physical cues on cell behavior remains challenging for a cell biology laboratory without experience in fabrication of biocompatible materials. Hydrogels are ideal biomaterials recapitulating the physical cues that natural Extracellular Matrices (ECM) delivers to cells. Here we streamlined protocols for the synthesis and functionalization of cell adhesive Polyacrylamide-based (PAA-OH) and fully-defined Polyethyleneglycol-based (PEG-RGD) hydrogels tuned at various rigidities for mechanobiology experiments, from 0.3 to >10kPa. We investigated the mechanosignaling properties of these hydrogels in distinct cell types by monitoring the activation state of YAP/TAZ. By independently modulating substrate stiffness and adhesiveness, we found that although ECM stiffness represents an overarching mechanical signal, the density of adhesive sites does impact on cellular mechanosignaling at least at intermediate rigidity values, corresponding to normal and pathological states of living tissues. Using these tools, we found that YAP/TAZ nuclear accumulation occurs when the projected area of the nucleus surpasses a critical threshold of approximatively 150 μm(2). This work suggests the existence of distinct checkpoints for cellular mechanosensing