Cell-Based Gene Therapies and Stem Cells for Regeneration of Ischemic Tissues

Coronary (CAD) and peripheral (PAD) artery disease are major causes of morbidity and mortality, requiring bypass surgery or angioplasty in approximately one million patients/year in the world (MERIT-HF Study Group, 1999). While collateral vessel formation as an alternative pathway for blood supply occurs in some of these patients, many do not form vascular networks adequate to compensate for the loss of the original blood supply (Hirsch et al., 2006). These patients might therefore benefit from stem cell transplantation therapies that would accelerate natural processes of postnatal collateral vessel formation, an approach referred to as therapeutic angiogenesis. On the other hand, recent seminal reports have indicated that the adult heart is self-healing and self-renewing. Specifically, these studies have demonstrated that there is a pool of resident cardiac stem cells (CSCs) that are clonogenic and multipotent and are capable of differentiating into new blood vessels or into new myocytes, and of cardiac progenitor cells (CPCs) (Marban, 2007). This suggests the possibility of using a therapeutic angiogenesis approach to complement other treatments (e.g., stem cell therapy) that facilitate myocardial repair. Such combined modalities may facilitate myocardial regeneration by inducing endogenous cardiac cells to migrate, differentiate, and proliferate in situ, replacing lost endothelial cells and cardiomyocytes (Urbaneket al., 2005). However, despite recent progress in applying the approaches of regenerative medicine to the treatment of such diseases, valid strategies aimed at repairing the infarcted heart and, in general, at treating end-organ ischemia continue to be elusive. Major obstacles are the difficulty in isolating and delivering stem cells that are specifically effective in myocardial repair, and in stimulating recruitment of endogenous stem cells to the ischemic tissue. To address these issues, there has been increasing focus on novel biotechnologies or pharmacological strategies to enhance the implantation of exogenous stem cells or to boost endogenous regeneration of myocardial tissue. By employing three fundamental “tools”, namely stem cells, biomaterials and growth factors (GFs) (Lavik & Langer, 2004; Mikos et al., 2006), such tissue engineering strategies may enhance the efficacy of stem cell therapy in several ways: by mobilizing endogenous stem/progenitor cells in vivo; by promoting cell proliferation and differentiation; and by augmenting cell engraftment and survival in the injured myocardium. In general, because of the short half-lives of GFs in the body and the necessity to deliver them to specific target sites, GF injections themselves do not always produce the anticipated therapeutic effect. At present, GF delivery in regenerative medicine basically relies upon two strategies: 1) delivery of the GF genes; 2) direct delivery of GFs by incorporating them into a vehicle. In the gene delivery approach, delivery of the GF gene may result in higher and more constant levels of protein produced, since the gene - rather than a degradable protein - is being delivered (Haastert & Grothe, 2007). Two major problems are associated however with this approach: 1) the complexity of cloning and integrating the gene into the target cells; 2) safety and efficiency of transduction. At present, there are insufficient well-controlled long-term studies in the preclinical area to make any conclusive statements about the clinical suitability/efficacy of gene delivery in humans. If resolved, cell-mediated synthesis of GFs should be associated with more efficient targeting of receptors and, consequently, a more robust and predictable approach in ischemic tissue regeneration

Is it the time of seno-therapeutics application in cardiovascular pathological conditions related to ageing?

It rates that in 2030, the cardiovascular diseases (CVD) will result in 40% of all deaths and rank as the leading cause. Thus, the research of appropriate therapies able to delay or retard their onset and progression is growing. Of particular interest is a new branch of the medical science, called anti-ageing medicine since CVD are the result of cardiovascular ageing. Senescent cells (SC) accumulate in cardiovascular system contributing to the onset of typical age-related cardiovascular conditions (i.e., atherosclerosis, medial aorta degeneration, vascular remodeling, stiffness). Such conditions progress in cardiovascular pathologies (i.e., heart failure, coronary artery disease, myocardial infarction, and aneurysms) by evocating the production of a proinflammatory and profibrotic senescence-associated secretory phenotype (SASP). Consequently, therapies able to specifically eliminate SC are in developing. The senotherapeutics represents an emerging anti-SC treatment, and comprises three therapeutic approaches: (a) molecules to selectively kill SC, defined senolytics; (b) compounds able in reducing evocated SC SASP, acting hence as SASP suppressors, or capable to change the senescent phenotype, called senomorphics; (c) inhibition of increase of the number of SC in the tissues. Here, it describes them and the emerging data about current investigations on their potential clinical application in CVD, stressing benefits and limitations, and suggesting potential solutions for applying them in near future as effective anti-CVD treatment

Transplantation of adipose tissue mesenchymal cells conjugated with VEGF-releasing microcarriers promotes repair in murine myocardial infarction

RATIONALE: Engraftment and survival of transplanted stem or stromal cells in the microenvironment of host tissues may be improved by combining such cells with scaffolds to delay apoptosis and enhance regenerative properties. OBJECTIVES: We examined whether poly(lactic-co-glycolic acid) (PLGA) pharmacologically active microcarriers (PAMs) releasing vascular endothelial growth factor (VEGF) enhance survival, differentiation and angiogenesis of adipose tissue-mesenchymal stromal cells (AT-MSCs). We analyzed the efficacy of transplanted AT-MSCs conjugated with PAMs in a murine model of acute myocardial infarction (AMI). METHODS: We used fibronectin-coated (empty) PAMs or VEGF-releasing PAMs covered with murine AT-MSCs. Twelve month-old C57 mice underwent coronary artery ligation (Lig) to induce AMI, and were randomized into 5 treatment groups: AMI control (saline 20 microL, n=7), AMI followed by intramyocardial injection with AT-MSCs (2.5x105 cells/20 microL, n=5), or concentrated medium from AT-MSCs (CM, 20 microL, n=8), or AT-MSCs (2.5x105 cells/20 microL) conjugated with empty PAMs (n=7), or VEGF-releasing PAMs (n=8). Sham-operated mice (n=7) were used as controls. RESULTS: VEGF-releasing PAMs increased proliferation and angiogenic potential of AT-MSCs, but did not impact their osteogenic or adipogenic differentiation. AT-MSCs conjugated with VEGF-releasing PAMs inhibited apoptosis, decreased fibrosis, increased arteriogenesis and the number of cardiac-resident Ki-67 positive cells, and improved myocardial fractional shortening compared with AT-MSCs alone when transplanted into the infarcted hearts of C57 mice. With the exception of fractional shortening, all such effects of AT-MSCs conjugated with VEGF-PAMs were paralleled by the injection of CM. CONCLUSIONS: AT-MSCs conjugated with VEGF-releasing PAMs exert paracrine effects that may have therapeutic applications

High glucose-induced hyperosmolarity impacts proliferation, cytoskeleton remodeling and migration of human induced pluripotent stem cells via aquaporin-1

Background and objective: Hyperglycemia leads to adaptive cell responses in part due to hyperosmolarity. In endothelial and epithelial cells, hyperosmolarity induces aquaporin-1 (AQP1) which plays a role in cytoskeletal remodeling, cell proliferation and migration. Whether such impairments also occur in human induced pluripotent stem cells (iPS) is not known. We therefore investigated whether high glucose-induced hyperosmolarity impacts proliferation, migration, expression of pluripotency markers and actin skeleton remodeling in iPS cells in an AQP1-dependent manner. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, c-Myc). After reprogramming, iPS cells were characterized by their adaptive responses to high glucose-induced hyperosmolarity by incubation with 5.5mmol/L glucose, high glucose (HG) at 30.5mM, or with the hyperosmolar control mannitol (HM). Exposure to either HG or HM increased the expression of AQP1. AQP1 co-immunoprecipitated with beta-catenin. HG and HM induced the expression of beta-catenin. Under these conditions, iPS cells showed increased ratios of F-actin to G-actin and formed increased tubing networks. Inhibition of AQP1 with small interfering RNA (siRNA) reverted the inducing effects of HG and HM. Conclusions: High glucose enhances human iPS cell proliferation and cytoskeletal remodeling due to hyperosmolarity-induced upregulation of AQP1

Impact of Sex Differences and Diabetes on Coronary Atherosclerosis and Ischemic Heart Disease

Cardiovascular diseases (CVD) including coronary artery disease (CAD) and ischemic heart disease (IHD) are the main cause of mortality in industrialized countries. Although it is well known that there is a difference in the risk of these diseases in women and men, current therapy does not consider the sexual dimorphism; i.e., differences in anatomical structures and metabolism of tissues. Here, we discuss how genetic, epigenetic, hormonal, cellular or molecular factors may explain the different CVD risk, especially in high-risk groups such as women with diabetes. We analyze whether sex may modify the effects of diabetes at risk of CAD. Finally, we discuss current diagnostic techniques in the evaluation of CAD and IHD in diabetic women

Co-expression of glycosylated aquaporin-1 and transcription factor NFAT5 contributes to aortic stiffness in diabetic and atherosclerosis-prone mice

Increased stiffness characterizes the early change in the arterial wall with subclinical atherosclerosis. Proteins inducing arterial stiffness in diabetes and hypercholesterolaemia are largely unknown. This study aimed at determining the pattern of protein expression in stiffening aorta of diabetic and hypercholesterolaemic mice. Male Ins2+/Akita mice were crossbred with ApoE-/- (Ins2+/Akita : ApoE-/- ) mice. Relative aortic distension (relD) values were determined by ultrasound analysis and arterial stiffness modulators by immunoblotting. Compared with age- and sex-matched C57/BL6 control mice, the aortas of Ins2+/Akita , ApoE-/- and Ins2+/Akita :ApoE-/- mice showed increased aortic stiffness. The aortas of Ins2+/Akita , ApoE-/- and Ins2+/Akita :ApoE-/- mice showed greater expression of VCAM-1, collagen type III, NADPH oxidase and iNOS, as well as reduced elastin, with increased collagen type III-to-elastin ratio. The aorta of Ins2+/Akita and Ins2+/Akita :ApoE-/- mice showed higher expression of eNOS and cytoskeletal remodelling proteins, such as F-actin and α-smooth muscle actin, in addition to increased glycosylated aquaporin (AQP)-1 and transcription factor NFAT5, which control the expression of genes activated by high glucose-induced hyperosmotic stress. Diabetic and hypercholesterolaemic mice have increased aortic stiffness. The association of AQP1 and NFAT5 co-expression with aortic stiffness in diabetes and hypercholesterolaemia may represent a novel molecular pathway or therapeutic target

Exogenous Nitric Oxide Protects Human Embryonic Stem Cell-Derived Cardiomyocytes against Ischemia/Reperfusion Injury

Background and Aims. Human embryonic stem cell- (hESC-) derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP) in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days) and then on more differentiated cardiomyocytes (6 + 24 days), both derived from hESCs. Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (10(-7), 10(-6), and 10(-5) M), BNP (10(-9), 10(-8), and 10(-7) M), and the nonspecific NO synthase inhibitor Nomega-nitro-L-arginine (L-NNA, 10(-5) M). Results. SNAP (10(-6), 10(-5) M) significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10(-6) M) also protected hESC-derived cardiomyocytes. Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms

Cardiac dysfunction in cancer patients : beyond direct cardiomyocyte damage of anticancer drugs. Novel cardio-oncology insights from the joint 2019 meeting of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions please email: [email protected] reviewedPostprin

Epigenomic and transcriptomic approaches in the post-genomic era: path to novel targets for diagnosis and therapy of the ischemic heart?

Despite advances in myocardial reperfusion therapies, acute myocardial ischemia/reperfusion injury and consequent ischemic heart failure represent the number one cause of morbidity and mortality in industrialized societies. Although different therapeutic interventions have been shown beneficial in preclinical settings, an effective cardioprotective or regenerative therapy has yet to be successfully introduced in the clinical arena. Given the complex pathophysiology of the ischemic heart, large scale, unbiased, global approaches capable of identifying multiple branches of the signaling networks activated in the ischemic/reperfused heart might be more successful in the search for novel diagnostic or therapeutic targets. High-throughput techniques allow high-resolution, genome-wide investigation of genetic variants, epigenetic modifications and associated gene expression profiles. Platforms such as proteomics and metabolomics (not described here in detail) also offer simultaneous readouts of hundreds of proteins and metabolites. Isolated omics analyses usually provide Big Data requiring large data storage, advanced computational resources and complex bioinformatics tools. The possibility of integrating different omics approaches gives new hope to better understand the molecular circuitry activated by myocardial ischemia, putting it in the context of the human "diseasome".Since modifications of cardiac gene expression have been consistently linked to pathophysiology of the ischemic heart, the integration of epigenomic and transcriptomic data seems a promising approach to identify crucial disease networks. Thus, the scope of this Position Paper will be to highlight potentials and limitations of these approaches, and to provide recommendations to optimize the search for novel diagnostic or therapeutic targets for acute ischemia/reperfusion injury and ischemic heart failure in the post-genomic era