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Background and Purpose: MicroRNA (miR)‐210 function in endothelial cells and its role in diabetes‐associated endothelial dysfunction are not fully understood. We aimed to characterize the miR‐210 function in endothelial cells and study its therapeutic potential in diabetes. Experimental Approach: Two different diabetic mouse models (db/db and Western diet‐induced), miR‐210 knockout and transgenic mice, isolated vessels and human endothelial cells were used. Key Results: miR‐210 levels were lower in aortas isolated from db/db than in control mice. Endothelium‐dependent relaxation (EDR) was impaired in aortas from miR‐210 knockout mice, and this was restored by inhibiting miR‐210 downstream protein tyrosine phosphatase 1B (PTP1B), mitochondrial glycerol‐3‐phosphate dehydrogenase 2 (GPD2), and mitochondrial oxidative stress. Inhibition of these pathways also improved EDR in both diabetic mouse models. High glucose reduced miR‐210 levels in endothelial cells and impaired EDR in mouse aortas, effects that were reversed by overexpressing miR‐210. However, plasma miR‐210 levels were not affected in individuals with type 2 diabetes (T2D) following improved glycaemic status. Of note, genetic overexpression using miR‐210 transgenic mice and pharmacological overexpression using miR‐210 mimic in vivo ameliorated endothelial dysfunction in both diabetic mouse models by decreasing PTP1B, GPD2 and oxidative stress. Genetic overexpression of miR‐210 altered the aortic transcriptome, decreasing genes in pathways involved in oxidative stress. miR‐210 mimic restored decreased nitric oxide production by high glucose in endothelial cells. Conclusion and Implications: This study unravels the mechanisms by which down‐regulated miR‐210 by high glucose induces endothelial dysfunction in T2D and demonstrates that miR‐210 serves as a novel therapeutic target. LINKED ARTICLES: This article is part of a themed issue Non‐coding RNA Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.2/issuetoc [ABSTRACT FROM AUTHOR]

Abstract Hypoxia-induced miR-210 is a crucial component of the tissue response to ischemia, stimulating angiogenesis and improving tissue regeneration. Previous analysis of miR-210 impact on the transcriptome in a mouse model of hindlimb ischemia showed that miR-210 regulated not only vascular regeneration functions, but also inflammation. To investigate this event, doxycycline-inducible miR-210 transgenic mice (Tg-210) and anti-miR-210 LNA-oligonucleotides were used. It was found that global miR-210 expression decreased inflammatory cells density and macrophages accumulation in the ischemic tissue. To dissect the underpinning cell mechanisms, Tg-210 mice were used in bone marrow (BM) transplantation experiments and chimeric mice underwent hindlimb ischemia. MiR-210 overexpression in the ischemic tissue was sufficient to increase capillary density and tissue repair, and to reduce inflammation in the presence of Wt-BM infiltrating cells. Conversely, when Tg-210-BM cells migrated in a Wt ischemic tissue, dysfunctional angiogenesis, inflammation, and impaired tissue repair, accompanied by fibrosis were observed. The fibrotic regions were positive for α-SMA, Vimentin, and Collagen V fibrotic markers and for phospho-Smad3, highlighting the activation of TGF-β1 pathway. Identification of Tg-210 cells by in situ hybridization showed that BM-derived cells contributed directly to fibrotic areas, where macrophages co-expressing fibrotic markers were observed. Cell cultures of Tg-210 BM-derived macrophages exhibited a pro-fibrotic phenotype and were enriched with myofibroblast-like cells, which expressed canonical fibrosis markers. Interestingly, inhibitors of TGF-β type-1-receptor completely abrogated this pro-fibrotic phenotype. In conclusion, a context-dependent regulation by miR-210 of the inflammatory response was identified. miR-210 expression in infiltrating macrophages is associated to improved angiogenesis and tissue repair when the ischemic recipient tissue also expresses high levels of miR-210. Conversely, when infiltrating an ischemic tissue with mismatched miR-210 levels, macrophages expressing high miR-210 levels display a pro-fibrotic phenotype, leading to impaired tissue repair, fibrosis, and dysfunctional angiogenesis.

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133+ cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133+ cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133+ cells. Validation of CD133+ cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as ‘proofs of principle’ of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133+ cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133+ cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies. [ABSTRACT FROM AUTHOR]

Objective: The antiapoptotic effect of p21Waf1/Cip1/Sdi1 (p21) was examined in human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress (SS) or to the nitric oxide donor sodium nitroprusside (SNP) and in a mouse model of hindlimb ischemia. Methods: In vitro: Cells were cultured without serum and in the presence of cobalt chloride to simulate hypoxia for 12 h (T0). Shear stress was applied to endothelial cells for additional 12 h. In vivo: Hindlimb ischemia was realized in mice by femoral artery ligation. SNP was acutely administered by subcutaneous injection or by Alzet osmotic pumps for a longer treatment. Results: At T0, HUVEC were either exposed to SS (15 dyn/cm2/s−1), treated with SNP or kept in static condition (ST) for 1–12 h; after additional 12 h in ST, 30–35% of cells still alive at T0 had died. In this condition, both SS and SNP treatments markedly increased p21 levels and reduced apoptosis in HUVEC. Recombinant adenoviruses carrying p21 (AdCMV.p21) or antisense p21 (AdCMV.ASp21) cDNA revealed that AdCMV.p21-infected HUVEC were protected from death while AdCMV.ASp21 reduced SS- and SNP-dependent protection from apoptosis. In mice, apoptosis was detected in endothelial cells of ischemic hindlimbs as early as 8 h after femoral artery ligation. Treatment with SNP enhanced p21 expression and protected ischemic tissue from damage. Remarkably, direct in vivo injection of AdCMV.p21 significantly reduced the number of apoptotic nuclei in the presence of ischemia. Conclusions: The present study establishes that, under our experimental conditions, (a) p21 plays an important role in SS and nitric oxide antiapoptotic effect in vitro, and (b) p21 gene transfer prevents apoptosis in vitro and in vivo, following acute interruption of blood flow. [Copyright &y& Elsevier]

Critical limb ischemia is the most serious form of peripheral artery disease, characterized by severe functional consequences, difficult clinical management and reduced life expectancy. The goal of this study was to investigate the miR-210 role in the neo-angiogenic response after acute limb ischemia. Complementary approaches were used in a mouse model of hindlimb ischemia: miR-210 loss-of-function was obtained by administration of LNA-oligonucleotides anti-miR-210; for miR-210 gain-of-function, a doxycycline-inducible miR-210 transgenic mouse was used. We tested miR-210 ability to stimulate vascular regeneration following ischemia. We found that miR-210 was necessary and sufficient to stimulate blood perfusion recovery, as well as arteriolar and capillary density increase, in the ischemic muscle. To clarify the molecular events underpinning miR-210 pro-angiogenic action, the transcriptomic changes in ischemic muscles upon miR-210 blocking were analyzed. We found that miR-210 impacted the transcriptome significantly, regulating pathways and functions linked to vascular regeneration. In agreement with a pro-angiogenic role, miR-210 also improved cardiac function and left ventricular remodeling after myocardial infarction. Moreover, miR-210 blocking decreased capillary density in a Matrigel plug assay, indicating that miR-210 is necessary for angiogenesis independently of ischemia. Collectively, these data indicate that miR-210 plays a pivotal role in promoting vascular regeneration. [ABSTRACT FROM AUTHOR]

Background: Myotonic dystrophy type 1 (DM1) is a genetic multisystemic disease, characterised by pleiotropic symptoms that exhibit notable variability in severity, nature and age of onset. The genetic cause of DM1 is the expansion of unstable CTG-repeats in the 3' untranslated region (UTR) of the DMPK gene, resulting in the accumulation of toxic CUG-transcripts that sequester RNA-binding proteins and form nuclear foci in DM1 affected tissues and, consequently, alter various cellular processes. Therapeutic gene editing for treatment of monogenic diseases is a powerful technology that could in principle remove definitively the disease-causing genetic defect. The precision and efficiency of the molecular mechanisms are still under investigation in view of a possible use in clinical practice., Methods: Here, we describe the application of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy to remove the CTG-expansion in the DMPK gene in a mouse model carrying the human transgene from a DM1 patient. To optimise the editing efficiency in vivo, we identified new tools that allowed to improve the expression levels and the activity of the CRISPR/Cas9 machinery. Newly designed guide RNA pairs were tested in DM1-patient derived cells before in vivo application. Edited cells were analysed to assess the occurrence of off-target and the accuracy of on-target genomic events. Gene editing-dependent and -independent mechanisms leading to decreased accumulation of the mutated DMPK transcripts were also evaluated., Results and Conclusion: Systemic delivery of CRISPR/Cas9 components in DM1 mice, through myotropic adeno-associated viral vectors, led to significant improvement of molecular alterations in the heart and skeletal muscle. Importantly, a persistent increase of body weight, improvement of muscle strength and body composition parameters were observed in treated animals. Accurate evaluation of CRISPR/Cas9-mediated-phenotypic recovery in vivo is a crucial preclinical step for the development of a gene therapy for DM1 patients., Key Points: In vivo application of a therapeutic gene editing strategy for permanent deletion of the pathogenetic CTG-repeat amplification in the DMPK gene that causes myotonic dystrophy type 1. Following treatment, diseased mice show a significant improvement of both molecular and phenotypic defects., (© 2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background: Oxidative stress plays a pivotal role in ischemia and ischemia/reperfusion injury. Because p66(ShcA)-null (p66(ShcA)-/-) mice exhibit both lower levels of intracellular reactive oxygen species and increased resistance to cell death induced by oxidative stress, we investigated whether tissue damage that follows acute ischemia or ischemia/reperfusion was altered in p66(ShcA)-/- mice., Methods and Results: Unilateral hindlimb ischemia was induced by femoral artery dissection, and ischemia/reperfusion was induced with an elastic tourniquet. Both procedures caused similar changes in blood perfusion in p66(ShcA) wild-type (p66(ShcA)wt) and p66(ShcA)-/- mice. However, significant differences in tissue damage were found: p66(ShcA)wt mice displayed marked capillary density decrease and muscle fiber necrosis. In contrast, in p66(ShcA)-/- mice, minimal capillary density decrease and myofiber death were present. When apoptosis after ischemia was assayed, significantly lower levels of apoptotic endothelial cells and myofibers were found in p66(ShcA)-/- mice. In agreement with these data, both satellite muscle cells and endothelial cells isolated from p66(ShcA)-/- mice were resistant to apoptosis induced by simulated ischemia in vitro. Lower apoptosis levels after ischemia in p66(ShcA)-/- cells correlated with decreased levels of oxidative stress both in vivo and in vitro., Conclusions: p66(ShcA) plays a crucial role in the cell death pathways activated by acute ischemia and ischemia/reperfusion, indicating p66(ShcA) as a potential therapeutic target for prevention and treatment of ischemic tissue damage.

Objective: The antiapoptotic effect of p21(Waf1/Cip1/Sdi1) (p21) was examined in human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress (SS) or to the nitric oxide donor sodium nitroprusside (SNP) and in a mouse model of hindlimb ischemia., Methods: In vitro: Cells were cultured without serum and in the presence of cobalt chloride to simulate hypoxia for 12 h (T0). Shear stress was applied to endothelial cells for additional 12 h. In vivo: Hindlimb ischemia was realized in mice by femoral artery ligation. SNP was acutely administered by subcutaneous injection or by Alzet osmotic pumps for a longer treatment., Results: At T0, HUVEC were either exposed to SS (15 dyn/cm2/s(-1)), treated with SNP or kept in static condition (ST) for 1-12 h; after additional 12 h in ST, 30-35% of cells still alive at T0 had died. In this condition, both SS and SNP treatments markedly increased p21 levels and reduced apoptosis in HUVEC. Recombinant adenoviruses carrying p21 (AdCMV.p21) or antisense p21 (AdCMV.ASp21) cDNA revealed that AdCMV.p21-infected HUVEC were protected from death while AdCMV.ASp21 reduced SS- and SNP-dependent protection from apoptosis. In mice, apoptosis was detected in endothelial cells of ischemic hindlimbs as early as 8 h after femoral artery ligation. Treatment with SNP enhanced p21 expression and protected ischemic tissue from damage. Remarkably, direct in vivo injection of AdCMV.p21 significantly reduced the number of apoptotic nuclei in the presence of ischemia., Conclusions: The present study establishes that, under our experimental conditions, (a) p21 plays an important role in SS and nitric oxide antiapoptotic effect in vitro, and (b) p21 gene transfer prevents apoptosis in vitro and in vivo, following acute interruption of blood flow.