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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.

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.

Therapies based on circulating proangiogenic cells (PACs) have shown promise in ischemic disease models but require further optimization to reach the bedside. Ischemia-associated hypoxia robustly increases microRNA-210 (miR-210) expression in several cell types, including endothelial cells (ECs). In ECs, miR-210 represses EphrinA3 (EFNA3), inducing proangiogenic responses. This study provides new mechanistic evidences for a role of miR-210 in PACs. PACs were obtained from either adult peripheral blood or cord blood. miR-210 expression was modulated with either an inhibitory complementary oligonucleotide (anti-miR-210) or a miRNA mimic (pre-miR-210). Scramble and absence of transfection served as controls. As expected, hypoxia increased miR-210 in PACs. In vivo, migration toward and adhesion to the ischemic endothelium facilitate the proangiogenic actions of transplanted PACs. In vitro, PAC migration toward SDF-1α/CXCL12 was impaired by anti-miR-210 and enhanced by pre-miR-210. Moreover, pre-miR-210 increased PAC adhesion to ECs and supported angiogenic responses in co-cultured ECs. These responses were not associated with changes in extracellular miR-210 and were abrogated by lentivirus-mediated EFNA3 overexpression. Finally, ex-vivo pre-miR-210 transfection predisposed PACs to induce post-ischemic therapeutic neovascularization and blood flow recovery in an immunodeficient mouse limb ischemia model. In conclusion, miR-210 modulates PAC functions and improves their therapeutic potential in limb ischemia., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Hypoxia-induced miR-210 displays a pro-survival, cytoprotective and pro-angiogenic role in several in vitro systems. In vivo, we previously found that miR-210 inhibition increases ischemic damage. Here we describe the generation of a versatile transgenic mouse model allowing the evaluation of miR-210 therapeutic potential in ischemic cardiovascular diseases. We generated a Tet-On miR-210 transgenic mouse strain (TG-210) by targeted transgenesis in the ROSA26 locus. To functionally validate miR-210 transgenic mice, hindlimb ischemia was induced by femoral artery dissection. Blood perfusion was evaluated by power Doppler while tissue damage and inflammation were assessed by histological evaluation. We found that miR-210 levels were rapidly increased in TG-210 mice upon doxycycline administration. miR-210 overexpression was maintained over time and remained within physiological levels in multiple tissues. When hindlimb ischemia was induced, miR-210 overexpression protected from both muscular and vascular ischemic damage, decreased inflammatory cells density and allowed to maintain a better calf perfusion. In conclusion, we generated and functionally validated a miR-210 transgenic mouse model. Albeit validated in the context of a specific cardiovascular ischemic disease, miR-210 transgenic mice may also represent a useful model to assess the function of miR-210 in other physio-pathological conditions.

Magnetic resonance imaging (MRI) provides non-invasive, repetitive measures in the same individual, allowing the study of a physio-pathological event over time. In this study, we tested the performance of 7 Tesla multi-parametric MRI to monitor the dynamic changes of mouse skeletal muscle injury and regeneration upon acute ischemia induced by femoral artery dissection. T2-mapping (T2 relaxation time), diffusion-tensor imaging (Fractional Anisotropy) and perfusion by Dynamic Contrast-Enhanced MRI (K-trans) were measured and imaging results were correlated with histological morphometric analysis in both Gastrocnemius and Tibialis anterior muscles. We found that tissue damage positively correlated with T2-relaxation time, while myofiber regeneration and capillary density positively correlated with Fractional Anisotropy. Interestingly, K-trans positively correlated with capillary density. Accordingly, repeated MRI measurements between day 1 and day 28 after surgery in ischemic muscles showed that: 1) T2-relaxation time rapidly increased upon ischemia and then gradually declined, returning almost to basal level in the last phases of the regeneration process; 2) Fractional Anisotropy dropped upon ischemic damage induction and then recovered along with muscle regeneration and neoangiogenesis; 3) K-trans reached a minimum upon ischemia, then progressively recovered. Overall, Gastrocnemius and Tibialis anterior muscles displayed similar patterns of MRI parameters dynamic, with more marked responses and less variability in Tibialis anterior. We conclude that MRI provides quantitative information about both tissue damage after ischemia and the subsequent vascular and muscle regeneration, accounting for the differences between subjects and, within the same individual, between different muscles.

Aims: Peripheral artery disease is caused by the restriction or occlusion of arteries supplying the leg. Better understanding of the molecular mechanisms underpinning tissue response to ischemia is urgently needed to improve therapeutic options. The aim of this study is to investigate hypoxia-induced miR-210 regulation and its role in a mouse model of hindlimb ischemia., Results: miR-210 expression was induced by femoral artery dissection. To study the role of miR-210, its function was inhibited by the systemic administration of a miR-210 complementary locked nucleic acid (LNA)-oligonucleotide (anti-miR-210). In the ischemic skeletal muscle, anti-miR-210 caused a marked decrease of miR-210 compared with LNA-scramble control, while miR-210 target expression increased accordingly. Histological evaluation of acute tissue damage showed that miR-210 inhibition increased both apoptosis at 1 day and necrosis at 3 days. Capillary density decrease caused by ischemia was significantly more pronounced in anti-miR-210-treated mice; residual limb perfusion decreased accordingly. To investigate the molecular mechanisms underpinning the increased damage triggered by miR-210 blockade, we tested the impact of anti-miR-210 treatment on the transcriptome. Gene expression analysis highlighted the deregulation of mitochondrial function and redox balance. Accordingly, oxidative damage was more severe in the ischemic limb of anti-miR-210-treated mice and miR-210 inhibition increased oxidative metabolism. Further, oxidative-stress resistant p66(Shc)-null mice displayed decreased tissue damage following ischemia., Innovation: This study identifies miR-210 as a crucial element in the adaptive mechanisms to acute peripheral ischemia., Conclusions: The physiopathological significance of miR-210 is context dependent. In the ischemic skeletal muscle it seems to be cytoprotective, regulating oxidative metabolism and oxidative stress.

The aim of this work was to identify micro-RNAs (miRNAs) involved in the pathological pathways activated in skeletal muscle damage and regeneration by both dystrophin absence and acute ischemia. Eleven miRNAs were deregulated both in MDX mice and in Duchenne muscular dystrophy patients (DMD signature). Therapeutic interventions ameliorating the mdx-phenotype rescued DMD-signature alterations. The significance of DMD-signature changes was characterized using a damage/regeneration mouse model of hind-limb ischemia and newborn mice. According to their expression, DMD-signature miRNAs were divided into 3 classes. 1) Regeneration miRNAs, miR-31, miR-34c, miR-206, miR-335, miR-449, and miR-494, which were induced in MDX mice and in DMD patients, but also in newborn mice and in newly formed myofibers during postischemic regeneration. Notably, miR-206, miR-34c, and miR-335 were up-regulated following myoblast differentiation in vitro. 2) Degenerative-miRNAs, miR-1, miR-29c, and miR-135a, that were down-modulated in MDX mice, in DMD patients, in the degenerative phase of the ischemia response, and in newborn mice. Their down-modulation was linked to myofiber loss and fibrosis. 3) Inflammatory miRNAs, miR-222 and miR-223, which were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. These findings show an important role of miRNAs in physiopathological pathways regulating muscle response to damage and regeneration.

Oxidative stress plays a pivotal role in ischemic injury, and p66(ShcA)ko mice exhibit both lower oxidative stress and decreased tissue damage following hind limb ischemia. Thus, it was investigated whether tissue regeneration following acute hind limb ischemia was altered in p66(ShcA)ko mice. Upon femoral artery dissection, muscle regeneration started earlier and was completed faster than in wild-type (WT) control. Moreover, faster regeneration was associated with decreased oxidative stress. Unlike ischemia, cardiotoxin injury induced similar skeletal muscle damage in both genotypes. However, p66(ShcA)ko mice regenerated faster, in agreement with the regenerative advantage upon ischemia. Since no difference between p66(ShcA)wt and knock-out (ko) mice was found in blood perfusion recovery after ischemia, satellite cells (SCs), a resident population of myogenic progenitors, were examined. Similar SCs numbers were present in WT and ko mice. However, in vitro cultured p66(ShcA)ko SCs displayed lower oxidative stress levels and higher proliferation rate and differentiated faster than WT. Furthermore, when exposed to sublethal H(2)O(2) doses, p66(ShcA)ko SCs were resistant to H(2)O(2)-induced inhibition of differentiation. Finally, myogenic conversion induced by MyoD overexpression was more efficient in p66(ShcA)ko fibroblasts compared with WT. The present work demonstrates that oxidative stress and p66(ShcA) play a crucial role in the regenerative pathways activated by acute ischemia.

Telomere dysfunction contributes to reduced cell viability, altered differentiation, and impaired regenerative/proliferative responses. Recent advances indicate that telomerase activity confers a pro-angiogenic phenotype to endothelial cells and their precursors. We have investigated whether telomerase contributes to tissue regeneration following hind limb ischemia and vascular endothelial growth factor 165 (VEGF(165)) treatment. VEGF delivery induced angiogenesis and increased expression of the telomerase reverse transcriptase (TERT) and telomerase activity in skeletal muscles and satellite and endothelial cells. Adenovirus-mediated transfer of wild type TERT but not of a dominant negative mutant, TERTdn, significantly induced capillary but not arteriole formation. However, when co-delivered with VEGF, TERTdn abrogated VEGF-dependent angiogenesis, arteriogenesis, and blood flow increase. This effect was paralleled by in vitro evidence that telomerase inhibition by 3'-azido-3'-deoxythymidine in VEGF-treated endothelial cells strongly reduced capillary density and promoted apoptosis in the absence of serum. Similar results were obtained with adenovirus-mediated expression of TERTdn and AKTdn, both reducing endogenous TERT activity and angiogenesis on Matrigel. Mechanistically, neo-angiogenesis in our system involved: (i) VEGF-dependent activation of telomerase through the nitric oxide pathway and (ii) telomerase-dependent activation of endothelial cell differentiation and protection from apoptosis. Furthermore, detection of TERT in activated satellite cells identified them as VEGF targets during muscle regeneration. Because TERT behaves as an angiogenic factor and a downstream effector of VEGF signaling, telomerase activity appears required for VEGF-dependent remodeling of ischemic tissue at the capillaries and arterioles level.

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.

We examined the effect of reactive oxygen species (ROS) on MicroRNAs (miRNAs) expression in endothelial cells in vitro, and in mouse skeletal muscle following acute hindlimb ischemia. Human umbilical vein endothelial cells (HUVEC) were exposed to 200 μM hydrogen peroxide (H2O2) for 8 to 24 h; miRNAs profiling showed that miR-200c and the co-transcribed miR-141 increased more than eightfold. The other miR-200 gene family members were also induced, albeit to a lower level. Furthermore, miR-200c upregulation was not endothelium restricted, and occurred also on exposure to an oxidative stress-inducing drug: 1,3-bis(2 chloroethyl)-1nitrosourea (BCNU). miR-200c overexpression induced HUVEC growth arrest, apoptosis and senescence; these phenomena were also induced by H2O2 and were partially rescued by miR-200c inhibition. Moreover, miR-200c target ZEB1 messenger RNA and protein were downmodulated by H2O2 and by miR-200c overexpression. ZEB1 knockdown recapitulated miR-200c-induced responses, and expression of a ZEB1 allele non-targeted by miR-200c, prevented miR-200c phenotype. The mechanism of H2O2-mediated miR-200c upregulation involves p53 and retinoblastoma proteins. Acute hindlimb ischemia enhanced miR-200c in wild-type mice skeletal muscle, whereas in p66ShcA −/− mice, which display lower levels of oxidative stress after ischemia, upregulation of miR-200c was markedly inhibited. In conclusion, ROS induce miR-200c and other miR-200 family members; the ensuing downmodulation of ZEB1 has a key role in ROS-induced apoptosis and senescence. [ABSTRACT FROM AUTHOR]