Ischemic Skeletal Muscle Research Articles

Aging exaggerates blood pressure response to ischemic rhythmic handgrip exercise in humans.

Ischemic skeletal muscle conditions are known to augment exercise‐induced increases in blood pressure (BP). Aging is also a factor that enhances the pressor response to exercise. However, the effects of aging on the BP response to ischemic exercise remain unclear. We, therefore, tested the hypothesis that aging enhances the BP response to rhythmic handgrip (RHG) exercise during postexercise muscle ischemia (PEMI). We divided the normotensive participants without cardiovascular diseases into three age groups: young (n = 26; age, 18–28 years), middle‐aged (n = 23; age, 35–59 years), and older adults (n = 23; age, 60–80 years). The participants performed RHG exercise with minimal effort for 1 min after rest with and without PEMI, which was induced by inflating a cuff on the upper arm just before the isometric handgrip exercise ended; the intensity was 30% of maximal voluntary contraction force. Under PEMI, the increase in diastolic BP (DBP) from rest to RHG exercise in the older adult group (Δ13 ± 2 mmHg) was significantly higher than that in the young (Δ5 ± 2 mmHg) and middle‐aged groups (Δ6 ± 1 mmHg), despite there being no significant difference between the groups in the DBP response from rest to RHG exercise without PEMI. Importantly, based on multiple regression analysis, age remained a significant independent determinant of both the SBP and DBP responses to RHG exercise during PEMI (p < 0.01). These findings indicate that aging enhances the pressor response to ischemic rhythmic exercise.

Differential Regulation of Damage-Associated Molecular Pattern Release in a Mouse Model of Skeletal Muscle Ischemia/Reperfusion Injury

BackgroundSkeletal muscle ischemia/reperfusion (I/R) injury is an important clinical issue that can cause remote organ injury. Although its pathogenesis has not been fully elucidated, recent studies have suggested that damage-associated molecular patterns (DAMPs) are mediators of remote organ injury in sterile inflammation. The purpose of this study was to investigate the possible involvement of DAMPs, including the nuclear proteins high-mobility group box 1 (HMGB1) and histone H3, in the pathogenesis of skeletal muscle I/R injury in mice.MethodsHindlimb ischemia was induced in mice through bilateral ligation of inguinal regions using rubber grommets. Reperfusion was induced by cutting the rubber grommets after 2–12 h of ischemic period. Survival rates, localization of HMGB1 and histone H3 in the gastrocnemius muscle, and circulating HMGB1 and histone H3 levels were analyzed. The effect of anti-HMGB1 and anti-histone H3 antibodies on survival was analyzed in mice with I/R injury.ResultsAll mice with hindlimb ischemia survived for at least 36 h, while all mice died within 24 h if the hindlimbs were reperfused after ischemia for 4–12 h. Immunohistochemical analysis revealed that HMGB1 translocated from the nucleus to the cytoplasm in the ischemic gastrocnemius muscle, while histone H3 was confined to the nucleus. Accordingly, serum HMGB1 levels were significantly elevated in mice with hindlimb I/R compared with normal mice or mice with hindlimb ischemia (P < 0.05). Serum histone H3 levels were not elevated after I/R. Treatment with anti-HMGB1 antibodies significantly improved survival of mice with hindlimb I/R injury compared with control antibodies (P < 0.05).ConclusionsHMGB1, but not histone H3, translocated to the cytoplasm during skeletal muscle ischemia, and was released into the systemic circulation after reperfusion in mice with I/R injury. Treatment with anti-HMGB1 antibodies partially improved survival.

Chronic ablation of TRPV1-sensitive skeletal muscle afferents attenuates the muscle metaboreflex.

Exercise intolerance is a hallmark symptom of cardiovascular disease and likely occurs via enhanced activation of muscle metaboreflex-induced vasoconstriction of the heart and active skeletal muscle which, thereby limits cardiac output and peripheral blood flow. Muscle metaboreflex vasoconstrictor responses occur via activation of metabolite-sensitive afferent fibers located in ischemic active skeletal muscle, some of which express transient receptor potential vanilloid 1 (TRPV1) cation channels. Local cardiac and intrathecal administration of an ultrapotent noncompetitive, dominant negative agonist resiniferatoxin (RTX) can ablate these TRPV1-sensitive afferents. This technique has been used to attenuate cardiac sympathetic afferents and nociceptive pain. We investigated whether intrathecal administration (L4-L6) of RTX (2 µg/kg) could chronically attenuate subsequent muscle metaboreflex responses elicited by reductions in hindlimb blood flow during mild exercise (3.2 km/h) in chronically instrumented conscious canines. RTX significantly attenuated metaboreflex-induced increases in mean arterial pressure (27 ± 5.0 mmHg vs. 6 ± 8.2 mmHg), cardiac output (1.40 ± 0.2 L/min vs. 0.28 ± 0.1 L/min), and stroke work (2.27 ± 0.2 L·mmHg vs. 1.01 ± 0.2 L·mmHg). Effects were maintained until 78 ± 14 days post-RTX at which point the efficacy of RTX injection was tested by intra-arterial administration of capsaicin (20 µg/kg). A significant reduction in the mean arterial pressure response (+45.7 ± 6.5 mmHg pre-RTX vs. +19.7 ± 3.1 mmHg post-RTX) was observed. We conclude that intrathecal administration of RTX can chronically attenuate the muscle metaboreflex and could potentially alleviate enhanced sympatho-activation observed in cardiovascular disease states.

Vascular permeability of skeletal muscle microvessels in rat arterial ligation model: in vivo analysis using two-photon laser scanning microscopy.

Peripheral artery disease (PAD) in the lower limb compromises oxygen supply due to arterial occlusion. Ischemic skeletal muscle is accompanied by capillary structural deformation. Therefore, using novel microscopy techniques, we tested the hypothesis that endothelial cell swelling temporally and quantitatively corresponds to enhanced microvascular permeability. Hindlimb ischemia was created in male Wistar rat's by iliac artery ligation (AL). The tibialis anterior (TA) muscle microcirculation was imaged using intravenously infused rhodamine B isothiocyanate dextran fluorescent dye via two-photon laser scanning microscopy (TPLSM) and dye extravasation at 3 and 7 days post-AL quantified to assess microvascular permeability. The TA microvascular endothelial ultrastructure was analyzed by transmission electron microscopy (TEM). Compared with control (0.40 ± 0.15 μm3 × 106), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 ± 0.40 μm3 × 106) and 7 (2.5 ± 0.8 μm3 × 106) days was increased (both P < 0.05). Capillary wall thickness was also elevated at 3 (0.21 ± 0.06 μm) and 7 (0.21 ± 0.08 μm) days versus control (0.11 ± 0.03 μm, both P < 0.05). Capillary endothelial cell swelling was temporally and quantitatively associated with elevated vascular permeability in the AL model of PAD but these changes occurred in the absence of elevations in protein levels of vascular endothelial growth factor (VEGF) its receptor (VEGFR2 which decreased by AL-7 day) or matrix metalloproteinase. The temporal coherence of endothelial cell swelling and increased vascular permeability supports a common upstream mediator. TPLSM, in combination with TEM, provides a sensitive and spatially discrete technique to assess the mechanistic bases for, and efficacy of, therapeutic countermeasures to the pernicious sequelae of compromised peripheral arterial function.

In a Mouse Model of Type 2 Diabetes and Peripheral Artery Disease, Modulation of MirR29a and ADAM12 Reduced Post -Ischemic Skeletal Muscle Injury, Improved Perfusion Recovery and Skeletal Muscle Function

Diabetes Mellitus (DM) is a major risk factor for developing peripheral arterial disease (PAD) and individuals with DM have worse PAD outcomes but the molecular mechanisms involved are poorly understood. Previously, in a hind limb ischemia (HLI) model of PAD, we identified a disintegrin and metalloproteinase gene 12 (ADAM12) as a key genetic modifier of post-ischemic perfusion recovery. Moreover, we showed that expression of ADAM12 in mouse and human tissue is regulated by miR29a. In non-diabetic mice, miR29a expression is downregulated after HLI that allows increased expression of ADAM12. However, upon HLI in high fat diet feed (HFD) mice, a model of type 2 diabetes, miR29a expression remains elevated that prevents ADAM12 increase and results in poor reperfusion recovery, increased skeletal muscle injury and decreased muscle function. Hence, we hypothesized that inhibition of miR29a or augmenting ADAM12 would improve these functional outcomes.Mice (male, 26–28 weeks old) were randomized into 3 treatment groups and their hind limbs were treated with saline (grp1), ADAM12 cDNA (grp 2) or mir29a-inhibitor (grp3), through targeted micro-bubble delivery. Mice were treated at -3 days and -1 pre-surgery, followed by post-surgery weekly boosting. HLI was achieved by unilateral ligation and excision of the femoral artery of the left hind limb. The right hind limb served as non-ischemic control. Gene expression analysis in the hind limbs 3 days post HLI showed decreased miR29a expression in normal chow fed B6, but elevated miR29a expression in HFD (B6 vs HFD; 0.5730±0.01 vs.1.02 ± 0.06, n=3–4, p= 0.001). Treatment with miR29a inhibitor decreased miR29a expression in HFD and increased ADAM12 expression compared to control untreated HFD mice (miR29a INH vs Control HFD: 0.70±0.06 vs 1.02±0.06, n= 4–5, p= 0.004) ADAM12 expression (miR29A INH vs Control: HFD 208.62±24.52 vs 11.75±4.94, n= 3–4 P<0.01). Although ADAM12 cDNA improved ADAM12 expression, miR29a inhibition increased ADAM12 expression to a greater extent (HFD vs ADAM12 vs miR29aINH; 11.75±4.94 vs 20.71±2.98 vs 208.62±24.52, n3-4, p=< 0.001). Accordingly, miR29a inhibition and ADAM12 augmentation decreased skeletal muscle injury assessed by the number of centralized nuclei/muscle fibre (Control vs ADAM12 vs miR29aINH: 0.252±0.043, vs 0.139±0.041 vs 0.040±0.012 n=4, p= 0.05), and improved skeletal muscle function assessed as maximum muscle contraction (Control vs ADAM12 vs miR29aINH: 0.17±0.06 vs 0.26±0.06, vs 0.54±0.08, n=6–7, p<0.01). It also improved perfusion recovery, (% ischemic to non-ischemic limb, control vs ADAM12 vs miR29aINH: 42.52±5.35, vs 58.45±4.87, vs 97.59±6.14, n= 5–10, p<0.01). Thus, our results show augmentation of ADAM12 and Inhibition of MiR29a improves outcomes in experimental PAD in diabetic mice but inhibiting miR29a is a more effective strategy.2414 characters now2500 characters allowed

Heat treatment attenuates activities of ET‐1 signaling pathway and enhances the protective HSP72‐SOD2 in ischemic skeletal muscle of rats with peripheral artery disease

Background The arterial blood pressure (BP) response is augmented during walking in patients with peripheral artery disease (PAD) and in simulated PAD animal models during static exercise. The exercise pressor response (EPR) is a contributor to the exaggerated BP response and heating treatment is effective to attenuate exaggeration of the EPR. However, the underlying mechanisms leading to the inhibitory effects of heat treatment on the EPR, especially on the essential alteration originating within the ischemic skeletal muscles of PAD, is poorly understood. Aims We targeted if the repeated heat treatment protocol (~1.5oC muscle temperature evaluation, 30 minutes per time, twice per day in 72 hours after the femoral artery occlusion) alters some of the fundamental molecular mediators involved in ischemic insult and oxidative stress in the skeletal muscles of the hindlimb. Based on the existing literature, we determined the levels of endothelin-1 (ET-1), and the protein expression of heat shock protein 72 (HSP72) and superoxide dismutase 2 (SOD2) in the skeletal muscles of control rats and PAD rats with 72 hours of femoral artery occlusion. Methods The red and white components of the gastrocnemius muscles were obtained in groups of control, PAD and PAD + heat rats. ET-1 was measured by the method of colorimetric immunometric enzyme immunoassay. The expression of HSP 72 and SOD2 was measured by western blotting. To elevate the muscle temperature, a heating pad was placed locally on the hindlimb and the Tm was increased by ~1.5 °C. Then the Tm was stabilized and maintained for 30 min. One way ANOVA with LSD post-hoc test was applied to determine the difference among groups. Results Compared with control rats, in PAD rats a significant increase in ET-1 was observed in both of the red muscle (0.91±0.28 pg/ml/µg total protein in PAD/n=5 vs. 0.52±0.05 pg/ml/µg total protein in control/n=4; P<0.05); and the white muscle (0.71±0.14 pg/ml/µg total protein in PAD rats/n=5 vs. 0.47±0.09 pg/ml/µg total protein in control rats/n=4; P<0.05). Notably, following heat treatment the amplification of ET-1 in the skeletal muscle of PAD rats was significantly attenuated (red muscle: 0.53±0.08 pg/ml/µg total protein in PAD + heat rats n=5 vs. 0.91±0.28 pg/ml/µg total protein in PAD rats/n=5; P<0.05; white muscle: 0.52±0.10 pg/ml/µg total protein in PAD + heat rats n=5 vs. 0.71±0.14 pg/ml/µg total protein in PAD rats/n=5; P<0.05). Note that no significant difference in the ET-1 levels was seen in skeletal muscle of control rats and PAD + heat rats (P>0.05). In addition, heat treatment increased expression of HSP72 and SOD2 in the red muscle of PAD rats (HSP72: 1.22±0.39 in PAD + heat rats/n=4 vs. 0.68±0.27 in PAD rats/n=4, P<0.05; SOD2: 1.42±0.45 in PAD + heat rats/n=5 vs. 0.92±0.12 in PAD rats/n=4, P=0.062). Conclusion Heat treatment attenuates the activities of ET-1 signaling pathway and enhances the levels of HSP 72 and SOD2, potential protective mediators, in the skeletal muscle of PAD rats under ischemic situations.

Estrogen-related receptor α is involved in angiogenesis and skeletal muscle revascularization in hindlimb ischemia.

Skeletal muscle ischemia is a major consequence of peripheral arterial disease (PAD) or critical limb ischemia (CLI). Although therapeutic options for resolving muscle ischemia in PAD/CLI are limited, the issue is compounded by poor understanding of the mechanisms driving muscle vascularization. We found that nuclear receptor estrogen-related receptor alpha (ERRα) expression is induced in murine skeletal muscle by hindlimb ischemia (HLI), and in cultured myotubes by hypoxia, suggesting a potential role for ERRα in ischemic response. To test this, we generated skeletal muscle-specific ERRα transgenic (TG) mice. In these mice, ERRα drives myofiber type switch from glycolytic type IIB to oxidative type IIA/IIX myofibers, which are typically associated with more vascular supply in muscle. Indeed, RNA sequencing and functional enrichment analysis of TG muscle revealed that "paracrine angiogenesis" is the top-ranked transcriptional program activated by ERRα in the skeletal muscle. Immunohistochemistry and angiography showed that ERRα overexpression increases baseline capillarity, arterioles and non-leaky blood vessel formation in the skeletal muscles. Moreover, ERRα overexpression facilitates ischemic neo-angiogenesis and perfusion recovery in hindlimb musculature of mice subjected to HLI. Therefore, ERRα is a hypoxia inducible nuclear receptor that is involved in skeletal muscle angiogenesis and could be potentially targeted for treating PAD/CLI.

Tourniquet-induced ischemia and reperfusion in subcutaneous tissue, skeletal muscle, and calcaneal cancellous bone.

This study aimed to evaluated ischemic metabolites in subcutaneous tissue, skeletal muscle, and calcaneal cancellous bone before, during, and after tourniquet application in a simultaneous paired comparison of tourniquet-exposed and non-tourniquet-exposed legs. Ten patients scheduled for hallux valgus or hallux rigidus surgery were included. Microdialysis catheters were placed to simultaneously and continuously sample the metabolites glucose, lactate, pyruvate, and glycerol bilaterally for 12h in subcutaneous tissue, skeletal muscle, and calcaneal cancellous bone. A tourniquet was applied on the leg planned for surgery (inflation time: 15min, mean tourniquet duration time (range): 65 (58;77) min). During tourniquet inflation, a 2- to 3-fold increase of the mean lactate/pyruvate ratio was found for all investigated tissues in the tourniquet-exposed leg compared with the non-tourniquet-exposed leg. The lactate/pyruvate ratio recovery time after tourniquet release was within 30min for skeletal muscle, 60min for subcutaneous tissue, and 130min for calcaneal cancellous bone. Only the tourniquet-exposed skeletal muscles were found to be ischemic during tourniquet inflation, defined by a significant increase of the lactate/pyruvate ratio exceeding the ischemic cutoff level of 25; however, this level decreased below 25 immediately after tourniquet release. The glycerol ratio increased instantly after inflation in the tourniquet-exposed leg in skeletal muscle and subcutaneous tissue, and recovered within 60 (skeletal muscle) and 130min (subcutaneous tissue) after tourniquet release. These findings suggest that applying tourniquet for approximately 1h results in limited tissue ischemia and cell damage in subcutaneous tissue, skeletal muscle, and calcaneal cancellous bone.

Suppressing Hippo signaling in the stem cell niche promotes skeletal muscle regeneration.

Lack of blood flow to the lower extremities in peripheral arterial disease causes oxygen and nutrient deprivation in ischemic skeletal muscles, leading to functional impairment. Treatment options for muscle regeneration in this scenario are lacking. Here, we selectively targeted the Hippo pathway in myofibers, which provide architectural support for muscle stem cell niches, to facilitate functional muscle recovery in ischemic extremities by promoting angiogenesis, neovascularization, and myogenesis. We knocked down the core Hippo pathway component, Salvador (SAV1), by using an adeno-associated virus 9 (AAV9) vector expressing a miR30-based triple short-hairpin RNA (shRNA), controlled by a muscle-specific promoter. In a mouse hindlimb-ischemia model, AAV9 SAV1 shRNA administration in ischemic muscles induced nuclear localization of the Hippo effector YAP, accelerated perfusion restoration, and increased exercise endurance. Intravascular lectin labeling of the vasculature revealed enhanced angiogenesis. Using 5-ethynyl-2'-deoxyuridine to label replicating cellular DNA in vivo, we found SAV1 knockdown concurrently increased paired box transcription factor Pax7+ muscle satellite cell and CD31+ endothelial cell proliferation in ischemic muscles. To further study Hippo suppression in skeletal muscle regeneration, we used a cardiotoxin-induced muscle damage model in adult (12-15 weeks old) and aged mice (26-month old). Two weeks after delivery of AAV9 SAV1 shRNA into injured muscles, the distribution of regenerative myofibers shifted toward a larger cross-sectional area and increased capillary density compared with mice receiving AAV9 control. Together, these findings suggest our approach may have clinical promise in regenerative therapy for leg ischemia and muscle injury.

Irisin Protects Against Hind Limb Ischemia Reperfusion Injury.

AimThe aim of this study was to evaluate the effects of irisin in a murine model of hind limb ischemia reperfusion (I/R).MethodsThe mice were divided into four groups (n = 6 in each group): control, irisin, ischemia reperfusion (I/R), and irisin-ischemia reperfusion (I–I/R). Irisin (0.5 µg.g−1, intraperitoneally [i.p.]) was administered 30 min before the I/R procedure. After 2 h of ischemia and 2.5 h of reperfusion, blood and tissue samples were taken for biochemical and histopathological analysis. The results were analyzed by Kruskal–Wallis and Mann–Whitney U-tests.ResultsThere was a statistically significant difference in the total antioxidant status (TAS) and total oxidant status (TOS) levels in all the groups. The TAS level in the I/R group was significantly lower than that in the control, irisin, and I–I/R groups, whereas the TOS level was significantly higher in the I/R group as compared with that in the other groups. Caspase-3 activity and caspase-8 activity, indicators of inflammation, were significantly higher in the I/R and I–I/R groups as compared with those in the control and irisin groups.ConclusionIrisin may have protective effects in skeletal muscle ischemia reperfusion injury.

Targeted Delivery of a Matrix Metalloproteinases-2 Specific Inhibitor Using Multifunctional Nanogels to Attenuate Ischemic Skeletal Muscle Degeneration and Promote Revascularization.

Critical limb ischemia (CLI) is a severe form of peripheral artery disease (PAD). It is featured by degenerated skeletal muscle and poor vascularization. During the development of CLI, the upregulated matrix metalloproteinase-2 (MMP-2) degrades muscle extracellular matrix to initiate the degeneration. Meanwhile, MMP-2 is necessary for blood vessel formation. It is thus hypothesized that appropriate MMP-2 bioactivity in ischemic limbs will not only attenuate muscle degeneration but also promote blood vessel formation. Herein, we developed ischemia-targeting poly(N-isopropylacrylamide)-based nanogels to specifically deliver an MMP-2 inhibitor CTTHWGFTLC (CTT) into ischemic limbs to tailor MMP-2 bioactivity. Besides acting as an MMP-2 inhibitor, CTT promoted endothelial cell migration under conditions mimicking the ischemic limbs. The nanogels were sensitive to the pH of ischemic tissues, allowing them to largely aggregate in the injured area. To help reduce nanogel uptake by macrophages and increase circulation time, the nanogels were cloaked with a platelet membrane. An ischemia-targeting peptide CSTSMLKA (CST) was further conjugated on the platelet membrane for targeted delivery of nanogels into the ischemic area. CTT gradually released from the nanogels for 4 weeks. The nanogels mostly accumulated in the ischemic area for 28 days. The released CTT preserved collagen in the muscle and promoted its regeneration. In addition, CTT stimulated angiogenesis. Four weeks after CLI, the blood flow and vessel density of the ischemic limbs treated with the nanogels were remarkably higher than the control groups without CTT release. These results demonstrate that the developed nanogel-based CTT release system has the potential to stimulate ischemic limb regeneration.

Oxygen-release microspheres capable of releasing oxygen in response to environmental oxygen level to improve stem cell survival and tissue regeneration in ischemic hindlimbs

Stem cell transplantation has been extensively explored to promote ischemic limb vascularization and skeletal muscle regeneration. Yet the therapeutic efficacy is low due to limited cell survival under low oxygen environment of the ischemic limbs. Therefore, continuously oxygenating the transplanted cells has potential to increase their survival. During tissue regeneration, the number of blood vessels are gradually increased, leading to the elevation of tissue oxygen content. Accordingly, less exogenous oxygen is needed for the transplanted cells. Excessive oxygen may induce reactive oxygen species (ROS) formation, causing cell apoptosis. Thus, it is attractive to develop oxygen-release biomaterials that are responsive to the environmental oxygen level. Herein, we developed oxygen-release microspheres whose oxygen release was controlled by oxygen-responsive shell. The shell hydrophilicity and degradation rate decreased as the environmental oxygen level increased, leading to slower oxygen release. The microspheres were capable of directly releasing molecular oxygen, which are safer than those oxygen-release biomaterials that release hydrogen peroxide and rely on its decomposition to form oxygen. The released oxygen significantly enhanced mesenchymal stem cell (MSC) survival without inducing ROS production under hypoxic condition. Co-delivery of MSCs and microspheres to the mouse ischemic limbs ameliorated MSC survival, proliferation and paracrine effects under ischemic conditions. It also significantly accelerated angiogenesis, blood flow restoration, and skeletal muscle regeneration without provoking tissue inflammation. The above results demonstrate that the developed microspheres have potential to augment cell survival in ischemic tissues, and promote ischemic tissue regeneration in a safer and more efficient manner.

Skeletal Muscle Mitochondrial Dysfunction and Oxidative Stress in Peripheral Arterial Disease: A Unifying Mechanism and Therapeutic Target.

Peripheral artery disease (PAD) is caused by atherosclerosis in the lower extremities, which leads to a spectrum of life-altering symptomatology, including claudication, ischemic rest pain, and gangrene requiring limb amputation. Current treatments for PAD are focused primarily on re-establishing blood flow to the ischemic tissue, implying that blood flow is the decisive factor that determines whether or not the tissue survives. Unfortunately, failure rates of endovascular and revascularization procedures remain unacceptably high and numerous cell- and gene-based vascular therapies have failed to demonstrate efficacy in clinical trials. The low success of vascular-focused therapies implies that non-vascular tissues, such as skeletal muscle and oxidative stress, may substantially contribute to PAD pathobiology. Clues toward the importance of skeletal muscle in PAD pathobiology stem from clinical observations that muscle function is a strong predictor of mortality. Mitochondrial impairments in muscle have been documented in PAD patients, although its potential role in clinical pathology is incompletely understood. In this review, we discuss the underlying mechanisms causing mitochondrial dysfunction in ischemic skeletal muscle, including causal evidence in rodent studies, and highlight emerging mitochondrial-targeted therapies that have potential to improve PAD outcomes. Particularly, we will analyze literature data on reactive oxygen species production and potential counteracting endogenous and exogenous antioxidants.

Therapeutic Angiogenesis by a "Dynamic Duo": Simultaneous Expression of HGF and VEGF165 by Novel Bicistronic Plasmid Restores Blood Flow in Ischemic Skeletal Muscle.

Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene delivery was used to stimulate blood vessels’ formation and growth. We have previously shown that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse ischemic limb, and efficacy of combined delivery was superior to each plasmid administered alone. In this work, we evaluated different approaches for co-expression of HGF and VEGF165 genes in a panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional promoter or two independent promoters for each gene of interest. Studies in HEK293T culture showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of expressed factors. Tests in skeletal muscle explants showed a dramatic difference and most plasmids failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two independent promoters (cytomegalovirus (CMV) for HGF and chicken b-actin (CAG) for VEGF165) provided expression of both grow factors in skeletal muscle at an equimolar ratio. Efficacy tests of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular administration of plasmid induced significant restoration of perfusion compared to an empty vector and saline. These findings were supported by increased CD31+ capillary density in animals that received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide basis for future development of treatment for an unmet medical need—peripheral artery disease and associated limb ischemia.

Mechanisms of ischemic skeletal muscle regeneration mediated by mechanically constrained human allogeneic mesenchymal stromal cells

Background and Hypothesis: Critical limb threatening ischemia (CLTI) is the end stage of peripheral arterial disease (PAD). CLTI presents a significant risk for lower extremity amputation, especially in diabetic patients with poor options for revascularization. Additionally, using a novel diabetic mouse model of CLTI, we have shown that IM injection of 3D cultured MSCs (spheroids) is more effective in promoting skeletal muscle regeneration of ischemic muscle than monolayer cultured MSC. We hypothesize that this result is due to the mechanical constrained nature of the MSC in spheroid form, which has been shown to alter the cellular phenotype. Alginate encapsulated cells are another form of mechanical confinement, and have additional benefits including resistance to immunological attack, making them a practical therapy for treatment of CLTI patients. &#x0D; &#x0D; Project Methods: In this work, cells were encapsulated in 2% alginate using a centrifugation method. Media from these cells along with others were analyzed for IL-10 and IL-33 using ELISA. Mouse tissue samples were stained with WGA-555 antibody and analyzed using a scanning microscope. Effects on tissue perfusion were measured with Laser Doppler Perfusion Imaging. At the time of this abstract, the media from encapsulated and naked MSCs is being used to culture myoblasts, looking at cell growth. &#x0D; &#x0D; Results: ELISA results did not show significant increases in IL-10 or IL-33 for encapsulated vs. non-encapsulated cells. LDPI has shown an increased perfusion rate for hindlimbs treated with encapsulated MSCs vs naked MSCs. Muscle fiber analysis is ongoing, but initial data appears promising. &#x0D; &#x0D; Conclusion and Potential Impact: This experiment provides a starting point for improving and expanding cell therapy for critical limb ischemia, potentially resulting in better outcomes for diabetic patients and preventing lower limb amputations. The encapsulation process also has value in other types of cell therapy, as it could protect cells from host defenses and increase dwell time.

Hmox1 (Heme Oxygenase-1) Protects Against Ischemia-Mediated Injury via Stabilization of HIF-1α (Hypoxia-Inducible Factor-1α).

Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body's response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and Results: Hmox1 deficient (Hmox1-/-) mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired in Hmox1-/- mice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O2) recapitulated these key findings. Metabolomics analyses indicated a failure of Hmox1-/- mice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α in Hmox1-/- fibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α in Hmox1-/- fibroblasts in response to hypoxia. Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1's protection against ischemic injury independent of neovascularization.

Effects of Hesperetin on the Serum Superoxide Dismutase, Glutathione Peroxidase, and Malondialdehyde Levels and Renal-Histopathological Alterations Following Skeletal Muscle Ischemia Reperfusion in Male Rats

Objectives: The aim of the current study was to assess the effects of hesperetin on damage to kidneys as remote organs following skeletal muscle ischemia-reperfusion (IR) in rats. Materials and Methods: In general, 30 male Wistar rats were randomized and placed into sham, IR, hesperetin, dimethyl sulfoxide (DMSO), and IR+hesperetin groups. The rats in the hesperetin and IR+hesperetin groups received a 50 mg/kg dose of hesperetin dissolved in DMSO intraperitoneally. In the IR+hesperetin group, hesperetin was injected exactly prior to reperfusion. To induce skeletal muscle ischemia, the femoral artery was clamped for two hours. Following a 24-hour period of reperfusion, the samples of blood were collected for renal function tests and oxidative stress measurements. Next, the rats were euthanized, and histological analyses were conducted on their removed kidneys. Results: Based on the results, urea and creatinine serum levels were significantly higher in the IR group (P&lt;0.05) whereas they significantly reduced following treatment with hesperetin (P&lt;0.05). The concentration of malondialdehyde (P&lt;0.05) increased for the IR group while those of superoxide dismutase (P&lt;0.05) and glutathione peroxidase (P&lt;0.05) activities were lower than the other groups. The analysis of renal tissues in the IR group showed glomerular necrosis, degeneration, and necrosis of the tubular epithelium, protein casts, interstitial edema, and inflammation. Finally, the degree of renal injury was significantly ameliorated (P&lt;0.05) in rats treated with hesperetin. Conclusions: Overall, the results indicated that in rats, hesperetin could reduce renal injury that has been induced through skeletal muscle IR.

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.

Compartment Syndrome: Evaluation of Skeletal Muscle Ischemia and Physiologic Biomarkers in Controlled Conditions Within Ex Vivo Isolated Muscle Bundles

To identify potential physiologic markers of muscle ischemia to serve as diagnostic indicators of compartment syndrome. We hypothesize that muscle bundles in hypoxic conditions will elicit decreases in potential hydrogen (pH) and increases in lactate and potassium that correlates with decreased muscle twitch forces. We performed an ex vivo evaluation of individual skeletal muscle bundles obtained from a swine's diaphragm that were exposed to hypoxic conditions and compared with control groups. Over a 4-hour period, we evaluated the following parameters for each muscle bundle: muscle twitch forces and levels of potassium, lactate, and pH. Comparisons between the hypoxic and control groups were calculated at each time point using the 2-tailed Wilcoxon rank sum test for nonparametric data. Longitudinal associations between biomarkers and muscle twitch forces were tested using repeated measures analyses. The hypoxic group elicited more significant decreases in normalized muscle twitch forces than the control group at all time points (0.15 g vs. 0.55 g at 4 hours, P < 0.001). Repeated measures analyses of the hypoxic group demonstrated a statistically significant association between potassium, lactate, and normalized peak force over the course of time. Potassium demonstrated the strongest association with a 1 mmol/L unit increase in potassium associated with a 2.9 g decrease in normalized peak force (95% confidence interval -3.3 to -2.4, P < 0.001). The pH of all muscle baths increased over the course of time at similar rates between the study groups. This study used an ex vivo ischemic skeletal muscle model as a representation for pathophysiologic pathways associated with compartment syndrome. In this experimental approach we were unable to evaluate the pH of the muscle bundles due to continuous applied gassing. Our findings support further evaluations of potassium and lactate levels as potential diagnostic markers.

Effects of Trans-Cinnamaldehyde on Reperfused Ischemic Skeletal Muscle and the Relationship to Laminin

Purpose Ischemia-reperfusion (I-R) injury is a serious problem caused by vascular trauma, tourniquet use and/or compartment syndrome. Studies have reported that skeletal muscle function is impaired due to the lower extremity I-R injury. There are insufficient studies on the treatment methods used for the recovery of dysfunction. This study is designed to investigate the effects of trans-cinnamaldehyde (TCA), a volatile oil of cinnamon structure, on the contractile dysfunction due to I-R injury of rat extensor-digitorum-longus (EDL) muscle. Materials and methods Sprague-Dawley rats were randomly divided into three groups. Except for the animals in the control group, all animals received saline (3-ml/kg) or TCA solution (30-mg/kg) which was administered orally three times with an 8-h interval before ischemia. After 24-hours, experimental groups were subjected to 3-h of lower extremity ischemia followed by 5-h reperfusion period. Then, the compound muscle action potential (CMAP) and mechanical activity of muscle were recorded using the standard electro-biophysical techniques. Results There was a decrease in the maximum contractile force in I-R group compared to the control group (p < 0.05). Oxidative damage indicator (MDA) and antioxidant indicator (CAT) increased in the EDL muscle and serum samples in the I-R group (p < 0.05). Laminin expression showed a reduction in the I-R group (p < 0.05). It was seen that TCA achieve again the maximum contraction force in the EDL muscle (p < 0.05) and maintain the expression of laminin (p > 0.05). Conclusion We concluded that TCA has a potential protective effect with antioxidant effects against I-R injury and may maintain laminin levels.