Induction Of Hindlimb Ischemia Research Articles

Tibial cortex transverse transport promotes ischemic diabetic foot ulcer healing via enhanced angiogenesis and inflammation modulation in a novel rat model

BackgroundTibial Cortex Transverse Transport (TTT) represents an innovative surgical method for treating lower extremity diabetic foot ulcers (DFUs), yet its underlying mechanisms remain elusive. Establishing an animal model that closely mirrors clinical scenarios is both critical and novel for elucidating the mechanisms of TTT.MethodsWe established a diabetic rat model with induced hindlimb ischemia to mimic the clinical manifestation of DFUs. TTT was applied using an external fixator for regulated bone movement. Treatment efficacy was evaluated through wound healing assessments, histological analyses, and immunohistochemical techniques to elucidate biological processes.ResultsThe TTT group demonstrated expedited wound healing, improved skin tissue regeneration, and diminished inflammation relative to controls. Marked neovascularization and upregulation of angiogenic factors were observed, with the HIF-1α/SDF-1/CXCR4 pathway and an increase in EPCs being pivotal in these processes. A transition toward anti-inflammatory M2 macrophages indicated TTT's immunomodulatory capacity.ConclusionOur innovative rat model effectively demonstrates the therapeutic potential of TTT in treating DFUs. We identified TTT's roles in promoting angiogenesis and modulating the immune system. This paves the way for further in-depth research and potential clinical applications to improve DFU management strategies.

Hypercholesterolemia impairs collateral artery enlargement by ten-eleven translocation 1-dependent hematopoietic stem cell autonomous mechanism in a murine model of limb ischemia

ObjectiveThe extent of collateral artery enlargement determines the risk of limb loss due to peripheral arterial disease. Hypercholesterolemia impairs collateral artery enlargement, but the underlying mechanism remains poorly characterized. This study tests the hypothesis that hypercholesterolemia impairs collateral artery enlargement through a ten-eleven translocation 1 (Tet1)-dependent hematopoietic stem cell (HSC)-autonomous mechanism that increases their differentiation into proinflammatory Ly6Chi monocytes and restricts their conversion into proangiogenic Ly6Clow monocytes. MethodsTo test our hypothesis, we induced limb ischemia and generated chimeric mouse models by transplanting HSCs from either wild-type (WT) mice or hypercholesterolemic mice into lethally irradiated WT recipient mice. ResultsWe found that the lethally irradiated WT recipient mice reconstituted with HSCs from hypercholesterolemic mice displayed lower blood flow recovery and collateral artery enlargement that was nearly identical to that observed in hypercholesterolemic mice, despite the absence of hypercholesterolemia and consistent with an HSC-autonomous mechanism. We showed that hypercholesterolemia impairs collateral artery enlargement by a Tet1-dependent mechanism that increases HSC differentiation toward proinflammatory Ly6Chi monocytes and restricts the conversion of Ly6Chi monocytes into proangiogenic Ly6Clow monocytes. Moreover, Tet1 epigenetically reprograms monocyte gene expression within the HSCs. Restoration of Tet1 expression in HSCs of hypercholesterolemic mice restores WT collateral artery enlargement and blood flow recovery after induction of hindlimb ischemia. ConclusionsThese results show that hypercholesterolemia impairs collateral artery enlargement by a novel Tet1-dependent HSC-autonomous mechanism that epigenetically reprograms monocyte gene expression within the HSCs.

Selective ROCK Inhibitor Enhances Blood Flow Recovery after Hindlimb Ischemia.

The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.

Chronic nicotine impairs the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells in a murine model of peripheral arterial disease

ObjectiveLifestyle choices such as tobacco and e-cigarette use are a risk factor for peripheral arterial disease (PAD) and may influence therapeutic outcomes. The effect of chronic nicotine exposure on the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) was assessed in a murine model of PAD. MethodsMice were exposed to nicotine or phosphate-buffered saline (PBS) for 28 days, followed by induction of limb ischemia and iPSC-EC transplantation. Cells were injected into the ischemic limb immediately after induction of hindlimb ischemia and again 7 days later. Limb perfusion was assessed by laser Doppler spectroscopy, and transplant cell survival was monitored for 14 days afterward using bioluminescence imaging, followed by histological analysis of angiogenesis. ResultsTransplant cell retention progressively decreased over time after implantation based on bioluminescence imaging, and there were no significant differences in cell survival between mice with chronic exposure to nicotine or PBS. However, compared with mice without nicotine exposure, mice with prior nicotine exposure had had an impaired therapeutic response to iPSC-EC therapy based on decreased vascular perfusion recovery. Mice with nicotine exposure, followed by cell transplantation, had significantly lower mean perfusion ratio after 14 days (0.47 ± 0.07) compared with mice undergoing cell transplantation without prior nicotine exposure (0.79 ± 0.11). This finding was further supported by histological analysis of capillary density, in which animals with prior nicotine exposure had a lower capillary density (45.9 ± 4.7 per mm2) compared with mice without nicotine exposure (66.5 ± 8.1 per mm2). Importantly, the ischemic limbs mice exposed to nicotine without cell therapy also showed significant impairment in perfusion recovery after 14 days, compared with mice that received PBS + iPSC-EC treatment. This result suggested that mice without chronic nicotine exposure could respond to iPSC-EC implantation into the ischemic limb by inducing perfusion recovery, whereas mice with chronic nicotine exposure did not respond to iPSC-EC therapy. ConclusionsTogether, these findings show that chronic nicotine exposure adversely affects the ability of iPSC-EC therapy to promote vascular perfusion recovery and angiogenesis in a murine PAD model.

The Efficacy of HGF/VEGF Gene Therapy for Limb Ischemia in Mice with Impaired Glucose Tolerance: Shift from Angiogenesis to Axonal Growth and Oxidative Potential in Skeletal Muscle

Combined non-viral gene therapy (GT) of ischemia and cardiovascular disease is a promising tool for potential clinical translation. In previous studies our group has developed combined gene therapy by vascular endothelial growth factor 165 (VEGF165) + hepatocyte growth factor (HGF). Our recent works have demonstrated that a bicistronic pDNA that carries both human HGF and VEGF165 coding sequences has a potential for clinical application in peripheral artery disease (PAD). The present study aimed to test HGF/VEGF combined plasmid efficacy in ischemic skeletal muscle comorbid with predominant complications of PAD-impaired glucose tolerance and type 2 diabetes mellitus (T2DM). Male C57BL mice were housed on low-fat (LFD) or high-fat diet (HFD) for 10 weeks and metabolic parameters including FBG level, ITT, and GTT were evaluated. Hindlimb ischemia induction and plasmid administration were performed at 10 weeks with 3 weeks for post-surgical follow-up. Limb blood flow was assessed by laser Doppler scanning at 7, 14, and 21 days after ischemia induction. The necrotic area of m.tibialis anterior, macrophage infiltration, angio- and neuritogenesis were evaluated in tissue sections. The mitochondrial status of skeletal muscle (total mitochondria content, ETC proteins content) was assessed by Western blotting of muscle lysates. At 10 weeks, the HFD group demonstrated impaired glucose tolerance in comparison with the LFD group. HGF/VEGF plasmid injection aggravated glucose intolerance in HFD conditions. Blood flow recovery was not changed by HGF/VEGF plasmid injection either in LFD or HFD conditions. GT in LFD, but not in HFD conditions, enlarged the necrotic area and CD68+ cells infiltration. However, HGF/VEGF plasmid enhanced neuritogenesis and enlarged NF200+ area on muscle sections. In HFD conditions, HGF/VEGF plasmid injection significantly increased mitochondria content and ETC proteins content. The current study demonstrated a significant role of dietary conditions in pre-clinical testing of non-viral GT drugs. HGF/VEGF combined plasmid demonstrated a novel aspect of potential participation in ischemic skeletal muscle regeneration, through regulation of innervation and bioenergetics of muscle. The obtained results made HGF/VEGF combined plasmid a very promising tool for PAD therapy in impaired glucose tolerance conditions.

Granulocyte Colony-Stimulating Factor Ameliorates Endothelial Activation and Thrombotic Diathesis Biomarkers in a Murine Model of Hind Limb Ischemia.

Novel therapies in peripheral arterial disease, such as granulocyte colony-stimulating factor (GCSF) administration, might result in anti-atherosclerotic effects. In this study, we used 10-week-old male ApoE−/− mice, which were fed an atherosclerosis-inducing diet for four weeks. At the end of the four weeks, hind limb ischemia was induced through left femoral artery ligation, the atherosclerosis-inducing diet was discontinued, and a normal diet was initiated. Mice were then randomized into a control group (intramuscular 0.4 mL normal saline 0.9% for 7 days) and a group in which GCSF was administrated intramuscularly in the left hind limb for 7 days (100 mg/kg). In the GCSF group, but not in the control group, we observed significant reductions in the soluble adhesion molecules (vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1)), sE-Selectin, and plasminogen activator inhibitor (PAI)-1 when they were measured through ELISA on the 1st and the 28th days after hind limb ischemia induction. Therefore, GCSF administration in an atherosclerotic mouse model of hind limb ischemia led to decreases in the biomarkers associated with endothelial activation and thrombosis. These findings warrant further validation in future preclinical studies.

Translational development of ABCB5+ dermal mesenchymal stem cells for therapeutic induction of angiogenesis in non-healing diabetic foot ulcers

BackgroundWhile rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5+ mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers.MethodsThe angiogenic potential of ABCB5+ MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5+ MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial.ResultsHypoxic incubation of ABCB5+ MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5+ MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5+ MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5+ MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5+ MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed.ConclusionsThe present observations identify GMP-manufactured ABCB5+ dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy.Trial registration: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784

Effects Of Exercise Training On Mitochondrial Function In A Large-animal Model Of Peripheral Artery Disease

PURPOSE: Our group has developed an endovascular porcine model of ischemic myopathy from peripheral artery disease (PAD). Herein, we tested the hypothesis that mitochondrial dysfunction and oxidative stress are attenuated by exercise training in this model. METHODS: Seven Yorkshire swine were exposed to acute right hindlimb ischemia (HLI) by endovascular external iliac artery occlusion. The swine were randomly separated into two groups: exercise trained (ET) (n = 4) and sedentary control (SED) (n = 3). Exercise training consisted of a structured exercise therapy program on a treadmill (GoPet USA- Model PR730) three days per week, starting 1 week prior to HLI induction and continuing for four weeks after HLI. Speed was increased weekly as tolerated by 1 km/h up to 3.5 km/h with a constant incline of 5%. Food lure was used to motivate animals to participate for as long as tolerated. The session was terminated when the animal reached severe fatigue preventing further exercise. SED animals were exposed to HLI with no ET (regular activity). After termination, mitochondrial respiration, and reactive oxygen species (ROS) production in permeabilized gastrocnemius fiber bundles were simultaneously measured by high-resolution respirometry. RESULTS: After training, the oxygen consumption rate was significantly increased in ischemic muscles, compared to SED during Complex III (CIII) respiration (ET: 14.92 ± 2.82; SED: 5.80 ± 2.26 pmols/sec/mg protein, p < 0.0001) and Complex IV (CIV) (ET: 39.16 ± 3.77; SED: 12.16 ± 1.40, p < 0.0001 pmols/sec/mg protein). In addition, ROS production was significantly attenuated after ET during CIII (ET: 3.21 ± 1.62; SED: 7.10 ± 0.94 pmols/s*ml, p < 0.0001) and CIV (ET: 4.14 ± 0.57; SED: 16.10 ± 0.44 pmols/s*ml, p < 0.0001). CONCLUSION: These data suggest that exercise therapy, which has established efficacy in PAD, is also effective in this new model. Thus, this model may be useful for testing therapeutics targeting the pathophysiological mechanisms of mitochondrial dysfunction and oxidative stress in PAD.

Repeated intravenous administration of hiPSC-MSCs enhance the efficacy of cell-based therapy in tissue regeneration

We seek to demonstrate whether therapeutic efficacy can be improved by combination of repeated intravenous administration and local transplantation of human induced pluripotential stem cell derived MSCs (hiPSC-MSCs). In this study, mice model of hind-limb ischemia is established by ligation of left femoral artery. hiPSC-MSCs (5 × 105) is intravenously administrated immediately after induction of hind limb ischemia with or without following intravenous administration of hiPSC-MSCs every week or every 3 days. Intramuscular transplantation of hiPSC-MSCs (3 × 106) is performed one week after induction of hind-limb ischemia. We compare the therapeutic efficacy and cell survival of intramuscular transplantation of hiPSC-MSCs with or without a single or repeated intravenous administration of hiPSC-MSCs. Repeated intravenous administration of hiPSC-MSCs can increase splenic regulatory T cells (Tregs) activation, decrease splenic natural killer (NK) cells expression, promote the polarization of M2 macrophages in the ischemic area and improved blood perfusion in the ischemic limbs. The improved therapeutic efficacy of MSC-based therapy is due to both increased engraftment of intramuscular transplanted hiPSC-MSCs and intravenous infused hiPSC-MSCs. In conclusion, our study support a combination of repeated systemic infusion and local transplantation of hiPSC-MSCs for cardiovascular disease.

Tissue ischemia induces mobilization of pro-angiogenic neutrophils from the spleen

Abstract Pro-angiogenic neutrophils (PAN) form a small subset of circulating neutrophils expressing CD49d+ VEGFR1hi CXCR4hi, which play a crucial role in the revascularization of transplanted islets. To determine the role of PANs in re-establishing tissue perfusion following a large ischemic event we used the mouse model for peripheral artery disease, the hindlimb ischemia (HLI) model. We found that numbers of PANs in the affected gastrocnemius muscle increased ≈4 times 3h following ischemia induction and reached its peak 2 days following ischemia onset. Similarly, in patients undergoing surgery where peripheral perfusion is transiently clamped, numbers of circulating PANs increase ≈3 times 30 min post-HLI. In healthy mice, PANs are found in high frequency in the spleen (spleen: 9,8% of total neutrophils, blood: 0,78%). Splenectomy prior to induction of HLI reduced recruitment of PANs to the ischemic muscle, which demonstrates that the spleen acts as a reservoir for PANs. Furthermore, HLI downregulated the CXCR4-ligand CXCL12α in the splenic red pulp. This was prevented by chemically induced sympathectomy, resulting in increased retention of PANs in spleen, reduced PAN accumulation in the ischemic muscle and reduced blood flow recovery. Therefore, we propose that the downregulation of CXCL12α controlled by sympathetic nerve signaling pathways drives retention of PANs in the spleen, thereby regulating their release in response to HLI. In summary, we identified that the spleen contains a peripheral pool of PANs and emphasize the pivotal role of the neuro-immune axis in the recruitment of PANs to the site of ischemia. These discoveries open new avenues for drug development to limit damage and accelerate healing following ischemic events. This study was supported by the Swedish Research Council, Knut and Alice Wallenberg foundation, The Swedish Cancer Society, and O. E. and Edla Johansson’s foundation.

Human Bone Marrow Mononuclear Cells Do Not Improve Limb Perfusion in the Hindlimb Ischemia Model.

Effective treatments for chronic limb-threatening ischemia are lacking. (Pre)clinical studies on administration of bone marrow (BM) mononuclear cells (MNCs) and BM-derived mesenchymal stromal cells (MSCs) have shown variable results and no studies have directly compared administration of human BM MNCs and BM MSCs in in vivo models. We studied the effect of intramuscular administration of human BM-derived MNCs and MSCs on limb perfusion in the murine hindlimb ischemia (HLI) model. Human BM MNCs and MSCs were obtained from healthy consenting donors. Both cell types were cryopreserved before use. Twenty-four hours after induction of HLI, nude NMRI mice were randomized to receive intramuscular administration of human BM MNCs (n = 13), or BM MSCs (n = 14), or vehicle control (n = 19) in various doses. Limb perfusion was measured using laser Doppler imaging on day 0, 1, 4, 7, 10, and 14. Intramuscular injection of human BM MNCs did not improve limb perfusion as compared with vehicle over the 2 weeks after cell administration (P = 0.88, mean relative perfusion for vehicle 0.56 ± 0.04 and 0.53 ± 0.04 for BM MNCs at day 14). Administration of human BM MSCs significantly improved limb perfusion as compared with both BM MNCs and vehicle (P ≤ 0.001, mean relative perfusion at day 14 0.79 ± 0.06). Our data suggest that BM MNCs are less suitable than BM MSCs for cell-based therapy that aims to restore perfusion.

E-Selectin-Overexpressing Mesenchymal Stem Cell Therapy Confers Improved Reperfusion, Repair, and Regeneration in a Murine Critical Limb Ischemia Model.

AimsNovel cell-based therapeutic angiogenic treatments for patients with critical limb ischemia may afford limb salvage. Mesenchymal stem cells (MSCs) do not overexpress E-selectin; however, we have previously demonstrated the cell-adhesion molecule's vital role in angiogenesis and wound healing. Thus, we created a viral vector to overexpress E-selectin on MSCs to increase their therapeutic profile.Methods and ResultsFemoral artery ligation induced hind limb ischemia in mice and intramuscular injections were administered of vehicle or syngeneic donor MSCs, transduced ex vivo with an adeno-associated viral vector to express either GFP+ (MSCGFP) or E-selectin-GFP+ (MSCE−selectin−GFP). Laser Doppler Imaging demonstrated significantly restored reperfusion in MSCE−selectin−GFP-treated mice vs. controls. After 3 weeks, the ischemic limbs in mice treated with MSCE−selectin−GFP had increased footpad blood vessel density, hematoxylin and eosin stain (H&E) ischemic calf muscle sections revealed mitigated muscular atrophy with restored muscle fiber size, and mice were able to run further before exhaustion. PCR array-based gene profiling analysis identified nine upregulated pro-angiogenic/pro-repair genes and downregulated Tumor necrosis factor (TNF) gene in MSCE−selectin−GFP-treated limb tissues, indicating that the therapeutic effect is likely achieved via upregulation of pro-angiogenic cytokines and downregulation of inflammation.ConclusionThis innovative cell therapy confers increased limb reperfusion, neovascularization, improved functional recovery, decreased muscle atrophy, and thus offers a potential therapeutic method for future clinical studies.

Cold-Inducible RNA-Binding Protein but Not Its Antisense lncRNA Is a Direct Negative Regulator of Angiogenesis In Vitro and In Vivo via Regulation of the 14q32 angiomiRs-microRNA-329-3p and microRNA-495-3p.

Inhibition of the 14q32 microRNAs, miR-329-3p and miR-495-3p, improves post-ischemic neovascularization. Cold-inducible RNA-binding protein (CIRBP) facilitates maturation of these microRNAs. We hypothesized that CIRBP deficiency improves post-ischemic angiogenesis via downregulation of 14q32 microRNA expression. We investigated these regulatory mechanisms both in vitro and in vivo. We induced hindlimb ischemia in Cirp−/− and C57Bl/6-J mice, monitored blood flow recovery with laser Doppler perfusion imaging, and assessed neovascularization via immunohistochemistry. Post-ischemic angiogenesis was enhanced in Cirp−/− mice by 34.3% with no effects on arteriogenesis. In vivo at day 7, miR-329-3p and miR-495-3p expression were downregulated in Cirp−/− mice by 40.6% and 36.2%. In HUVECs, CIRBP expression was upregulated under hypothermia, while miR-329-3p and miR-495-3p expression remained unaffected. siRNA-mediated CIRBP knockdown led to the downregulation of CIRBP-splice-variant-1 (CIRBP-SV1), CIRBP antisense long noncoding RNA (lncRNA-CIRBP-AS1), and miR-495-3p with no effects on the expression of CIRBP-SV2-4 or miR-329-3p. siRNA-mediated CIRBP knockdown improved HUVEC migration and tube formation. SiRNA-mediated lncRNA-CIRBP-AS1 knockdown had similar long-term effects. After short incubation times, however, only CIRBP knockdown affected angiogenesis, indicating that the effects of lncRNA-CIRBP-AS1 knockdown were secondary to CIRBP-SV1 downregulation. CIRBP is a negative regulator of angiogenesis in vitro and in vivo and acts, at least in part, through the regulation of miR-329-3p and miR-495-3p.

Single Intraosseous Simvastatin Application Induces Endothelial Progenitor Cell Mobilization and Therapeutic Angiogenesis in a Diabetic Hindlimb Ischemia Rat Model.

Endothelial progenitor cells have shown the ability to enhance neovascularization. In this study, the authors tested whether intraosseous delivery of simvastatin could mobilize endothelial progenitor cells and enhance recovery in a hindlimb ischemia model. There are eight groups of rats in this study: normal control; type 1 diabetes mellitus control group control without drug intervention; and type 1 diabetes mellitus rats that randomly received intraosseous simvastatin (0, 0.5, or 1 mg) or oral simvastatin administration (0, 20, or 400 mg). All type 1 diabetes mellitus rats had induced hindlimb ischemia. The number of endothelial progenitor cells in peripheral blood, and serum markers, were detected. The recovery of blood flow at 21 days after treatment was used as the main outcome. The authors demonstrated that endothelial progenitor cell mobilization was increased in the simvastatin 0.5- and 1-mg groups compared with the type 1 diabetes mellitus control and simvastatin 0-mg groups at 1, 2, and 3 weeks. Serum vascular endothelial growth factor levels were significantly increased at 2 weeks in the simvastatin 0.5- and 1-mg groups, in addition to the increase of the blood flow and the gastrocnemius weight at 3 weeks. Similar increase can also been seen in simvastatin 400 mg orally but not in simvastatin 20 mg orally. These findings demonstrate that a single intraosseous administration of simvastatin mobilized endothelial progenitor cells at a dose one-hundredth of the required daily oral dose in rats, and this potent mobilization of endothelial progenitor cells markedly improved diabetic limb ischemia by means of neovascularization.

Pharmacological blockade of histone methyltransferase SETD7 restores angiogenic response in experimental diabetes

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in diabetic patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in the ischemic limb of diabetic patients. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the methyltransferase SETD7 - favours a chromatin active and open state thus enabling the gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate post-ischemic vascularization in experimental diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro angiogenic assays like migration assay &amp; tube formation assay were performed. Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor called (R)-PFI-2. T1D mice (streptozotocin-induced diabetes) was orally treated with (R)-PFI-2 and with vehicle for 21 days and followed by induction of hindlimb ischemia. Blood flow recovery was analyzed at 30 minutes, 7 and 14 days by laser doppler imaging. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with an increase of H3K4-mono-methylation levels as well as with impaired endothelial cell migration and tube formation. Of interest, both gene silencing (SETD7-siRNA) and pharmacological blockade of SETD7 by (R)-PFI-2 rescued hyperglycemia-induced impairment of angiogenic properties in HAECs. RNA-seq in HG-treated HAECs with and without SETD7 depletion unveiled an array of differentially expressed genes, which were mainly involved in blood vessel growth and angiogenic response. Among dysregulated genes, Chromatin immunoprecipitation (ChIP) assays showed that SETD7 specifically mono-methylates H3K4m1 in proximity of Semaphorin-3G (SEMA3G) promoter, thus regulating its expression. Treatment of T1D mice with (R)-PFI-2 improved blood flow reperfusion at 14 days as compared to vehicle-treated animals. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich

Release of basic fibroblast growth factor from acoustically-responsive scaffolds promotes therapeutic angiogenesis in the hind limb ischemia model

Pro-angiogenic growth factors have been studied as potential therapeutics for cardiovascular diseases like critical limb ischemia (CLI). However, the translation of these factors has remained a challenge, in part, due to problems associated with safe and effective delivery. Here, we describe a hydrogel-based delivery system for growth factors where release is modulated by focused ultrasound (FUS), specifically a mechanism termed acoustic droplet vaporization. With these fibrin-based, acoustically-responsive scaffolds (ARSs), release of a growth factor is non-invasively and spatiotemporally-controlled in an on-demand manner using non-thermal FUS. In vitro studies demonstrated sustained release of basic fibroblast growth factor (bFGF) from the ARSs using repeated applications of FUS. In in vivo studies, ARSs containing bFGF were implanted in mice following induction of hind limb ischemia, a preclinical model of CLI. During the 4-week study, mice in the ARS + FUS group longitudinally exhibited significantly more perfusion and less visible necrosis compared to other experimental groups. Additionally, significantly greater angiogenesis and less fibrosis were observed for the ARS + FUS group. Overall, these results highlight a promising, FUS-based method of delivering a pro-angiogenic growth factor for stimulating angiogenesis and reperfusion in a cardiovascular disease model. More broadly, these results could be used to personalize the delivery of therapeutics in different regenerative applications by actively controlling the release of a growth factor.

Exercise Prior to Lower Extremity Peripheral Artery Disease Improves Endurance Capacity and Hindlimb Blood Flow by Inhibiting Muscle Inflammation.

Lower extremity peripheral artery disease (PAD) is associated with functional decline. Physical exercise has been proven to be an effective therapeutic strategy for PAD; however the effect of exercise initiated before PAD remains unknown. Here, we investigated the preventive effects of exercise on endurance capacity, hindlimb perfusion, and on polarization profile of circulating monocytes and limb muscle macrophages. ApoE−/− mice were subjected to 5-week running wheel exercise or remained sedentary before induction of hindlimb ischemia. The two groups were thereafter kept sedentary. Exercised mice prior to PAD showed higher exhaustive treadmill running distance and time than sedentary mice. Preventive exercise also increased perfusion, arteriole density, and muscle regeneration in the ischemic hindlimb. Moreover, preventive exercise prevented ischemia-induced increased gene expression of pro-inflammatory M1 macrophages markers and cytokines in the ischemic muscle, while no changes were observed for anti-inflammatory M2 macrophage markers. Flow cytometry analysis showed that the proportion of circulating pro-inflammatory monocyte subtype decreased whereas that of anti-inflammatory monocytes increased with preventive exercise. Overall, we show that exercise initiated before PAD improves endurance performance and hindlimb perfusion in mice probably via inhibition of M1 macrophage polarization and inflammation in the ischemic muscle. Our study provides experimental evidence for a role of regular exercise in primary prevention of PAD.

Chronic intermittent hypobaric hypoxia attenuates ischemic limb injury by promoting angiogenesis in mice.

This study aimed to evaluate the protective effect of chronic intermittent hypobaric hypoxia (CIHH) against limb ischemic injury. C57BL/6 mice were randomly divided into three groups: limb ischemic injury group (Ischemia, induced by ligation and excision of the left femoral artery), limb ischemia following CIHH pretreatment group (CIHH+Ischemia, simulated a 5000 m altitude hypoxia, 6h per day for 28days, before induction of hind-limb ischemia), and sham group (Sham). The blood flow in the mouse models of hind-limb ischemia was examined using laser doppler imaging. The functional and morphological performance of ischemic muscle was evaluated using contraction force and hematoxylin-eosin and Masson's trichrome staining. Angiogenesis was determined by immunohistochemistry staining of the endothelial markers CD31 and CD34. The protein expressions of angiogenesis-related genes were detected using Western blot assay. Chronic ischemia resulted in reduced blood perfusion, decreased contraction tension, and morphological destruction in gastrocnemius muscle. CIHH pretreatment increased the contractile force and muscle fiber diameter and decreased necrosis and fibrosis of the ischemic muscle. Also, CIHH significantly increased the density of CD31+ and CD34+ cells and promoted the expression of angiogenesis-related molecules in ischemic muscle. These data demonstrate that CIHH has a protective effect against chronic limb ischemia by promoting angiogenesis.

Human Placenta Derived Exosomes Enhance Ischemia Repair in a Mouse Model of Hind-Limb Ischemia

Background Ischemic cardiovascular diseases are the major causes of death in the world. Recently, mesenchymal or tissue progenitor cell derived exosomes are being investigated as an emerging therapeutic agent for treating cardiovascular diseases due to their potentials in restoring the damaged vasculature via promoting angiogenesis. Exosome therapy has the advantages in low immunogenicity and stability features comparing to cells 1. Tissue and organ derived exosomes could have a broader functionality than those derived from a homogeneous cell population as they contain multiple cell types. Placenta is a large and highly vascularized organ and placenta exosomes are known to play essential roles during pregnancy in supporting fetus development 2. Placenta is abundant with endothelial cells, epithelial cells, cytotrophoblast, syncytiotrophoblasts and stem cells including both hematopoietic and non-hematopoietic stem cells. It is perceivable that exosomes isolated from a placenta possess the functions of the cells in a placenta. Materials and Methods Full-term human placentas were obtained under the full consent of healthy donors. Placenta tissues were cultured in serum-free DMEM medium supplemented with antibiotics. After culturing 8 to 16 hours, supernatant was harvested, medium was changed every 8 to 12 hours for up to two days. Placenta derived exosomes (pExo) were isolated through sequential centrifugation of the supernatant of the placenta culture and final preparation were suspended in PBS and stored at -80oC. Quantification of pExo was performed with Bicinchoninic Acid protein assay kit. pExo size was determined using NanoSight. MILLIPLEX-MAP human cytokine/chemokine-PX41 was used to analyze the cytokine composition of pExo. In vitro cell migration assays were performed with seeding 1x10e5/mL human umbilical vessel endothelial cells (HUVEC) in basal media (BM) on the top chamber of an 8-um transwell on a 24-well plate with 500uL BM with or without pExo in the bottom chamber. After 6-24 hours of culture, transwells were observed under an inverted microscope after staining. In vitro cell proliferation assay was performed by seeding HUVEC at 2-4x10e3 cells/96-well with a water-soluble tetrazolium salt-based cell viability assay. To evaluate the pro-angiogenic activity of pExo in vivo, a hind limb ischemia (HLI) model in db/db diabetic mice was used. After surgical induction of HLI on Day 0, pExo from two different donors or vehicle (n=15 each group) was injected via i.v. at 100ug/mice or 100uL on Days 1, 6 and 11. The blood flow through hind paws was measured by Speckle Doppler. Results Utilizing the established cultivation and isolation methods, we achieved the average yield of pExo at 327±91 mg per placenta (n=10). pExo has an average size of 118±15 nm (n=10) as determined with nano-particle tracking analysis, consistent with consensus exosome size ranging 50nm to 200nm 1. Flow cytometry and Western blot analyses confirmed that pExo exhibits characteristic markers known for exosomes including CD9, CD63 and CD81. pExo contained abundant IL-8, HGF, FGF2, PDGF-BB, RANTES, MCP-1 and GM-CSF which are known to play roles in supporting angiogenesis and chemotaxis. In co-culture assays, pExo promoted the proliferation of HUVECs by 149±13% (n=5) comparing to basal medium controls. pExo also demonstated chemotactic activities comparable to that of complete HUVEC growth medium in stimulating the migration of HUVECs across membrane of transwell. Chlorpromazine and Pitstop2 abolished pExo-induced transwell-migration of HUVECs, suggesting that HUVECs may uptake pExo through endocytosis. In the HLI study, pExo treated mice showed significantly higher (50% to 250%) blood flow on Days 14, 28 and 35 comparing to the vehicle group. Histology analysis showed pExo treated mice had 40% to 140% higher density of CD34+ capillary on Day 35, suggesting a significant increase of angiogenesis in the pExo treated groups comparing to the vehicle group. Conclusion: In summary, pExo contains proangiogenic cytokines and chemokines and demonstrates the promising pro-angiogenic activities in vitro and in HLI mice model. These results support further development of pExo as a potential therapeutic agent for the treatment of cardiosvascular diseases.

Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease.

Cell therapy for treatment of peripheral arterial disease (PAD) is a promising approach but is limited by poor cell survival when cells are delivered using saline. The objective of this study was to examine the feasibility of aligned nanofibrillar scaffolds as a vehicle for the delivery of human stromal vascular fraction (SVF), and then to assess the efficacy of the cell-seeded scaffolds in a murine model of PAD. Flow cytometric analysis was performed to characterize the phenotype of SVF cells from freshly isolated lipoaspirate, as well as after attachment onto aligned nanofibrillar scaffolds. Flow cytometry results demonstrated that the SVF consisted of 33.1 ± 9.6% CD45+ cells, a small fraction of CD45–/CD31+ (4.5 ± 3.1%) and 45.4 ± 20.0% of CD45–/CD31–/CD34+ cells. Although the subpopulations of SVF did not change significantly after attachment to the aligned nanofibrillar scaffolds, protein secretion of vascular endothelial growth factor (VEGF) significantly increased by six-fold, compared to SVF cultured in suspension. Importantly, when SVF-seeded scaffolds were transplanted into immunodeficient mice with induced hindlimb ischemia, the cell-seeded scaffolds induced a significant higher mean perfusion ratio after 14 days, compared to cells delivered using saline. Together, these results show that aligned nanofibrillar scaffolds promoted cellular attachment, enhanced the secretion of VEGF from attached SVF cells, and their implantation with attached SVF cells stimulated blood perfusion recovery. These findings have important therapeutic implications for the treatment of PAD using SVF.