Mouse Model Of Hindlimb Ischemia Research Articles

Microchannel network hydrogel induced ischemic blood perfusion connection

Angiogenesis induction into damaged sites has long been an unresolved issue. Local treatment with pro-angiogenic molecules has been the most common approach. However, this approach has critical side effects including inflammatory coupling, tumorous vascular activation, and off-target circulation. Here, the concept that a structure can guide desirable biological function is applied to physically engineer three-dimensional channel networks in implant sites, without any therapeutic treatment. Microchannel networks are generated in a gelatin hydrogel to overcome the diffusion limit of nutrients and oxygen three-dimensionally. Hydrogel implantation in mouse and porcine models of hindlimb ischemia rescues severely damaged tissues by the ingrowth of neighboring host vessels with microchannel perfusion. This effect is guided by microchannel size-specific regenerative macrophage polarization with the consequent functional recovery of endothelial cells. Multiple-site implantation reveals hypoxia and neighboring vessels as major causative factors of the beneficial function. This technique may contribute to the development of therapeutics for hypoxia/inflammatory-related diseases.

Mesenchymal Stem Cell-Mediated Therapy of Peripheral Artery Disease Is Stimulated by a Lamin A-Progerin Binding Inhibitor.

ObjectiveHuman adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to promote angiogenesis and tissue repair. However, poor survival and engraftment efficiency of transplanted ASCs are the major bottlenecks for therapeutic application. The present study aims to improve the therapeutic efficacy of ASCs for peripheral artery diseases.MethodsHydrogen peroxide (H2O2) was used to induce apoptotic cell death in ASCs. To measure apoptosis, we used flow cytometry-based apoptosis analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. A murine hindlimb ischemia model was established to measure the ASC-mediated therapeutic angiogenesis and in vivo survival ability of ASCs.ResultsWe identified that the inhibitor of lamin A-progerin binding, JH4, protects ASCs against H2O2-induced oxidative stress and apoptosis. Co-administration of ASCs with JH4 improved ASC-mediated blood reperfusion recovery and limb salvage compared to that of the control group in a mouse hind limb ischemia model. Immunofluorescence showed that JH4 treatment potentiated ASC-mediated vascular regeneration via reducing ASC apoptosis post transplantation.ConclusionJH4 exerts anti-apoptotic effects in ASCs in conditions of oxidative stress, and contributes to the repair of ischemic hind limb injury by improving cell survival.

Arrb2 promotes endothelial progenitor cell-mediated postischemic neovascularization.

Background and aim: Modulating biological functions of endothelial progenitor cells (EPCs) is essential for therapeutic angiogenesis in ischemic vascular diseases. This study aimed to explore the role and molecular mechanisms of β-arrestin 2 (Arrb2) in EPCs biology and angiogenic therapy.Methods: The influence of Arrb2 on postischemic neovascularization was evaluated in Arrb2-deficient mice. The proliferation, apoptosis, and various functions of EPCs were analyzed in vitro by manipulating the expression of Arrb2. Finally, the in vivo effect of Arrb2 on EPC-mediated neovascularization was investigated in a mouse model of hind-limb ischemia (HLI).Results: Arrb2-deficient mice exhibited impaired blood flow recovery based on laser Doppler measurements and reduced capillary density in the adductor muscle after unilateral HLI. Arrb2-deficient mice also showed restricted intraplug angiogenesis in subcutaneously implanted Matrigel plugs. In vitro, lentivirus-mediated Arrb2 overexpression promoted EPC proliferation, migration, adhesion, and tube formation, whereas Arrb2 knockdown had opposite effects. In addition, the overexpression of Arrb2 in EPCs protected them from hypoxia-induced apoptosis and improved intraplug angiogenesis ex vivo. Mechanistically, Arrb2 interacted with and activated extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (Akt) signaling pathways. Finally, the transplantation of EPCs overexpressing Arrb2 resulted in a significantly higher blood flow restoration in ischemic hind limb and higher capillary density during histological analysis compared with control or Arrb2-knockdown EPC-treated nude mice.Conclusions: The data indicated that Arrb2 augmented EPC-mediated neovascularization through the activation of ERK and Akt signaling pathways. This novel biological function of Arrb2 might provide a potential therapeutic option to promote EPCs in the treatment of ischemic vascular diseases.

Dose optimization of decellularized skeletal muscle extracellular matrix hydrogels for improving perfusion and subsequent validation in an aged hindlimb ischemia model.

Peripheral artery disease (PAD) affects more than 27 million individuals in North America and Europe, and current treatment strategies mainly aim to restore blood perfusion. However, many patients are ineligible for existing procedures, and these therapies are often ineffective. Previous studies have demonstrated success of an injectable decellularized skeletal muscle extracellular matrix (ECM) hydrogel in a young rat hindlimb ischemia model of PAD, but further pre-clinical studies are necessary prior to clinical translation. In this study, varying concentrations of a skeletal muscle ECM hydrogel were investigated for material properties and in vivo effects on restoring blood perfusion. Rheological measurements indicated an increase in viscosity and mechanical strength with the higher concentrations of the ECM hydrogels. When injecting dye-labelled ECM hydrogels into a healthy rat, differences were also observed for the spreading and degradation rate of the various concentrations. The three concentrations for the ECM hydrogel were then further examined in a young rat hindlimb ischemia model. The efficacy of the optimal ECM hydrogel concentration was then further confirmed in an aged mouse hindlimb ischemia model. These results further validate the use of decellularized skeletal muscle ECM hydrogels for improving blood perfusion in small animal models of PAD.

Hypoxia-Induced miR-210 Is Necessary for Vascular Regeneration upon Acute Limb Ischemia.

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.

Extracellular Vesicles From Adipose Stem Cells Prevent Muscle Damage and Inflammation in a Mouse Model of Hind Limb Ischemia: Role of Neuregulin-1.

Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of hindlimb ischemia. Approach and Results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxygenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of NRG1 (neuregulin 1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection, vascular growth, and recruitment of inflammatory cells. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration/protection. This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We, therefore, propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle protection.

P3495Long-term engraftment of human CD271-positive adipose-derived stem cells with pericytic and less-aged gene profile in a mouse model of hindlimb ischemia

Abstract Background Therapeutic angiogenesis using adipose-derived stem cells (ADSCs) is an attractive strategy for ischemic cardiovascular diseases. We previously reported that human CD271+ population of adipose-derived stem cells (ADSCs) promoted neovascularization with enhanced engraftment in a mouse model of hindlimb ischemia. However, whether and how CD271+ ADSCs promote the long-term engraftment is still uncertain. Purpose We aimed to examine whether the angiogenic effect and cell engraftment capacity of CD271+ ADSCs would be sustained in long-term period. Then, comparative gene profiling between CD271+ and CD271- ADSCs were analyzed. Finally, cell proliferation and endothelial differentiation assays were conducted. Methods ADSCs were isolated from subcutaneous adipose tissue of 5 patients received cardiovascular surgery. CD271+ and CD271- ADSCs were sorted from CD45-CD31-CD34+ ADSCs fraction by FACS sorting (Fig. A). Cultured CD271+ and CD271- ADSCs at passage 6 were labeled by PKH26 cell linker dye and used for xenograft experiments. Briefly, athymic nude mice were subjected to hindlimb ischemia and one million of human ADSCs were injected into the ischemic muscles. In control group, PBS was solely injected. At 2 and 5 weeks, neovascularization was evaluated by immunohistochemistry (capillary density using lectin perfusion). Cell engraftment was assessed by counting PKH26-positive cells. Furthermore, we compared gene profiling between CD271+ and CD271- ADSCs by microarray. Proliferative capacity was evaluated by colony-forming unit (CFU) assay with Giemsa staining. In endothelial differentiation assay, CD271+ and CD271- ADSCs were cultured in differentiation induction medium containing vascular endothelial growth factor for 2 weeks and stained with anti-human CD31 antibody. Results Cell therapy using CD271+ ADSCs demonstrated approximately 3-fold more enhanced neovascularization than those using CD271- ADSCs or PBS in histological analysis of capillary density at 2 weeks from cell therapy (Fig. B and C). At 5 weeks, mice treated with CD271+ ADSCs were significantly rescued from limb ischemia and this was accompanied by sustained engraftment of ADSCs (Fig. D). In microarray analysis, the differentially expressed 2167 genes were extracted to classify CD271+ and CD271- ADSCs. Pathway analysis demonstrated CD271 expression on ADSCs was associated with the pathways related to stemness and cell differentiation. Indeed, we found that genes related to cell proliferation (PI3K, Cyclin D, and Cyclin D2) were up-regulated in CD271+ ADSCs. Additionally, we found the pericytic marker nestin which was significantly up-regulated in CD271+ ADSCs. Consistent with these findings, CD271+ ADSCs were more proliferative and capable for endothelial differentiation while CD271- ADSCs were not. FACS and cell therapy experiments Conclusion These results suggest that CD271+ ADSCs possess long-term engraftment and angiogenic capacity due to their less-aged and more pericytic gene profile. Acknowledgement/Funding Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan) Grant Number JP16H06828

P5384Postnatal mouse aorta contains yolk sac-derived haemangioblasts with myeloid and endothelial plasticity and vasculogenic capacity

Abstract Background Macrophages and endothelial cells share an intimate relationship during neovessel formation in different pathophysiological conditions. Recent studies have determined that in some tissues, both cell types are derived embryonically from yolk sac (YS) progenitor cells and are maintained postnatally without contribution from circulating sources. The mechanism by which this local “self-maintenance” occurs is unknown. Purpose We previously identified that mouse arteries contain macrophage and endothelial progenitor cells in their adventitial Sca-1+CD45+ compartment. Here we investigated at a clonal level for the existence of postnatal adventitial haemangioblasts and studied their developmental origins. Methods and results Single cell digests were prepared from murine aortas to perform colony-forming unit (CFU) assays in methylcellulose. Aortic cells from C57BL/6J mice selectively generated macrophage colonies (CFU-M) which contained progenitor cells that displayed >95% positive for expression of CD45, Sca-1, c-Kit, CX3CR1 and CSF1R, but negative for Lineage markers, as well as mature monocyte/macrophage (CD11b, F4/80) and endothelial (CD144) markers. Secondary replating of CFU-M progenitors from adult aortas revealed their self-renewal capacity, with 1 in 10 cells forming new CFU-M. Lineage mapping using Flt3CrexRosamT/mG mice demonstrated that aortic CFU-M progenitors were FLT3-ve, indicating that they were not derived from definitive bone marrow haematopoiesis. CFU-M prevalence in C57BL/6J aortas was highest in neonatal mice and diminished progressively with increasing age (∼100 per 105 cells at P1, ∼15 at 12w, ∼5 at 52w, P<0.01, n>4/gp), consistent with prenatal seeding. Embryonic profiling determined that CFU-M progenitors first appeared in extra-embryonic yolk sac around E9.5 and in aorta-gonad-mesonephros at E10.5, before the emergence of definitive haematopoietic stem cells. Inducible fate-mapping then confirmed that aortic CFU-M progenitors originated from CX3CR1+ and CSF1R+ cells in E9.5 yolk sac. Both yolk sac and postnatal aortic CFU-M progenitors generated vascular-like networks when cultured in Matrigel in vitro, containing M2-like macrophages (CD11b+F4/80+CD206+) and endothelial cells (CD31+CD144+). They produced similar progeny and rescued adventitial vascular sprouting when seeded around aortic rings whose adventitia had been stripped. Finally, adoptive transfer of CFU-M progenitors into a mouse model of hindlimb ischaemia resulted in 80% augmentation in hindlimb perfusion compared to cell-free control, with de novo transformation of donor cells into macrophages, endothelial cells and perfused neovessels (n=6). Conclusion To the best of our knowledge, this is the first ever definitive proof at a clonal level for the existence of haemangioblasts in postnatal tissue. Adventitial haemangioblasts originate from extra-embryonic YS and are a source of vasculogenesis in the arterial wall, relevant to vasa vasorum formation. Acknowledgement/Funding NHMRC of Australia (GNT1086796, CDF1161506), NHFA (FLF100412, FLF102056) Royal Australasian College of Physicians

Growth hormone-releasing hormone promotes therapeutic effects of peripheral blood endothelial progenitor cells in ischemic repair.

In peripheral artery disease, blockage of the blood supply to the limbs leads to blood flow attenuation and tissue ischemia. We investigated whether growth hormone-releasing hormone (GHRH) could enhance the biological functions and therapeutic effects of endothelial progenitor cells (EPCs) derived from adult human peripheral blood (PB). EPCs were isolated from human PB (PB-EPCs) and cord blood and expanded in vitro. PB-EPCs incubated with or without GHRH were evaluated for proliferation, migration, and angiogenesis capacity and apoptosis rates under oxidative stress conditions. Activation of STAT3 and Akt pathways was evaluated using Western blot. A hind-limb ischemia (HLI) mouse model was used to study the efficacy of GHRH in improving EPC therapy in vivo. GHRH enhanced the proliferation, migration, and angiogenesis capacity of PB-EPCs and reduced apoptosis under H2O2 stimulation. These beneficial effects were GHRH receptor-dependent and were paralleled by increased phosphorylation of STAT3 and Akt. Transplantation of GHRH-preconditioned EPCs into HLI model mice enhanced blood flow recovery by increasing vascular formation density and enhanced tissue regeneration at the lesion site. Our studies demonstrate a novel role for GHRH in dramatically improving therapeutic angiogenesis in HLI by enhancing the biological functions of EPCs. These findings support additional studies to explore the full potential of GHRH in augmenting cell therapy for the management of ischemia.

Capillary-resident EphA7+ pericytes are multipotent cells with anti-ischemic effects through capillary formation.

The presence of pericytes (PCs) with multipotency and broad distribution along capillary suggests that microvasculature plays a role not only as a duct for blood fluid transport but also as a stem cell niche that contributes to tissue maintenance and regeneration. The lack of an appropriate marker for multipotent PCs still limits our understanding of their pathophysiological roles. We identified the novel marker EphA7 to detect multipotent PCs using microarray analysis of an immortalized PC library. PCs were isolated from microvessels of mouse subcutaneous adipose tissues, then EphA7+ PCs called capillary stem cells (CapSCs) were separated from EphA7− control PCs (ctPCs) using fluorescence‐activated cell sorting system. CapSCs had highly multipotency that enabled them to differentiate into mesenchymal and neuronal lineages compared with ctPCs. CapSCs also differentiated into endothelial cells and PCs to form capillary‐like structures by themselves. Transplantation of CapSCs into ischemic tissues significantly improved blood flow recovery in hind limb ischemia mouse model due to vascular formation compared with that of ctPCs and adipose stromal cells. These data demonstrate that EphA7 identifies a subpopulation of multipotent PCs that have high angiogenesis and regenerative potency and are an attractive target for regenerative therapies.

Forced expression of microRNA-146b reduces TRAF6-dependent inflammation and improves ischemia-induced neovascularization in hypercholesterolemic conditions

Background and aimsMicroRNA (miR)-146 is a key regulator of inflammation, endothelial activation and atherosclerosis. This study sought to define its potential role for the modulation of ischemia-induced neovascularization in atherosclerotic conditions. MethodsNext generation sequencing and qRT-PCR analyses were used to compare microRNA expression in the ischemic muscles of hypercholesterolemic ApoE-deficient (ApoE−/−) mice vs. wild type mice, and in HUVECs exposed or not to oxLDL. Neovascularization was investigated in a mouse model of hindlimb ischemia and the functional activities of HUVECs and pro-angiogenic cells (PACs) were assessed in vitro. ResultsWe found that miR-146b (but not miR-146a) is significantly reduced in the ischemic muscles of ApoE−/− mice, and in HUVECs exposed to oxLDL. Inhibition of miR-146b reduces angiogenesis in vitro, whereas forced expression of miR-146b rescues oxLDL-mediated impairment of endothelial cell proliferation and tube formation. Mechanistically, miR146b directly targets tumor necrosis factor-alpha (TNFa) Receptor Associated Factor 6 (TRAF6) to inhibit inflammation. We found that hypercholesterolemia and oxLDL exposure are associated with higher levels of TRAF6, and increased expression of TNFa. However, forced expression of miR-146b in high cholesterol conditions reduces the expression of these inflammatory factors. In vivo, intramuscular injection of miR-146b mimic reduces ischemic damages and restores blood flow recuperation and capillary density in the ischemic muscles of ApoE−/− mice. Treatment with miR-146b also increases the number and functional activities of pro-angiogenic cells (PACs). ConclusionsHypercholesterolemia is associated with reduced expression of miR-146b, which increases TRAF6-dependent inflammation and is associated with poor neovascularization in response to ischemia. Forced expression of miR-146b using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

Reproducible hindlimb ischemia model based on photochemically induced thrombosis to evaluate angiogenic effects

Critical limb ischemia is one of the most common types of peripheral arterial disease. Preclinical development of ischemia therapeutics relies on the availability of a relevant and reproducible in vivo disease model. Thus, establishing appropriate animal disease models is essential for the development of new therapeutic strategies. Currently, the most commonly employed model of hindlimb ischemia is the surgical induction method with ligation of the femoral artery and its branches after skin incision. However, the efficiency of the method is highly variable depending on the availability of skilled technicians. In addition, after surgical procedures, animals can quickly and spontaneously recover from damage, limiting observations of the therapeutic effect of potential agents. The aim of this study was to develop a hindlimb ischemia mouse model with similarities to human ischemic disease. To that end, a photochemical reaction was used to induce thrombosis in the hindlimb. After the photochemical reaction was induced by light irradiation, thrombotic plugs and adjacent red blood cell stasis were observed in hindlimb vessels in the light-irradiated zone. Additionally, the photochemically induced thrombosis maintained the ischemic condition and did not cause notable side effects in mice.

Abstract 413: Adipose-derived Stem Cells With Thymosin Beta 4 Enhanced Neovascularization in Mouse Ischemic Hind Limb Model

Adipose-derived stem cells (ASCs) have potential of differentiating into endothelial lineage and are easily obtainable, therefore they are promising candidates for stem cell therapy. Thymosin beta 4 (Tb4) is G-actin sequestering protein that exerts a broad range of functions such as cell migration and angiogenesis. In this study, we investigated the effects of Tb4 on angiogenic and endothelial differentiation potential of ASCs. Exogenous treatment of Tb4 (100 ng/mL) induced a significant increase in endogenous mRNA expression of Tb4, and morphological changes with increased cell length in ASCs. In addition, Tb4-treated ASCs showed significantly higher endothelial differentiation potential than untreated ASCs after the induction of endothelial differentiation for 3 weeks. The expression of endothelial markers such as PDGFRb and CD144 were significantly enhanced by the treatment of Tb4 with the endothelial differentiation induction media compared to untreated ASCs. Indeed, Tb4-treated ASCs expressed higher expression of angiogenic genes including Ang-1, vWF, Tie1, CXCR4, uPAR, FGFR4, IGFR2, and VE-Cadherin than untreated ASCs, both cultured in the endothelial differentiation induction media. Moreover, a scratch wound healing assay revealed that the treatment of Tb4 significantly increased cell migration compared to untreated and Tb4-siRNA transfected ASCs. In the microbead sprouting assay, the treatment of Tb4 significantly augmented the number of sprouts per bead and the length of sprouts compared to untreated and Tb4-siRNA transfected ASCs. Futhermore, transplantation of ASCs with Tb4 induced neovascularization improved the blood flow in mouse ischemic hind limb models, compared to sham, ASC-transplanted, and Tb4-transplanted groups. Taken together, therapeutic application of ASCs with Tb4 could be useful to enhance endothelial differentiation and neovascularization.

Abstract 142: New Approach for Directly Reprogrammed Endothelial Cells

Rationale: For therapeutic neovascularization through cell therapy to treat cardiovascular diseases, recently, we successfully generated reprogrammed ECs, directly from human dermal fibroblasts (HDF) with the ETV2 transcription factor. Although functional ECs were generated, ETV2 was delivered lentiviral vector, which is clinically unsuitable. Objective: In this study, we aimed to generate clinically compatible rECs, by employing an adenoviral vector to minimize genetic integration. Furthermore, to prolong the cell retention and improve cell survival, we applied synthetic biomaterial, polypeptide amphiphile (PA) nanomatrix gel and tested for experimental cell therapy in animal models. Methods and Results: After we transduced HDFs with Ad- ETV2 (Ad- ETV2 -HDF), EC gene expression was measured by qRT-PCR and flow cytometry. We found that Ad- ETV2- HDFs displayed a cobblestone-like morphology and showed notable induction of EC genes (CDH5, KDR, PECAM1). Flow cytometry analysis showed peak expression of CDH5 and KDR at D6 and continuous increase of PECAM1 over 9 days. Also, Ad- ETV2 -HDFs took up acetylated LDL and bound lectin, suggesting that these cells possess functional EC characteristics. To enrich cells showing EC characters, Ad- ETV 2-HDFs were sorted for KDR by FACS at day 6. qRT-PCR showed substantial enrichment of EC-specific gene expression in KDR + cells. Only KDR + cells (reprogrammed ECs, rECs) formed tube-like structures on Matrigel. In addition, immunostaining confirmed high expression of EC marker proteins in rECs. Next, we tested the functionality of rECs in hindlimb ischemia mouse model. At 4 weeks after injection of 4 x 10 5 rECs into the ischemic hindlimb, we observed rECs incorporated into host blood vessels or resided perivascular regions. Furthermore, we found more rECs in the tissues and were directly incorporated into the vessels when they were encapsulated in PA nanomatrix gel compared to bare rEC-injected mice. Conclusions: Together, these data strongly support the generation of Ad- ETV2 -induced rECs (Ad-rECs) and enhanced retention of rECs by PA nanomatrix gel in tissue. With this study, we can develop advanced concept and techniques for clinical application of rECs and establish a new platform for future cell therapy.

Transplantation of Adipose Stromal Cell Sheet Producing Hepatocyte Growth Factor Induces Pleiotropic Effect in Ischemic Skeletal Muscle.

Cell therapy remains a promising approach for the treatment of cardiovascular diseases. In this regard, the contemporary trend is the development of methods to overcome low cell viability and enhance their regenerative potential. In the present study, we evaluated the therapeutic potential of gene-modified adipose-derived stromal cells (ADSC) that overexpress hepatocyte growth factor (HGF) in a mice hind limb ischemia model. Angiogenic and neuroprotective effects were assessed following ADSC transplantation in suspension or in the form of cell sheet. We found superior blood flow restoration, tissue vascularization and innervation, and fibrosis reduction after transplantation of HGF-producing ADSC sheet compared to other groups. We suggest that the observed effects are determined by pleiotropic effects of HGF, along with the multifactorial paracrine action of ADSC which remain viable and functionally active within the engineered cell construct. Thus, we demonstrated the high therapeutic potential of the utilized approach for skeletal muscle recovery after ischemic damage associated with complex tissue degenerative effects.

Human Endothelial Colony Forming Cells Express Intracellular CD133 that Modulates their Vasculogenic Properties.

Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n= 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n= 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p< 0.001) and mature endothelial cells (p< 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p= 0.03), capillary density (CD31) (p= 0.01) and myofiber regeneration (p= 0.04) was observed. Also, a significant necrosis (p= 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.

Important roles of endothelial caveolin-1 in endothelium-dependent hyperpolarization and ischemic angiogenesis in mice.

Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. We have previously demonstrated that endothelium-derived H2O2 is an endothelium-dependent hyperpolarization (EDH) factor and that loss of endothelial caveolin-1 reduces EDH/H2O2 in the microcirculation. Caveolin-1 (Cav-1) is a scaffolding/regulatory protein that interacts with diverse signaling pathways, including angiogenesis. However, it remains unclear whether endothelial Cav-1 plays a role in ischemic angiogenesis by modulating EDH/H2O2. In the present study, we thus addressed this issue in a mouse model of hindlimb ischemia using male endothelium-specific Cav-1 (eCav-1) knockout (KO) mice. In isometric tension experiments with femoral arteries from eCav-1-KO mice, reduced EDH-mediated relaxations to acetylcholine and desensitization of sodium nitroprusside-mediated endothelium-independent relaxations were noted ( n = 4~6). An ex vivo aortic ring assay also showed that the extent of microvessel sprouting was significantly reduced in eCav-1-KO mice compared with wild-type (WT) littermates ( n = 12 each). Blood flow recovery at 4 wk assessed with a laser speckle flowmeter after femoral artery ligation was significantly impaired in eCav-1-KO mice compared with WT littermates ( n = 10 each) and was associated with reduced capillary density and muscle fibrosis in the legs ( n = 6 each). Importantly, posttranslational protein modifications by reactive nitrogen species and ROS, as evaluated by thiol glutathione adducts and nitrotyrosine, respectively, were both increased in eCav-1-KO mice ( n = 6~7 each). These results indicate that endothelial Cav-1 plays an important role in EDH-mediated vasodilatation and ischemic angiogenesis through posttranslational protein modifications by nitrooxidative stress in mice in vivo. NEW & NOTEWORTHY Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. The present study provides a line of novel evidence that endothelial caveolin-1 plays important roles in endothelium-dependent hyperpolarization and ischemic angiogenesis in hindlimb ischemia in mice through posttranslational protein modifications by reactive nitrogen species and ROS in mice in vivo.

Gelatin-based micro-hydrogel carrying genetically engineered human endothelial cells for neovascularization

Cell delivery systems based on micro-hydrogels may facilitate the long-term survival of cells upon transplantation. Micro-hydrogels may effectively support cell proliferation, attachment, and migration in ischemic environments. In this study, we report the fabrication of a gelatin methacrylate (GelMA)-based micro-hydrogel for efficient in vivo delivery of genetically engineered endothelial cells. Micro-hydrogels were initially processed via electrospraying of GelMA and alginate (ALG) mixtures (at different ratios) on to calcium chloride (CaCl2) solution. Electrospraying of the GelMA/ALG mixture resulted in the formation of a micro-hydrogel, owing to ALG crosslinking. Secondary crosslinking of GelMA with UV light and ALG hydrogel chelation using sodium citrate solution resulted in GelMA-based micro-hydrogel formation. We observed the angiogenic response of human umbilical vein endothelial cells (HUVECs) in GelMA concentration-dependent manner. The seeding of HUVECs engineered to express human vascular endothelial growth factor on to the GelMA micro-hydrogel and the subsequent transplantation of the micro-hydrogel into a hindlimb ischemia model effectively attenuated the ischemia condition. This facile and simple micro-hydrogel fabrication strategy may serve as a robust method to fabricate efficient cell carriers for various ischemic diseases. Statement of SignificanceFor the therapeutic angiogenesis, it is important to provide the therapeutic cells with a carrier that could stabilize therapeutic cells and facilitate long-term survival of cells. Furthermore, it is also important to administer as many therapeutic cells as possible in a fixed volume. From these cues, we fabricated ECM-based micro-hydrogel produced by the high through-put system. And we intended to facilitate activation of therapeutic cells by coating the therapeutic cells onto the micro-hydrogel. In this manuscript, we fabricated methacrylate gelatin (GelMA) based micro-hydrogels using the electro-spraying method and coated HUVECs engineered to express hVEGF onto the micro-hydrogels. Then, we identified that GelMA concentration-dependent angiogenic response of HUVECs. Furthermore, we demonstrated that the VEGF secreting HUVEC-GelMA micro-hydrogels induced the restoration of blood flow and neovascularization in a hind-limb ischemia mouse model. These findings demonstrate that the high-throughput fabrication of ECM micro-hydrogels could be a novel platform to apply in neovascularization and tissue engineering.

Phorbol ester-induced angiogenesis of endothelial progenitor cells: The role of NADPH oxidase-mediated, redox-related matrix metalloproteinase pathways.

Endothelial progenitor cells (EPCs) may contribute to ischemia-induced angiogenesis in atherosclerotic diseases. The protein kinase C (PKC) family is involved in the regulation of angiogenesis, however the role of PKCα in EPCs during angiogenesis is unclear. The aim of this study was to evaluate the role of PKCα in EPCs during angiogenesis. Phorbol-12-myristate-13-acetate (PMA), a PKCα activator, significantly increased the activity and expression of matrix metalloproteinases (MMP) -2 and -9 in human (late outgrowth) EPCs in vitro. The MMPs promoted the migratory function and vascular formation of EPCs, which then contributed to neovascularization in a mouse hindlimb-ischemia model. Reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enhanced the expression of MMPs to increase the bioactivity of EPCs during angiogenesis. The mitogen-activated protein kinase (MAPK) signal pathway was associated with the activation of NADPH oxidase. PMA extensively activated the extracellular signal–regulated kinase (Erk) signal pathway to increase the expression of MMP-9. PMA also activated the p38, Erk, and c-Jun N-terminal kinase signal pathways to increase the expression of MMP-2. PMA-stimulated EPCs enhanced neovascularization in a mouse model of hindlimb ischemia via nuclear factor-κB translocation to up-regulation of the expression of MMP-2 and MMP-9. PMA could activate PKCα and promote the angiogenesis capacity of human EPCs via NADPH oxidase-mediated, redox-related, MMP-2 and MMP-9 pathways. The PKCα-activated, NADPH oxidase-mediated, redox-related MMP pathways could contribute to the function of human EPCs for ischemia-induced neovascularization, which may provide novel insights into the potential modification of EPCs for therapeutic angiogenesis.

Noninvasive monitoring of the development and treatment response of ischemic hindlimb by targeting matrix metalloproteinase-2 (MMP-2).

Critical limb ischemia (CLI) is a common cause of high vascular morbidity and mortality. Monitoring the development and treatment response of hindlimb ischemia (HI) in an animal model enables a better understanding of the pathological mechanisms underlying CLI, and evaluation of the efficacy of novel therapeutic approaches. Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue including extracellular matrix degradation and angiogenesis. Herein, a mouse HI model is established and subjected to noninvasive optical imaging with a novel and ultra-sensitive MMP activatable probe, termed MMP-P12, for analyzing the development and treatment response of HI. Our results show that angiogenesis development during HI was well correlated with MMP-2 activity alteration as examined by western blot, histological staining and MMP-P12 fluorescence signal recovery. Moreover, vascular endothelial growth factor (VEGF) mediated HI treatment was also monitored by MMP-P12. Up-regulated MMP-2 expression and an enhancement of angiogenesis were observed after VEGF treatment, which peaked at 7 days after the treatment. Overall, our results showed that MMP-2 plays an important role in the monitoring of angiogenesis during HI development and therapy. Application of MMP-P12 to visualize MMP-2 activity alteration can serve as a promising noninvasive optical imaging strategy to monitor angiogenesis and its response to therapy in CLI.