Mouse Model Of Hindlimb Ischemia Research Articles

Engineering a Dual-Layer Chitosan–Lactide Hydrogel To Create Endothelial Cell Aggregate-Induced Microvascular Networks In Vitro and Increase Blood Perfusion In Vivo

Here, we report the use of chemically cross-linked and photo-cross-linked hydrogels to engineer human umbilical vein endothelial cell (HUVEC) aggregate-induced microvascular networks to increase blood perfusion in vivo. First, we studied the effect of chemically cross-linked and photo-cross-linked chitosan-lactide hydrogels on stiffness, degradation rates, and HUVEC behaviors. The photo-cross-linked hydrogel was relatively stiff (E = ∼15 kPa) and possessed more compact networks, denser surface texture, and lower enzymatic degradation rates than the relatively soft, chemically cross-linked hydrogel (E = ∼2 kPa). While both hydrogels exhibited nontoxicity, the soft chemically cross-linked hydrogels expedited the formation of cell aggregates compared to the photo-cross-linked hydrogels. Cells on the less stiff, chemically cross-linked hydrogels expressed more matrix metalloproteinase (MMP) activity than the stiffer, photo-cross-linked hydrogel. This difference in MMP activity resulted in a more dramatic decrease in mechanical stiffness after 3 days of incubation for the chemically cross-linked hydrogel, as compared to the photo-cross-linked one. After determining the physical and biological properties of each hydrogel, we accordingly engineered a dual-layer hydrogel construct consisting of the relatively soft, chemically cross-linked hydrogel layer for HUVEC encapsulation, and the relatively stiff, acellular, photo-cross-linked hydrogel for retention of cell-laden microvasculature above. This dual-layer hydrogel construct enabled a lasting HUVEC aggregate-induced microvascular network due to the combination of stable substrate, enriched cell adhesion molecules, and extracellular matrix proteins. We tested the dual-layer hydrogel construct in a mouse model of hind-limb ischemia, where the HUVEC aggregate-induced microvascular networks significantly enhanced blood perfusion rate to ischemic legs and decreased tissue necrosis compared with both no treatment and nonaggregated HUVEC-loaded hydrogels within 2 weeks. This study suggests an effective means for regulating hydrogel properties to facilitate a stable, HUVEC aggregate-induced microvascular network for a variety of vascularized tissue applications.

Inducible HGF-secreting Human Umbilical Cord Blood-derived MSCs Produced via TALEN-mediated Genome Editing Promoted Angiogenesis.

Mesenchymal stem cells (MSCs) promote therapeutic angiogenesis to cure serious vascular disorders. However, their survival period and cytokine-secretory capacity are limited. Although hepatocyte growth factor (HGF) can accelerate the rate of angiogenesis, recombinant HGF is limited because of its very short half-life (<3–5 minutes). Thus, continuous treatment with HGF is required to obtain an effective therapeutic response. To overcome these limitations, we produced genome-edited MSCs that secreted HGF upon drug-specific induction. The inducible HGF expression cassette was integrated into a safe harbor site in an MSC chromosome using the TALEN system, resulting in the production of TetOn-HGF/human umbilical cord blood-derived (hUCB)-MSCs. Functional assessment of the TetOn-HGF/hUCB-MSCs showed that they had enhanced mobility upon the induction of HGF expression. Moreover, long-term exposure by doxycycline (Dox)-treated TetOn-HGF/hUCB-MSCs enhanced the anti-apoptotic responses of genome-edited MSCs subjected to oxidative stress and improved the tube-formation ability. Furthermore, TetOn-HGF/hUCB-MSCs encapsulated by arginine-glycine-aspartic acid (RGD)-alginate microgel induced to express HGF improved in vivo angiogenesis in a mouse hindlimb ischemia model. This study showed that the inducible HGF-expressing hUCB-MSCs are competent to continuously express and secrete HGF in a controlled manner. Thus, the MSCs that express HGF in an inducible manner are a useful therapeutic modality for the treatment of vascular diseases requiring angiogenesis.

Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

AimsThe aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.Methods and ResultsMSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.ConclusionIn two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

Methods for Acute and Subacute Murine Hindlimb Ischemia.

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators' ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Curcumin Ameliorates Ischemia-Induced Limb Injury Through Immunomodulation.

BackgroundThe prevalence of peripheral arterial disease (PAD) is increasing worldwide. Currently, there is no effective treatment for PAD. Curcumin is an ingredient of turmeric that has antioxidant, anti-inflammation, and anticancer properties. In the present study we investigated the potential effect of curcumin in protecting against ischemic limb injury.Material/MethodsWe used an established hindlimb ischemia mouse model in our study. Curcumin was administrated through intraperitoneal (I.P.) injection. Immunohistochemical staining and ELISA assays were performed. Treadmill training was used to evaluate skeletal muscle functions of animals.ResultsOur experiments using in vivo treadmill training showed that curcumin treatment improved the running capacity of animals after ischemic injury. Histological analysis revealed that curcumin treatment significantly reduced the skeletal muscle damage and fibrosis associated with ischemic injury. In order to determine the cellular and molecular mechanisms underlying curcumin-mediated tissue protection, immunohistochemical staining and ELISA assays were performed. The results showed that curcumin treatment led to less macrophage infiltration and less local inflammatory responses as demonstrated by decreasing TNF-α, IL-1, and IL-6 levels. Further immunofluorescent staining of tissue slides indicated that curcumin treatment inhibited the NF-κB signaling pathway. Finally, curcumin can inhibit NF-κB activation induced by LPS in macrophages.ConclusionsOur study results show that curcumin treatment can ameliorate hindlimb injury following ischemic surgery, which suggests that curcumin could be used for PAD treatment.

Platelet-derived factor V promotes angiogenesis in a mouse hind limb ischemia model

Coagulation factor V (FV) is distributed in plasma and platelet pools, which are distinguished by physical and functional differences. FV has been extensively studied for its roles in coagulation. The roles of FV in other physiologic pathways remain understudied. Hind limb ischemia was produced in transgenic mice by femoral artery ligation, with different levels of FV gene expression restricted to the plasma or platelets. Hind limb blood flow perfusion in mice with higher platelet FV was significantly increased. The expression of major angiogenesis-related factors was correlated with the level of FV during ischemia. Furthermore, a platelet depletion and transfusion procedure showed that the transfusion of platelets with higher levels of FV into transgenic mice with undetectable platelet FV significantly rescued the ischemia-mediated impairments in blood flow perfusion. Immunohistochemistry analysis also indicated markedly increased capillary formation in the ischemic muscle of mice with higher platelet FV. Moreover, thrombin activity was significantly higher in the mice with higher platelet FV. Platelets expressing higher levels of FV stimulated increased endothelial cell migration. Hind limb blood flow perfusion was significantly blocked by thrombin inhibitor. These findings suggest that platelet-derived FV contributes to the control of angiogenesis and is likely associated with thrombin generation.

Soluble CD146 boosts therapeutic effect of endothelial progenitors through proteolytic processing of short CD146 isoform.

Endothelial colony-forming cells (ECFC) constitute an endothelial progenitor fraction with a promising interest for the treatment of ischaemic cardiovascular diseases. As soluble CD146 (sCD146) is a new factor promoting angiogenesis, we examined whether sCD146 priming could improve the therapeutic potential of ECFC and defined the involved mechanism. We investigated the effects of sCD146 priming on regenerative properties of ECFC in vivo. In a mouse model of hindlimb ischaemia, the homing of radiolabelled cells to ischaemic tissue was assessed by SPECT-CT imaging. Soluble CD146 priming did not modify the number of engrafted ECFC but improved their survival capacity, leading to an enhanced revascularization. The mechanism of action of sCD146 on ECFC was studied in vitro. We showed that sCD146 acts in ECFC through a signalosome, located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. Soluble CD146 induced a sequential proteolytic cleavage of shCD146, with an extracellular shedding followed by an intramembrane cleavage mediated by matrix metalloprotease (MMP)/ADAM and presenilin-1, respectively. The generated intracellular part of shCD146 was directed towards the nucleus where it associated with the transcription factor CSL and modulated the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). This effect was dependent on both VEGFR1 and VEGFR2, which were rapidly phosphorylated by sCD146. These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases.

Abstract 536: Platelet-derived Factor V Promotes Angiogenesis in a Mouse Hindlimb Ischemia Model

Objective: Coagulation factor V (fV) is distributed in plasma and platelet pools distinguished by physical and functional differences. fV has been extensively studied for its roles in coagulation. The roles of fV in other physiological pathways remain understudied. In the current study, we report that platelet fV is critical for the regulation of angiogenesis in a mouse model of hind-limb ischemia. Methods and Results: Hindlimb ischemia was produced in mice by femoral artery ligation in transgenic mice, with different level of fV gene expression restricted to either the plasma or platelets. The hindlimb blood flow perfusion in mice with higher platelet fV was significantly greater. Furthermore, using a platelet depletion/transfusion procedure, transfusion of platelets with higher level of fV into transgenic mice with undetectable platelet fV significantly rescued the ischemia-mediated impairments in blood flow perfusion. Immunohistochemical analysis also indicated markedly increased capillary formation in ischemic muscle of mice with higher platelet fV. Moreover, thrombin activity was significantly higher in the mice with higher platelet fV. Platelet with higher level of fV demonstrated significantly higher pro-migratory activity in an endothelial cells migration assay. Conclusion: These findings suggest that platelet-derived fV contributes to the control of angiogenesis, in addition to its role in hemostasis. The stimulation is likely associated with thrombin generation due to platelet-derived fV.

Abstract 6: The RNA-binding Protein ADAR2 Controls Interleukin-6-induced Endothelial Cell Pro-inflammatory Response by Regulating MicroRNA Biogenesis

Background: RNA binding proteins (RBPs) are key players in posttranscriptional regulation of mRNAs. Adenosine deaminase acting on RNA-2 (ADAR2) enzyme binds to double-stranded RNAs and controls brain development. The role of ADAR2 in endothelial cell function has not been described so far. Methods and Results: ADAR2 is expressed in human and murine endothelial cells and is 2-fold induced by hypoxia or hind limb ischemia in mice (P&lt;0.05 for all). ADAR2 deficiency resulted in 73±12% impairment of leukocyte infiltration, in 53±4% reduced neovascularization, and a 40±6% decreased blood-flow recovery of ischemic muscle tissues in a hindlimb ischemia mouse model (P&lt;0.001 for all). Mechanistically, ADAR2 knockdown causes a 2-fold downregulation of 522 transcripts, whereas 113 transcripts are found to be upregulated in RNA-seq experiments. Among the endothelial cell enriched, highly ADAR2-regulated transcripts was interleukin-6 signal transducer (IL6ST also known as glycoprotein-130, gp130), the receptor of interleukin-6 (IL-6). Silencing of ADAR2 resulted in a downregulation of gp130 mRNA and protein expression in endothelial cells by 65±5% and 50±5%, respectively (P&lt;0.001 for both). Similarly, the expression of gp130 mRNA was decreased by 50±25% (P&lt;0.05) in murine lung endothelial cells, which derived from transgenic ADAR2-null mice. Silencing of ADAR2 reduced by at least 2-fold the IL-6-mediated STAT3 phosphorylation, the mRNA expression of the IL-6-induced downstream genes MCP-1, VCAM-1 and E-selectin, and the IL-6-induced platelet and leukocyte adhesion on endothelial cells and endothelial cell network formation (P&lt;0.05 for all). Of interest, ADAR2 regulated gp130 mRNA stability. Co-silencing of ADAR2 and Drosha completely restored the ADAR2-regulated gp130 mRNA expression indicating that ADAR2 regulates the maturation of specific microRNAs (miRs). Indeed, ADAR2 regulates by at least 2-fold the expression of 20 microRNAs, including of miR-579 and miR-320e, which are predicted to bind to the 3’-untranslated region of IL6ST mRNA. Conclusions: This study shows for the first time that ADAR2 controls miR biogenesis and thus IL-6/STAT3 signalling in endothelial cells.

Implication of Long noncoding RNAs in the endothelial cell response to hypoxia revealed by RNA-sequencing.

Long noncoding RNAs (lncRNAs) are non-protein coding RNAs regulating gene expression. Although for some lncRNAs a relevant role in hypoxic endothelium has been shown, the regulation and function of lncRNAs is still largely unknown in the vascular physio-pathology. Taking advantage of next-generation sequencing techniques, transcriptomic changes induced by endothelial cell exposure to hypoxia were investigated. Paired-end sequencing of polyadenylated RNA derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% O2 or normoxia was performed. Bioinformatics analysis identified ≈2000 differentially expressed genes, including 122 lncRNAs. Extensive validation was performed by both microarray and qPCR. Among the validated lncRNAs, H19, MIR210HG, MEG9, MALAT1 and MIR22HG were also induced in a mouse model of hindlimb ischemia. To test the functional relevance of lncRNAs in endothelial cells, knockdown of H19 expression was performed. H19 inhibition decreased HUVEC growth, inducing their accumulation in G1 phase of the cell cycle; accordingly, p21 (CDKN1A) expression was increased. Additionally, H19 knockdown also diminished HUVEC ability to form capillary like structures when plated on matrigel. In conclusion, a high-confidence signature of lncRNAs modulated by hypoxia in HUVEC was identified and a significant impact of H19 lncRNA was shown.

Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia

Background and aimsHeparin/protamine micro/nanoparticles (LH/P-MPs) were recently developed as low-molecular weight, biodegradable carriers for adipose-derived stromal cells (ADSCs). These particles can be used for a locally delivered stem cell therapy that promotes angiogenesis. LH/P-MPs bind to the cell surface of ADSCs and promote cell-to-cell interaction and aggregation of ADSCs. Cultured ADSC/LH/P-MP aggregates remain viable. Here, we examined the ability of these aggregates to rescue limb loss in a mouse model of hindlimb ischemia. MethodsUnilateral hindlimb ischemia was induced in adult male BALB/c mice by ligation of the iliac artery and hindlimb vein. For allotransplantation of ADSCs from the same inbred strain, we injected ADSC alone or ADSC/LH/P-MP aggregates or control medium (sham-treated) directly into the ischemic muscles. Ischemic limb blood perfusion, vessel density, and vessel area were recorded. The extent of ischemic limb necrosis or limb loss was assessed on postoperative days 2, 7, and 14. ResultsCompared with the sham-treatment control, treatment with ADSCs alone showed modest effects on blood perfusion recovery and increased the number of α-SMA-positive vessels. Response to ADSC/LH/P-MP aggregates was significantly greater than ADSCs alone for every endpoint. ADSC/LH/P-MP aggregates more effectively prevented the loss of ischemic hindlimbs than ADSCs alone or the sham-treatment. ConclusionThe LH/P-MPs augmented the effects of ADSCs on angiogenesis and reversal of limb ischemia. Use of ADSC/LH/P-MP aggregates offers a novel and convenient treatment method and potentially represents a promising new therapeutic approach to inducing angiogenesis in ischemic diseases.

TRNA-Derived Small Non-Coding RNAs in Response to Ischemia Inhibit Angiogenesis.

Ischemic injuries will lead to necrotic tissue damage, and post-ischemia angiogenesis plays critical roles in blood flow restoration and tissue recovery. Recently, several types of small RNAs have been reported to be involved in this process. In this study, we first generated a rat brain ischemic model to investigate the involvement of new types of small RNAs in ischemia. We utilized deep sequencing and bioinformatics analyses to demonstrate that the level of small RNA fragments derived from tRNAs strikingly increased in the ischemic rat brain. Among these sequences, tRNAVal- and tRNAGly-derived small RNAs account for the most abundant segments. The up-regulation of tRNAVal- and tRNAGly-derived fragments was verified through northern blot and quantitative PCR analyses. The levels of these two fragments also increased in a mouse hindlimb ischemia model and cellular hypoxia model. Importantly, up-regulation of the tRNAVal- and tRNAGly-derived fragments in endothelial cells inhibited cell proliferation, migration and tube formation. Furthermore, we showed that these small RNAs are generated by angiogenin cleavage. Our results indicate that tRNA-derived fragments are involved in tissue ischemia, and we demonstrate for the first time that tRNAVal- and tRNAGly-derived fragments inhibit angiogenesis by modulating the function of endothelial cells.

Therapeutic angiogenesis using tumor cell-conditioned medium.

Stem cell-conditioned medium (CM), which contains angiogenic factors that are secreted by stem cells, represents a potential therapy for ischemic diseases. Along with stem cells, tumor cells also secrete various angiogenic factors. Here, tumor cells as a cell source of CM for therapeutic angiogenesis was evaluated and the therapeutic efficacy of tumor cell CM in mouse hindlimb ischemia models was demonstrated. CM obtained from a human fibrosarcoma HT1080 cell line culture was compared with CM obtained from a human bone marrow-derived mesenchymal stem cell (MSC) culture. HT1080 CM contained higher concentrations of angiogenic factors compared with MSC CM, which was attributable to the higher cell density that resulted from a much faster growth rate of HT1080 cells compared with MSCs. For use in in vitro and in vivo angiogenesis studies, HT1080 CM was diluted such that HT1080 CM and MSC CM would have the same cell number basis. The two types of CMs induced the same extent of human umbilical vein endothelial cell (HUVEC) proliferation in vitro. The injection of HT1080 CM into mouse ischemic limbs significantly improved capillary density and blood perfusion compared with the injection of fresh medium. Although the therapeutic outcome of HT1080 CM was similar to that of MSC CM, the preparation of CM by tumor cell line culture would be much more efficient due to the faster growth and unlimited life-time of the tumor cell line. These data suggest the potential application of tumor cell CM as a therapeutic modality for angiogenesis and ischemic diseases. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:456-464, 2016.

Anti-vascular endothelial growth factor treatment induces blood flow recovery through vascular remodeling in high-fat diet induced diabetic mice

Diabetes mellitus (DM) leads to the development of microvascular diseases and is associated with impaired angiogenesis. The presence of vascular endothelial growth factor (VEGF) can block PDGF-BB dependent regulation of neovascularization and vessel normalization. We tested the hypothesis that the inhibition of VEGF improves blood flow in a mouse hindlimb ischemia model produced by femoral artery ligation. In this study, we examined the effect of bevacizumab, a humanized monoclonal antibody against VEGF-A, on blood perfusion and angiogenesis after hindlimb ischemia. We showed that bevacizumab induces functional blood flow in high fat chow (HFC)-fed diabetic mice. Treatment with bevacizumab increased the expression of platelet derived growth factor-BB (PDGF-BB) in ischemic muscle, and led to vascular normalization. It also blocked vascular leakage by improving the recruitment of pericytes associated with nascent blood vessels, but it did not affect capillary formation. Furthermore, treatment with an anti-PDGF drug significantly inhibited blood flow perfusion in diabetic mice treated with bevacizumab. These results indicate that bevacizumab improves blood flow recovery through the induction of PDGF-BB in a diabetic mouse hindlimb ischemia model, and that vessel normalization may represent a useful strategy for the prevention and treatment of diabetic peripheral arterial disease.

MicroRNA-134 Contributes to Glucose-Induced Endothelial Cell Dysfunction and This Effect Can Be Reversed by Far-Infrared Irradiation

Diabetes mellitus (DM) is a metabolic disease that is increasing worldwide. Furthermore, it is associated with the deregulation of vascular-related functions, which can develop into major complications among DM patients. Endothelial colony forming cells (ECFCs) have the potential to bring about medical repairs because of their post-natal angiogenic activities; however, such activities are impaired by high glucose- (HG) and the DM-associated conditions. Far-infrared radiation (FIR) transfers energy as heat that is perceived by the thermoreceptors in human skin. Several studies have revealed that FIR improves vascular endothelial functioning and boost angiogenesis. FIR has been used as anti-inflammatory therapy and as a clinical treatment for peripheral circulation improvement. In addition to vascular repair, there is increasing evidence to show that FIR can be applied to a variety of diseases, including cardiovascular disorders, hypertension and arthritis. Yet mechanism of action of FIR and the biomarkers that indicate FIR effects remain unclear. MicroRNA-134 (miR-134-5p) was identified by small RNA sequencing as being increased in high glucose (HG) treated dfECFCs (HG-dfECFCs). Highly expressed miR-134 was also validated in dmECFCs by RT-qPCR and it is associated with impaired angiogenic activities of ECFCs. The functioning of ECFCs is improved by FIR treatment and this occurs via a reduction in the level of miR-134 and an increase in the NRIP1 transcript, a direct target of miR-134. Using a mouse ischemic hindlimb model, the recovery of impaired blood flow in the presence of HG-dfECFCs was improved by FIR pretreatment and this enhanced functionality was decreased when there was miR-134 overexpression in the FIR pretreated HG-dfECFCs. In conclusion, our results reveal that the deregulation of miR-134 is involved in angiogenic defects found in DM patients. FIR treatment improves the angiogenic activity of HG-dfECFCs and dmECFCs and FIR has potential as a treatment for DM. Detection of miR-134 expression in FIR-treated ECFCs should help us to explore further the effectiveness of FIR therapy.

Anthrax Toxin Receptor 1 Is Essential for Arteriogenesis in a Mouse Model of Hindlimb Ischemia.

Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1-/- mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases.

Induction of Angiogenesis by a Type III Phosphodiesterase Inhibitor, Cilostazol, Through Activation of Peroxisome Proliferator-Activated Receptor-γ and cAMP Pathways in Vascular Cells.

Peripheral arterial disease is highly prevalent in the elderly and in the subjects with cardiovascular risk factors such as diabetes. Approximately 2% to 4% of those affected with peripheral arterial disease commonly complain of intermittent claudication. Cilostazol, a type III phosphodiesterase inhibitor, is the only Food and Drug Administration-approved drug for the treatment of intermittent claudication. Cilostazol has been shown to be beneficial for the improvement of pain-free walking distance in patients with intermittent claudication in a series of randomized clinical trials. However, the underlying mechanism how cilostazol improved intermittent claudication symptoms is still unclear. In this study, the effect of cilostazol on ischemic leg was investigated in mouse ischemic hindlimb model. Administration of cilostazol significantly increased the expression of hepatocyte growth factor (HGF), vascular endothelial growth factor, angiopoietin-1, and peroxisome proliferator-activated receptor-γ in vasculature. The capillary density in ischemic leg was also significantly increased in cilostazol treatment group when compared with control and aspirin treatment group. However, an increase in capillary density and the expression of growth factors was almost completely abolished by coadministration of HGF-neutralizing antibody, suggesting that cilostazol enhanced angiogenesis mainly through HGF. In vitro experiment revealed that cilostazol treatment increased HGF production in vascular smooth muscle cells via 2 major pathways: peroxisome proliferator-activated receptor-γ and cAMP pathways. Our data suggest that the favorable effects of cilostazol on ischemic leg might be through the angiogenesis through the induction of HGF via peroxisome proliferator-activated receptor-γ and cAMP pathways.

Polymer-DNA Nanoparticle-Induced CXCR4 Overexpression Improves Stem Cell Engraftment and Tissue Regeneration in a Mouse Hindlimb Ischemia Model.

Peripheral arterial disease affects nearly 202 million individuals worldwide, sometimes leading to non-healing ulcers or limb amputations in severe cases. Genetically modified stem cells offer potential advantages for therapeutically inducing angiogenesis via augmented paracrine release mechanisms and tuned dynamic responses to environmental stimuli at disease sites. Here, we report the application of nanoparticle-induced CXCR4-overexpressing stem cells in a mouse hindlimb ischemia model. We found that CXCR4 overexpression improved stem cell survival, modulated inflammation in situ, and accelerated blood reperfusion. These effects, unexpectedly, led to complete limb salvage and skeletal muscle repair, markedly outperforming the efficacy of the conventional angiogenic factor control, VEGF. Importantly, assessment of CXCR4-overexpressing stem cells in vitro revealed that CXCR4 overexpression induced changes in paracrine signaling of stem cells, promoting a therapeutically desirable pro-angiogenic and anti-inflammatory phenotype. These results suggest that nanoparticle-induced CXCR4 overexpression may promote favorable phenotypic changes and therapeutic efficacy of stem cells in response to the ischemic environment.

Angio-Vasculogenic Properties of Endothelial-Induced Mesenchymal Stem Cells Derived From Human Adipose Tissue.

Although stem cells have been regarded as a promising therapeutic option, the marginal therapeutic effects of stem cells are limitations that must be overcome for the development of effective cell therapy. This study sought to identify the angio-vasculogenic properties of endothelial differentiated mesenchymal stem cells (MSCs) and to determine whether these cells are effective for vascular repair. Adipose MSCs were cultured for 10 days under endothelial cell (EC) culture conditions. These endothelial cell differentiated adipose MSCs (EA) and undifferentiated adipose MSCs (UA) were characterized via angiogenesis and adhesion assays. These cells were transplanted into a hindlimb ischemia (HLI) model to determine therapeutic effects and their underlying mechanisms. EA displayed low adhesion and angiogenic properties in vitro compared with UA. When implanted into mouse HLI models, EA exhibited the decreased recovery of blood perfusion in limb ischemia than uncultured UA. Histology data showed that injected EA exhibited lower retention, angiogenic cytokine levels, and neovascularization in vivo than did UA. Short-term differentiated EA display less cell engraftment and angio-vasculogenic potential, and are less effective for peripheral vascular repair than UA. EC differentiation of MSCs may not present an effective strategy for the promotion of therapeutic neovascularization.

A shape-controlled tunable microgel cell delivery platform for low-dose delivery of primed stem cells for in vivo therapeutic neovascularization

Event Abstract Back to Event A shape-controlled tunable microgel cell delivery platform for low-dose delivery of primed stem cells for in vivo therapeutic neovascularization Dilip Thomas1, 2, Grazia Marsico1, Arun Thirumaran2, Bartlomiej Lukasz3, Kerry Thompson4, Peter Dockery4, Brian Rodriguez3, Martina Marchetti-Deschmann5, Timothy O'Brien1, 2 and Abhay Pandit1 1 National University of Ireland Galway, CÚRAM, Ireland 2 National University of Ireland, REMEDI, Ireland 3 University College Dublin, Conway Institute, Ireland 4 National University of Ireland Galway, Anatomy, Ireland 5 Vienna University of Technology, Institute of Chemical Technologies and Analytics, Austria Introduction: Hydrogel-based cell delivery platforms fabricated from synthetic or natural polymers have evolved from a cytoprotective design to a functional biomaterial design, which not only offers better cell survival but also modulates cellular behaviour via intrinsic biophysical or biochemical cues. Tissue regenerative processes are driven by reparative mesenchymal stem cells (MSCs) under the influence of biochemical cues dictated by the microenvironment. Prolonged ischemia and lack of blood supply drive cellular apoptosis and tissue death. However, delivery of primed stem cells via a tunable microenvironment triggers an ‘angiocrine’ response, driving therapeutic angiogenesis[1]. Hence, in this study, it is hypothesised that delivery of primed human mesenchymal stem cells on a shape-controlled microgel platform at a low-cell dose promotes angiogenesis in a murine model of hind-limb ischemia (HLI). The specific objectives of the study were to investigate the cellular behaviour on tunable parameters such as matrix concentration, cross-linker concentration and cell densities and therapeutic efficacy of the optimized group in a hind-limb ischemia model. Materials and Methods: Collagen-based microgels of concentrations 1, 2 and 3mg/ml were fabricated by neutralizing type-I collagen and dispensing a mixture composed of cells and cross-linker 4S-Star-PEG Mw 10000 KDa (Jenkem Tech, U.S.A) on to a hydrophobic surface. hMSCs at an optimized cell density of 0.8x106 were embedded within the microgels[2]. Cell embedded microgels were tested for changes in morphology by confocal microscopy; protein and gene expression quantitation by multiplex ELISA and gene arrays; and changes in matrix stiffness with atomic force microscopy and confocal microscopy. In vivo experiments were performed in a Balb/c nude hind-limb ischemia mouse model. Animal groups (n=12/group) were PBS; microgels alone; microgels with 50,000 hMSCs; 50,000 and 1,000,000 cells alone. Laser-Doppler perfusion and clinical signs of disease severity were assessed along with molecular evaluation using; multiplex ELISA, gene expression arrays and MALDI-imaging mass spectrometry. Statistical analysis was performed using two-way ANOVA with p<0.05. Results and Discussion: The use of 4S-StarPEG combined with the high surface hydrophobicity allowed gelation after 40 minutes, conferring a spherical shape to the microgels. Over 80% viability and low apoptosis was observed using flow-cytometry. 2mg/ml microgels at 0.8x106 cell density showed up-regulation of paracrine factors involved in angiogenesis and distinguishing integrin expression profile (Figure1). AFM analysis revealed insignificant changes in matrix stiffness and unique cell morphology in 2mg/ml microgels compared to 1 and 3mg/ml microgels. Results from the in vivo hind-limb ischemia studies (Figure 2) showed increased blood perfusion, up-regulation of protein and gene markers related to angiogenesis, and changes in glycan environment between the treatment and control groups. Conclusion: Tunable shape-controlled collagen based microgel platform can be used to deliver hMSCs at a low cell dose for angiogenesis in vivo. This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant Number 13/RC/2073. Science Foundation Ireland (SFI) under grant no.09/SRC/B1794.