Ischemic Muscle Tissue Research Articles

An evaluation of rivaroxaban and clopidogrel in a rat lower extremity ischemia-reperfusion model: An experimental study.

This study aims to compare clopidogrel and rivaroxaban against ischemia-reperfusion injury after a long reperfusion time and to investigate its effects on various tissues. A total of 40 Wistar rats were included in the study and were randomly divided into four groups (n=10 per group). Groups were defined as follows: control (Group 1), sham (Group 2), clopidogrel pre-treatment (Group 3), and rivaroxaban pre-treatment (Group 4). Ischemia (6 h) and reperfusion (8 h) were induced at the lower hind limb in Groups 2, 3, and 4. The ischemic muscle, heart, kidney, liver, and plasma tissues of the subjects were obtained to test for the oxidant (malondialdehyde) and antioxidants (glutathione, superoxide dismutase, and nitric oxide). Malondialdehyde levels were significantly higher in the sham group, compared to the controls in all tissues. Clopidogrel and rivaroxaban pre-treatment significantly decreased malondialdehyde levels, compared to the heart, ischemic muscle, liver, and blood tissues of the sham group. Kidney malondialdehyde levels were reduced only by rivaroxaban. Group 4 had significantly decreased malondialdehyde levels, compared to Group 3 in ischemic muscle (p<0.010). The glutathione reduction, compared to sham group, in the kidney was only significant for Group 4 (p<0.050). With clopidogrel and rivaroxaban pretreatment, nitric oxide levels significantly decreased only in the heart tissue, compared to sham group (p<0.001 and p<0.050, respectively). The study results suggest that rivaroxaban and clopidogrel are effective in reducing ischemia-reperfusion injury in the heart, ischemic muscle, liver, and blood. Rivaroxaban also protects the kidneys and is superior to clopidogrel in ischemic muscle protection.

Sodium Tanshinone IIA sulfonate improves post-ischemic angiogenesis in hyperglycemia

BackgroundDiabetes is a strong risk factor of peripheral arterial disease (PAD), and also leads to impaired perfusion recovery in the ischemic limb, which eventually results in poor outcomes in PAD patients. Sodium Tanshinone IIA Sulfonate (STS), a monomer from herbs, has been shown to improve the outcomes in a variety of ischemic disease including myocardial infarction. However, the effects of STS treatment in PAD is not known. Methods and resultsUnilateral femoral artery was ligated in mice as experimental PAD models, STS treatment improved perfusion recovery, increased capillary densities, decreased reactive oxygen species (ROS) level and microRNA-133a (miR-133a) expression in the ischemic hindlimb in diabetic mice; however, STS did not change perfusion recovery in non-diabetic C57BL/6 mice. Ischemic muscle tissue from diabetic mice was harvested 7 days after femoral ligation for biochemical test, STS resulted in reduced malondialdehyde (MDA), and increased GTP cyclohydrolase 1 (GCH1) and cyclic guanine monophosphate (cGMP) levels. In addition, STS treatment increased miR-133a expression in endothelial cells isolated from ischemic muscle tissue of diabetic mice. In endothelial cells cultured in high glucose medium, STS increased tube formation and nitric oxide (NO) production, and reduced cellular ROS level and miR-133a expression under simulated ischemic condition. In addition, GCH1 inhibitor or miR-133a overexpression using exogenous microRNA mimic blunted STS-induced angiogenic effects and ROS neutralization in cultured endothelial cells under hyperglycemic and hypoxic conditions. ConclusionThese findings demonstrate STS improves angiogenesis via inhibiting miR-133a expression and increasing GCH-1 protein levels in experimental PAD with diabetes.

1438MicroRNA editing is integral for interleukin-6 trans-signalling and leukocyte trafficking to ischemic tissues

Abstract Background/Aim Adenosine to inosine RNA editing is an essential post-transcriptional RNA modification catalysed by adenosine deaminase acting on RNA-1 and -2 (ADAR1; ADAR2). Endothelial cells (ECs) attract and guide leukocytes to sites of ischemic tissue injury. Here we studied the role of RNA editing in ischemic disease. Methods Primary human and murine vascular endothelial cell cultures were used to assess the EC responses to interleukin-6 (IL-6) or ischemia. For the animal studies, the effect of ADAR2 in acute and chronic ischemic disease was evaluated in cremaster muscle microcirculation by intravital microscopy, in peritoneal cavity after sterile peritonitis and in gastrocnemius muscle after hind-limb ischemia by 8-colour flow cytometry and immunohistochemistry (IHC) studies of Adar2−/−/tg as well as of i(nducible)EC-ADAR2 knockout (KO) mice. For the mechanistic studies, deep RNA sequencing, qRT-PCR, western blot, confocal microscopy, target-specific microRNA (miRNA) editing studies, RNA-immunoprecipitation, miRNA/plasmid silencing/overexpression and luciferase reporter assays were used among others. For human studies, ischemic tissues derived from patients with acute or chronic ischemic heart disease were processed. Results ADAR2, but not ADAR1, expression is induced by &gt;2-fold in hypoxic ECs and in ischemic vascular ECs in mice and humans. Unbiased gene ontology analysis of the EC transcriptome indicated that ADAR2 controls inflammatory responses and predominantly the expression of interleukin-6-signal transducer (IL6ST), the co-receptor of IL-6. Subsequently, ADAR2 controls IL-6 trans-signalling in ECs as documented by the STAT3 phosphorylation and expression of the downstream leukocyte adhesion molecules, E-selectin and VCAM-1. IL-6-inflamed cremaster muscles showed that rolling and adhesion of leukocyte subsets to vascular wall were severely impaired in Adar2−/−/tg mice. Leukocyte transmigration was also diminished by &gt;2-fold in Adar2−/−/tg and in iEC-ADAR2 KO mice in response to IL-6 or ischemia. Similar results were obtained for leukocyte rolling, adhesion and infiltration after acute (4h) and chronic (3d; 21d) ischemia from iEC-ADAR2 KO mice and human ischemic muscle tissues. Next we studied how ADAR2 controls IL6ST expression. ADAR2-deficient vascular EC miRNAome revealed the upregulation of a conserved group of miRNAs targeting the IL6ST mRNA including miR-199a-5p and miR-335-3p. At a single-nucleotide level, ADAR2-induced RNA editing of the stem loops of the primary miR-199a1/2 and miR-335 directly disrupted Drosha recruitment to both and thus inhibited their maturation process. Accordingly, rescue experiments using miRNA-inhibitors restored IL6ST levels after ADAR2 deficiency. Conclusion Taking together, inhibition of the microRNA maturation process by ADAR2-mediated RNA editing is integral for IL-6 trans-signalling in vascular endothelium and subsequent leukocyte trafficking to ischemic tissues in mice and humans.

In experimental peripheral arterial disease, type 2 diabetes alters post-ischemic gene expression

Peripheral arterial disease is characterized by impaired blood flow to tissues outside the heart due to atherosclerosis and it most frequently occurs in the lower extremities. Type 2 diabetes (T2D) is a well-known risk factor that accelerate the course and contributes to poor clinical outcomes of PAD. While there is some evidence that T2D is associated with altered expression of genes involved in regulating PAD severity, our knowledge about the specific genes and pathways involved remains incomplete.We induced experimental PAD or hind limb ischemia in T2D and non-diabetic mice and subjected the ischemic gastrocnemius muscle tissues to genome-wide mRNA transcriptome analysis. We subsequently performed pathway analysis on the top 500 genes that showed the most significant expression differences between the ischemic diabetic and ischemic non-diabetic muscle tissues. Pathway analysis of the differentially expressed genes identified pathways involved in essential biological processes such as “metabolic pathways,” “phagosomes,” “lysosomes,” and “regulation of actin cytoskeleton”. Overall, our data provides the opportunity to test hypotheses on the potential role of the altered genes/molecular pathways in poor PAD outcomes in diabetes.

Type 1 diabetes alters ischemia-induced gene expression

Peripheral Artery Disease (PAD) is a chronic, activity-limiting disease that is caused by atherosclerotic occlusion of blood vessels outside the heart. Type 1 Diabetes (T1D) not only increases an individual’s likelihood of developing PAD, but also contributes to poor clinical outcomes after PAD manifestation. Although there is some evidence suggesting that hyperglycemia might alter expression of genes involved in regulating PAD severity or outcomes, our knowledge about the specific genes and pathways involved remains incomplete.We induced experimental PAD or hind limb ischemia in T1D and non-diabetic mice and subjected the ischemic gastrocnemius muscle tissues to genome-wide mRNA transcriptome and pathway analysis. We identified 513 probe sets that represented 443 different genes with highly significant expression differences (p < 0.005) between the ischemic diabetic and ischemic non-diabetic muscle tissues. Moreover, pathway analysis of the differentially expressed genes identified pathways involved in essential biological processes such as “cell cycle,” “DNA replication,” “metabolic pathways,” “focal adhesion,” “regulation of actin cytoskeleton,” and “nucleotide excision repair”. Taken together, our data offer the opportunity to test hypotheses on the roles played by the altered genes/molecular pathways in poor PAD outcomes in diabetes. Such studies may lead to the development of specific therapies to improve PAD outcomes in patients with comorbid diabetes.

Curcumin improves perfusion recovery in experimental peripheral arterial disease by upregulating microRNA-93 expression.

In peripheral arterial disease (PAD), angiogenesis is the major process involved in repairing the microvasculature in the ischemic lower limb. Curcumin, a monomer isolated from turmeric roots, has been demonstrated to have pro- and anti-angiogenic effects under different circumstances. Previous studies have indicated that curcumin treatment improves tissue repair and perfusion recovery in a mouse model of diabetic PAD. However, the effects of curcumin on PAD under non-diabetic conditions has remained elusive, In the present study, mice with PAD and a normal glycaemic profile were treated with curcumin, which improved perfusion recovery, increased capillary density and elevated microRNA (miR)-93 expression in ischemic muscle tissue. In cultured endothelial cells under simulated ischemia, curcumin improved endothelial cell viability and enhanced tube formation. However, following miR-93 knockdown using a microRNA inhibitor, endothelial cell tube formation was inhibited. Furthermore, in the presence of the miR-93 inhibitor, curcumin did not alter endothelial cell viability or tube formation. These results demonstrate that curcumin had beneficial effects in non-diabetic PAD by improving angiogenesis, which may have been achieved partially via the promotion of miR-93 expression.

Umbilical cord-derived mesenchymal stromal/stem cells enhance recovery of surgically induced skeletal muscle ischemia in a rat model.

This study delves into possible mechanisms underlying the stimulating influence of UC-MSCs transplantation on functional and structural recovery of ischemic skeletal muscles. Limb ischemia was created in Sprague-Dawley rats by excision of femoral and popliteal arteries. Allogeneic rat PKH26-labeled UC-MSCs were administered by direct intramuscular injection. Animals of experimental group responded to the transplantation by improvement in their locomotor function as assessed by the rotarod performance test on day 9 and 29 after transplantation. Histomorphometric analysis showed that relative area of the lesions in the experimental group was significantly smaller than in the control group at all time points during the observation. Calculated densities of microcirculation vessels within the lesions were significantly higher in the experimental group than in the control group on day 10 after transplantation. Only a part of the transplanted allogeneic UC-MSCs survived within the ischemic muscle tissue, and a considerable portion of these surviving cells were found alongside the VEGF-producing preserved muscle fibers. The PKH26 label was not found within the walls of capillaries or larger blood vessels. The administration of allogeneic UC-MSCs significantly increased the proportion of M2 macrophages, exhibiting proangiogenic and anti-inflammatory properties, for at least 10 days following the transplantation.

Hydrogen molecules (H2) improve perfusion recovery via antioxidant effects in experimental peripheral arterial disease.

Reactive oxygen species (ROS) impair neovascularization and perfusion recovery following limb ischemia in patients with peripheral arterial disease (PAD). Hydrogen molecules (H2) comprise an antioxidant gas that has been reported to neutralize cytotoxic ROS. The present study investigated whether H2 may serve as a novel therapeutic strategy for PAD. H2-saturated water or dehydrogenized water was supplied to mice with experimental PAD. Laser Doppler perfusion imaging demonstrated that H2-saturated water improved perfusion recovery, decreased the rate of necrosis, increased the capillary density in the gastrocnemius muscle and increased the artery density in the abductor muscle in the ischemic limbs, at 14 and 21 days post-hindlimb ischemia. Ischemic muscle tissue was harvested 7 days after experimental PAD for biochemical testing and H2 was observed to reduce the levels of malondialdehyde and increase the levels of cyclic guanine monophosphate (cGMP). In cultured endothelial cells, H2-saturated culture medium resulted in reduced ROS levels, increased tube formation and increased cGMP levels. In macrophages, H2 decreased cellular ROS levels and promoted M2 polarization. H2-saturated water increases angiogenesis and arteriogenesis and subsequently improves perfusion recovery in a mouse PAD model via reduction of ROS levels.

Interleukin-22 receptor 1 upregulation and activation in hypoxic endothelial cells improves perfusion recovery in experimental peripheral arterial disease

ObjectiveInflammation induced by muscle ischemia is involved in tissue repair and perfusion recovery in peripheral arterial disease (PAD) patients. Interleukin (IL)-22 is an inflammatory cytokine discovered in recent years and shows versatile functions; however, its role in PAD remains unknown. Here, we test whether IL-22 and its receptors are involved in angiogenesis in experimental PAD. Methods and resultsBoth IL-22 and its receptor—IL-22 receptor 1(IL-22R1) were upregulated in muscle and endothelial cells after ischemia. In experimental PAD models, blocking IL-22 using IL-22 monoclonal antibody impaired perfusion recovery and angiogenesis; on the other hand, IL-22 treatment improved perfusion recovery. Ischemic muscle tissue was harvested 3 days after experimental PAD for biochemical test, IL-22 antagonism resulted in decreased Signal Transducer and Activator of Transcription (STAT3) phosphorylation, but did not alter the levels of VEGF-A or cyclic guanine monophosphate (cGMP) levels in ischemic muscle. In cultured endothelial cells, IL-22R1 was upregulated under simulated ischemic conditions, and IL-22 treatment increased STAT3 phosphorylation, endothelial cell survival and tube formation. Knock down of IL-22R1 or treatment with STAT3 inhibitor blunted IL-22-induced endothelial cell survival or tube formation. ConculsionIschemia-induced IL-22 and IL-22R1 upregulation improves angiogenesis in PAD by inducing STAT3 phosphorylation in endothelial cells. IL-22R1 may serve as a new therapeutic target for PAD.

Ager Deletion Enhances Ischemic Muscle Inflammation, Angiogenesis, and Blood Flow Recovery in Diabetic Mice.

Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block angiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated angiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1 (early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, angiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. These findings indicate that RAGE attenuates adaptive inflammation in hindlimb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic peripheral vascular disease.

Abstract 8: Endothelial TFEB Regulates Postischemic Angiogenesis via AMPKalpha Activation

Rationale: Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of angiogenesis are not fully unraveled. Transcription factor-EB (TFEB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in the vascular disease is less understood. Objective: We aim to determine the role of endothelial TFEB in postischemic angiogenesis and underlying molecular mechanism. Methods and Results: In a murine hindlimb ischemic model, we demonstrated that TFEB was upregulated in the ischemic skeletal muscle tissue. Utilizing genetically-engineered endothelial cell (EC) specific TFEB transgenic mice, we investigated the function of TFEB in postischemic angiogenesis. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates (n = 8-9 for each group, p &lt; 0.01). Furthermore, we found that blood flow recovery was attenuated in EC-selective TFEB deficient mice compared with control mice (n =8-9 for each group, p&lt; 0.01). In aortic ring cultures, we found that TFEB transgene significantly increased the vessel sprouting. Adenovirus-mediated TFEB overexpression promoted EC tube formation whereas small interfering RNA (siRNA)-mediated TFEB knockdown suppressed tube formation in ECs. Mechanistically, TFEB activated calcium/calmodulin-dependent protein kinase kinase-β and AMP-activated protein kinase (AMPK)-α signaling pathway. Through pharmacological inactivation and siRNA-mediated knockdown of AMPKα, we demonstrated that AMPKα is necessary for TFEB to regulate tube formation in ECs. Conclusions: In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα signaling, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

Trimodal rescue of hind limb ischemia with growth factors, cells, and nanocarriers: fundamentals to clinical trials.

Peripheral artery disease is a severe medical condition commonly characterized by critical or acute limb ischemia. Gradual accumulation of thrombotic plaques in peripheral arteries of the lower limb may lead to intermittent claudication or ischemia in muscle tissue. Ischemic muscle tissue with lesions may become infected, resulting in a non-healing wound. Stable progression of the non-healing wound associated with severe ischemia might lead to functional deterioration of the limb, which, depending on the severity, can result in amputation. Immediate rescue of ischemic muscles through revascularization strategies is considered the gold standard to treat critical limb ischemia. Growth factors offer multiple levels of protection in revascularization of ischemic tissue. In this review, the basic mechanism through which growth factors exert their beneficial properties to rescue the ischemic limb is extensively discussed. Moreover, clinical trials based on growth factor and stem cell therapy to treat critical limb ischemia are considered. The clinical utility of stem cell therapy for the treatment of limb ischemia is explained and recent advances in nanocarrier technology for selective growth factor and stem cell supplementation are summarized.

Administration of FTY720 during Tourniquet-Induced Limb Ischemia Reperfusion Injury Attenuates Systemic Inflammation.

Acute ischemia-reperfusion injury (IRI) of the extremities leads to local and systemic inflammatory changes which can hinder limb function and can be life threatening. This study examined whether the administration of the T-cell sequestration agent, FTY720, following hind limb tourniquet-induced skeletal muscle IRI in a rat model would attenuate systemic inflammation and multiple end organ injury. Sprague-Dawley rats were subjected to 1 hr of ischemia via application of a rubber band tourniquet. Animals were randomized to receive an intravenous bolus of either vehicle control or FTY720 15 min after band placement. Rats (n = 10/time point) were euthanized at 6, 24, and 72 hr post-IRI. Peripheral blood as well as lung, liver, kidney, and ischemic muscle tissue was analyzed and compared between groups. FTY720 treatment markedly decreased the number of peripheral blood T cells (p < 0.05) resulting in a decreased systemic inflammatory response and lower serum creatinine levels and had a modest but significant effect in decreasing the transcription of injury-associated target genes in multiple end organs. These findings suggest that early intervention with FTY720 may benefit the treatment of IRI of the limb. Further preclinical studies are necessary to characterize the short-term and long-term beneficial effects of FTY720 following tourniquet-induced IRI.

Dietary glutamine supplementation enhances endothelial progenitor cell mobilization in streptozotocin-induced diabetic mice subjected to limb ischemia

Diabetes is a metabolic disorder with increased risk of vascular diseases. Tissue ischemia may occur with diabetic vascular complications. Bone marrow-derived endothelial progenitor cells (EPCs) constitute a reparative response to ischemic injury. This study investigated the effects of oral glutamine (GLN) supplementation on circulating EPC mobilization and expression of tissue EPC-releasing markers in diabetic mice subjected to limb ischemia. Diabetes was induced by a daily intraperitoneal injection of streptozotocin for 5 days. Diabetic mice were divided into 2 nonischemic groups and 6 ischemic groups. One of the nonischemic and 3 ischemic groups were fed the control diet, while the remaining 4 groups received diets with identical components except that part of the casein was replaced by GLN. The respective diets were fed to the mice for 3 weeks, and then the nonischemic mice were sacrificed. Unilateral hindlimb ischemia was created in the ischemic groups, and mice were sacrificed at 1, 7 or 21 days after ischemia. Their blood and ischemic muscle tissues were collected for further analyses. Results showed that plasma matrix metallopeptidase (MMP)-9 and the circulating EPC percentage increased after limb ischemia in a diabetic condition. Compared to groups without GLN, GLN supplementation up-regulated plasma stromal cell-derived factor (SDF)-1 and muscle MMP-9, SDF-1, hypoxia-inducible factor-1 and vascular endothelial growth factor gene expression. The CD31-immunoreactive intensities were also higher in the ischemic limb. These findings suggest that GLN supplementation enhanced circulating EPC mobilization that may promote endothelium repair at ischemic tissue in diabetic mice subjected to limb ischemia.

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.

Abstract 61: Deficiency of the RNA Editing Enzyme ADAR2 Impairs Inflammation, Neovascularization and Functional Recovery of Ischemic Tissues

Background: Adenosine deaminase acting on RNA-2 (ADAR2) enzyme catalyzes adenosine-to-inosine (A-to-I) RNA editing of mRNAs and microRNAs and controls brain development. However, the role of endothelial cell ADAR2 in vascular biology and inflammation 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, among the highly ADAR2-regulated transcripts was interleukin-6 signal transducer (IL6ST or 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. In order to investigate the physiological relevance we treated endothelial cells, which were transfected with a siRNA against ADAR2 or scrambled control, with IL-6 and sIL6Ra. 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 miRNAs. 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: Taken together, ADAR2 constitutes a potential candidate for therapeutic modulation of angiogenesis and inflammation in ischemic tissues.

Local delivery of VEGF and SDF enhances endothelial progenitor cell recruitment and resultant recovery from ischemia.

Biomaterials may improve outcomes of endothelial progenitor-based therapies for the treatment of ischemic cardiovascular disease, due to their ability to direct cell behavior. We hypothesized that local, sustained delivery of exogenous vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF) from alginate hydrogels could increase recruitment of systemically infused endothelial progenitors to ischemic tissue, and subsequent neovascularization. VEGF and SDF were found to enhance in vitro adhesion and migration of outgrowth endothelial cells (OECs) and circulating angiogenic cells (CACs), two populations of endothelial progenitors, by twofold to sixfold, and nearly doubled recruitment to both ischemic and nonischemic muscle tissue in vivo. Local delivery of VEGF and SDF to ischemic hind-limbs in combination with systemic CAC delivery significantly improved functional perfusion recovery over OEC delivery, or either treatment alone. Compared with OECs, CACs were more responsive to VEGF and SDF treatment, promoted in vitro endothelial sprout formation in a paracrine manner more potently, and demonstrated greater influence on infiltrating inflammatory cells in vivo. These studies demonstrate that accumulation of infused endothelial progenitors can be enriched using biomaterial-based delivery of VEGF and SDF, and emphasize the therapeutic benefit of using CACs for the treatment of ischemia.

Nanoparticle oxygen delivery to the ischemic heart.

Currently there are no medications that can be administered to help deliver more oxygen to the myocardial region experiencing abnormal perfusion. The purpose of this study was to look at the nanoparticle dodecafluoropentane in an emulsion as an oxygen carrier. Using nanoparticles as an oxygen carrier is advantageous because they are able to carry oxygen past blockages that are obstructing red blood cells (6-8 µm) due to their smaller size (250 nm). With the reintroduction of oxygen to the ischemic muscle tissue, a reduced infarct size should be seen. Male C57BL/6J mice underwent left anterior descending artery (LAD) ligation using 8-0 monofilament nylon suture. Immediately after ligation of the LAD, the control group received a 200-µl intravenous injection of phosphate buffered saline (PBS). The treated group received a dose of 0.6 ml/kg of dodecafluoropentane diluted to a total volume of 200 µl in PBS. The mice were then allowed to recover from anesthesia and were sacrificed 24 hours after the time of ligation. After the mice were sacrificed, the heart was excised and placed at -20°C for 20 minutes. The heart was then sliced into 1-mm sections and stained with tetrazolium red to identify the infarcted area. The area of infarct and ventricle were then analyzed using ImageJ software. The average area of infarct in comparison to the ventricle for the control mice was 29.3±0.04% compared to 11.7±0.02% for the dodecafluoropentane-treated mice. The use of dodecafluoropentane in this murine model of myocardial infarction showed a 60% reduction in infarct size (p<0.01). The possibility of using nanoparticles to deliver oxygen to hypoxic tissues has interesting implications and justifies further study.

Neuron-derived Neurotrophic Factor Functions as a Novel Modulator That Enhances Endothelial Cell Function and Revascularization Processes

Strategies to stimulate revascularization are valuable for cardiovascular diseases. Here we identify neuron-derived neurotrophic factor (NDNF)/epidermacan as a secreted molecule that is up-regulated in endothelial cells in ischemic limbs of mice. NDNF was secreted from cultured human endothelial cells, and its secretion was stimulated by hypoxia. NDNF promoted endothelial cell network formation and survival in vitro through activation of Akt/endothelial NOS (eNOS) signaling involving integrin αvβ3. Conversely, siRNA-mediated knockdown of NDNF in endothelial cells led to reduction of cellular responses and basal Akt signaling. Intramuscular overexpression of NDNF led to enhanced blood flow recovery and capillary density in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of Akt and eNOS. The stimulatory actions of NDNF on perfusion recovery in ischemic muscles of mice were abolished by eNOS deficiency or NOS inhibition. Furthermore, siRNA-mediated reduction of NDNF in muscles of mice resulted in reduction of perfusion recovery and phosphorylation of Akt and eNOS in response to ischemia. Our data indicate that NDNF acts as an endogenous modulator that promotes endothelial cell function and ischemia-induced revascularization through eNOS-dependent mechanisms. Thus, NDNF can represent a therapeutic target for the manipulation of ischemic vascular disorders.

Abstract 606: Pharmacological Inhibition of PAI-1 Rescues Blood Flow Perfusion Through Promoting Capillary Arterialization in Diabetic Microangiopathy in Ischemic Limb

OBJECTIVE: Type II diabetes mellitus (DM) leads to the development of microvascular diseases and is associated with impaired angiogenesis. Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with DM. However, the role of PAI-1 in the development of DM-associated microvascular disease is poorly defined. RESEARCH DESIGN AND METHODS: Hind-limb ischemia was generated by femoral artery occlusion in diet-induced diabetic mice and age-matched normal chow (NC)-fed mice. Plasma was analyzed for glucose, non-esterified fatty acids, and PAI-1. PAI-039, a specific, small molecule pharmacological inhibitor of PAI-1, was orally administered to mice to determine if PAI-1 attenuates neovasculature formation in DM mice. RESULTS: PAI-1 activity and antigen were significantly increased within 2 weeks of DM onset and remained elevated throughout the experimental period. PAI-039 normalizes elevated plasma and ischemic muscles PAI-1 levels in the DM group. Pharmacologic inhibition of PAI-1 with PAI-039 rescued the ischemia-mediated impairments in blood flow perfusion and accelerated arterialization of capillaries in ischemic muscles in DM group compared to vehicle-DM group. Immunohistochemical analysis indicated increased expression of smooth muscle α-actin on capillary-sized vessels, and increased infiltration by CD45 leukocytes in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated mice. Furthermore, in these mice, VEGF-A was significantly down-regulated, and conversely, the expression of the PDGF-B and MMP 2, 9 were up-regulated in ischemic muscles in the inhibition of PAI-1 after induced ischemia. CONCLUSIONS: Taken together, these findings illustrate that the pharmacologic inhibition of elevated PAI-1 rescues the impairments in ischemia-mediated flow perfusion and capillary arterializations observed in DM, and may have efficacy as a therapeutic strategy to prevent diabetic microangiopathy.