LOX-1 Plays an Important Role in Ischemia-Induced Angiogenesis of Limbs

LOX-1, lectin-like oxidized low-density lipoprotein (LDL) receptor-1, is a single transmembrane receptor mainly expressed on endothelial cells. LOX-1 mediates the uptake of oxidized LDL, an early step in atherosclerosis; however, little is known about whether LOX-1 is involved in angiogenesis during tissue ischemia. Therefore, we examined the role of LOX-1 in ischemia-induced angiogenesis in the hindlimbs of LOX-1 knockout (KO) mice. Angiogenesis was evaluated in a surgically induced hindlimb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD) and arteriole density (AD). After right hindlimb ischemia, the ischemic/nonischemic hindlimb blood flow ratio was persistently lower in LOX-1 KO mice than in wild-type (WT) mice. CD and AD were significantly smaller in LOX-1 KO mice than in WT mice on postoperative day 14. Immunohistochemical analysis revealed that the number of macrophages infiltrating ischemic tissues was significantly smaller in LOX-1 KO mice than in WT mice. The number of infiltrated macrophages expressing VEGF was also significantly smaller in LOX-1 KO mice than in WT mice. Western blot analysis and ROS production assay revealed that LOX- KO mice show significant decrease in Nox2 expression, ROS production and HIF-1α expression, the phosphorylation of p38 MAPK and NF-κB p65 subunit as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and LOX-1 itself in ischemic muscles, which is supposed to be required for macrophage infiltration expressing angiogenic factor VEGF. Reduction of VEGF expression successively suppressed the phosphorylation of Akt and eNOS, which accelerated angiogenesis, in the ischemic leg of LOX-1 KO mice. Our findings indicate that LOX-1 plays an important role in ischemia-induced angiogenesis by 1) Nox2-ROS-NF-κB activation, 2) upregulated expression of adhesion molecules: VCAM-1 and LOX-1 and 3) promoting macrophage infiltration, which expresses angiogenic factor VEGF.     

Heat shock factor 1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of bone marrow stem/progenitor cells

Bone marrow (BM)-derived stem/progenitor cells play an important role in ischemia-induced angiogenesis in cardiovascular diseases. Heat shock factor 1 (HSF1) is known to be induced in response to hypoxia and ischemia. We examined whether HSF1 contributes to ischemia-induced angiogenesis through the mobilization and recruitment of BM-derived stem/progenitor cells using HSF1-knockout (KO) mice. After the induction of ischemia, blood flow and microvessel density in the ischemic hindlimb were significantly lower in the HSF1-KO mice than in the wild-type (WT) mice. The mobilization of BM-derived Sca-1- and c-kit-positive cells in peripheral blood after ischemia was significantly lower in the HSF1-KO mice than in the WT mice. BM stem/progenitor cells from HSF1-KO mice showed a significant decrease in their recruitment to ischemic tissue and in migration, adhesion, and survival when compared with WT mice. Blood flow recovery in the ischemic hindlimb significantly decreased in WT mice receiving BM reconstitution with donor cells from HSF1-KO mice. Conversely, blood flow recovery in the ischemic hindlimb significantly increased in HSF1-KO mice receiving BM reconstitution with donor cells from WT mice. These findings suggest that HSF1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of BM-derived stem/progenitor cells.     

Tie2-Dependent Neovascularization of the Ischemic Hindlimb Is Mediated by Angiopoietin-2

The angiopoietins (ANGPT) are ligands for the endothelial cell (EC) receptor tyrosine kinase, Tie2. Angpt-1 is a Tie2 agonist that promotes vascular maturation and stabilization, whereas Angpt-2 is a partial agonist/antagonist involved in the initiation of postnatal angiogenesis. Therefore, we hypothesized that overexpression of Angpt-2 would be more effective than Angpt-1 for enhancing the perfusion recovery in the ischemic hindlimb. Perfusion recovery was markedly impaired in Tie2-deficient animals at day 35 in a model of chronic hindlimb ischemia. Injections of Angpt-2 or VEGFA plasmid at 7 days post femoral artery resection enhanced recovery and improved arteriogenesis as assessed by angiographic scores, whereas Angpt-1 or null plasmid had no effect. In addition, Angpt-2 together with VEGF resulted in greater improvement in perfusion and collateral vessel formation than VEGF alone. Similarly, conditional overexpression of Angpt-2 in mice improved ischemic limb blood flow recovery, while Angpt-1 overexpression was ineffective. These data from Tie2 heterozygote deficient mice demonstrate, for the first time, the importance of the Tie2 pathway in spontaneous neovascularization in response to chronic hindlimb ischemia. Moreover, they show that overexpression of the partial agonist, Angpt-2, but not Angpt-1, enhanced ischemic hind limb perfusion recovery and collateralization, suggesting that a coordinated sequence antagonist and agonist activity is required for effective therapeutic revascularization.     

Critical Role of Endothelial Hydrogen Peroxide in Post-Ischemic Neovascularization

Background

Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) in vitro and neovascularization in vivo. However, little is known about the role of endogenous vascular hydrogen peroxide (H₂O₂) in postnatal neovascularization.

Methodology/Principal Findings

We used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H₂O₂ in ECs in post-ischemic neovascularization in vivo. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H₂O₂ in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice.

Conclusions/Significance

Endogenous ECs-derived H₂O₂ plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.     

Comparisons between the beneficial effects of different sulphonylurea treatments on ischemia-induced retinal neovascularization

The aim of this study was to assess the potential beneficial effects of gliclazide and other sulphonylureas on ischemia-induced retinal neovascularization. To produce an animal model of oxygen-induced ischemic retinopathy, 7-day-old (P7) mice were exposed to a 75% oxygen environment for 5 days. On their return to ambient air at P12, these mice were then treated with gliclazide, glibenclamide, glimepiride, or N-acetylcysteine. Gliclazide, but not glibenclamide or glimepiride, markedly suppresses retinal neovascularization. N-Acetylcysteine, however, only moderately suppresses retinal neovascularization. The number of neovascular nuclei in the retinal cross sections decreased by 29% in the gliclazide-treated mice (P<0.05 vs control). The induction of VEGF mRNA expression at P13 is significantly suppressed in the gliclazide group, relative to the control group (-44%, P<0.05). The VEGF protein expression levels at P15 were also suppressed in the gliclazide group (-43%, P<0.01). The 8-isoprostane production levels at P15 were suppressed in both the gliclazide group (-20%, P<0.05) and the N-acetylcysteine-treated group (-31%, P<0.01). Gliclazide inhibits ischemia-induced retinal neovascularization, and this is likely to be mediated in part through the downregulation of VEGF and the suppression of oxidative stress.     

Silencing of PTGS2 exerts promoting effects on angiogenesis endothelial progenitor cells in mice with ischemic stroke via repression of the NF-κB signaling pathway

The objective of the current study is to investigate the effect of PTGS2 on proliferation, migration, angiogenesis and apoptosis of endothelial progenitor cells (EPCs) in mice with ischemic stroke through the NF-κB signaling pathway. Middle cerebral artery occlusion (MCAO) model was established in mice. EPCs were identified, in which ectopic expression and depletion experiments were conducted. The mRNA and protein expression of related factors in tissues and cells were measured. Besides, proliferation, migration, angiogenesis, and apoptosis, as well as cell cycle distribution, of cells were determined. MCAO mice showed overexpression of interleukin-6 (IL-6), IL-17, and IL-23, and increased positive protein expression of PTGS2, as well as expression of PTGS2, nuclear factor-κB (NF-κB), tumor suppressor region 1 (TSP-1) and Bcl-2-associated X protein (Bax), but underexpression of vascular endothelial growth factor (VEGF), S-phase kinase associated protein 2 (Skp2), and B-cell lymphoma 2 (Bcl-2). Moreover, ectopic expression of tumor necrosis factor-α significantly elevated the expression of PTGS2, NF-κB, TSP-1, and Bax, as well as cell apoptosis and cell cycle arrest, but decreased the expression of VEGF, Skp2, and Bcl-2, as well as proliferation, migration and angiogenesis of EPCs, and the PTGS2-siRNA group showed an opposite trend. Taken together, we conclude that the specific knockdown of PTGS2 expression could repress the NF-κB signaling pathway, thereby inhibits apoptosis and promotes proliferation, migration and angiogenesis of EPCs, providing protective effect on mice with ischemic stroke.     

Ischemia-induced angiogenesis is impaired in aminopeptidase A deficient mice via down-regulation of HIF-1α

Aminopeptidase A (APA; EC 3.4.11.7) is a transmembrane metalloprotease with several functions in tumor angiogenesis. To investigate the role of APA in the process of ischemia-induced angiogenesis, we evaluated the cellular angiogenic responses under hypoxic conditions and the process of perfusion recovery in the hindlimb ischemia model of APA-deficient (APA-KO; C57Bl6/J strain) mice.Western blotting of endothelial cells (ECs) isolated from the aorta of APA-KO mice revealed that the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein in response to hypoxic challenge was blunted. Regarding the proteasomal ubiquitination, a proteasome inhibitor MG-132 restored the reduced accumulation of HIF-1α in ECs from APA-KO mice similar to control mice under hypoxic conditions. These were associated with decreased growth factor secretion and capillary formation in APA-KO mice. In the hindlimb ischemia model, perfusion recovery in APA-KO mice was decreased in accordance with a significantly lower capillary density at 2 weeks. Regarding vasculogenesis, no differences were observed in cell populations and distribution patterns between wild type and APA-KO mice in relation to endothelial progenitor cells.Our results suggested that Ischemia-induced angiogenesis is impaired in APA-KO mice partly through decreased HIF-1α stability by proteasomal degradation and subsequent suppression of HIF-1α-driven target protein expression such as growth factors. APA is a functional target for ischemia-induced angiogenesis.     

Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia

Blood flow restoration to ischemic tissue is affected by various risk factors. The aim of this study was to examine gender effects on arteriogenesis and angiogenesis in a mouse ischemic hindlimb model. C57BL/6J mice were subjected to unilateral hindlimb ischemia. Flow recovery was less and hindlimb use impairment was greater in females. No gender difference in vessel number was found at baseline, although 7 days postsurgery females had fewer α-smooth muscle actin-positive vessels in the midpoint of the adductor region. Females had higher hindlimb vascular resistance, were less responsive to vasodilators, and were more sensitive to vasoconstrictors postligation. Western blotting showed that females had higher baseline levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the calf, while 7 days postligation males had higher levels of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein. Females had less angiogenesis in a Matrigel plug assay and less endothelial cell proliferation in vitro. Females have impaired recovery of flow, a finding presumably caused by multiple factors including decreased collateral remodeling, less angiogenesis, impaired vasodilator response, and increased vasoconstrictor activity; our results also suggest the possibility that new collateral formation, from capillaries, is impaired in females.     

Improved angiogenic potency by implantation of ex vivo hypoxia prestimulated bone marrow cells in rats

Therapeutic angiogenesis can be induced by local implantation of bone marrow cells. We tried to enhance the angiogenic potential of this treatment by ex vivo hypoxia stimulation of bone marrow cells before implantation. Bone marrow cells were collected and cultured at 33 degrees C under 2% O(2)-5% CO(2)-90% N(2) (hypoxia) or 95% air-5% CO(2) (normoxia). Cells were also injected into the ischemic hindlimb of rats after 24 h of culture. Hypoxia culture increased the mRNA expression of vascular endothelial growth factor (VEGF), vascular endothelial (VE)-cadherin, and fetal liver kinase-1 (Flk-1) from 2.5- to fivefold in bone marrow cells. The levels of VEGF protein in the ischemic hindlimb were significantly higher 1 and 3 days after implantation with hypoxia-cultured cells than with normoxia-cultured or noncultured cells. The microvessel density and blood flow rate in the ischemic hindlimbs were also significantly (P < 0.001) higher 2 wk after implantation with hypoxia-cultured cells (89.7 +/- 5.5%) than with normoxia-cultured cells (67.0 +/- 9.6%) or noncultured cells (70.4 +/- 7.7%). Ex vivo hypoxia stimulation increased the VEGF mRNA expression and endothelial differentiation of bone marrow cells, which together contributed to improved therapeutic angiogenesis in the ischemic hindlimb after implantation.     

Adipose Stromal Cells Amplify Angiogenic Signaling via the VEGF/mTOR/Akt Pathway in a Murine Hindlimb Ischemia Model: A 3D Multimodality Imaging Study

Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.0×10⁷) were isolated from Tg(Fluc-egfp) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc⁺-eGFP⁺, mADSCs^Fluc+GFP+), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (∼5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and bioluminescence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (3D) imaging of the cells’ distribution and kinetics in vivo. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/mTOR/Akt pathway in vivo, even though the cells failed to incorporate into the host microvasculature as functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using in vivo 3D multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification via a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.     

Fiber type-specific differential expression of angiogenic factors in response to chronic hindlimb ischemia

Alterations in endogenous levels of the angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were assessed in rabbit hindlimb muscles subjected to 1, 5, or 21 days of ischemia. In the glycolytic [tibialis anterior (TA)] and the oxidative [soleus (SOL)] muscles from the ischemic and contralateral (control) hindlimb, bFGF and VEGF protein expression was determined by ELISA and immunoblot analysis. Total VEGF protein expression was greater in oxidative than in glycolytic muscles after 5 days of hindlimb ischemia. In SOL muscle, total VEGF detected by ELISA in ischemic limbs was increased to 137, 300, and 220% of control at 1, 5, and 21 days, respectively. However, in TA, total VEGF expression by ELISA was increased only at 1 and 5 days of ischemia to 140 and 134% of control, respectively. By immunoblotting, the expression of the 165-amino acid isoform (VEGF(165)) was initially decreased to 55% of control in ischemic SOL at 1 day but was increased to 250% of control at day 5 and remained at 155% at day 21. In TA, VEGF(165) was increased to 260% of control at 1 day of ischemia but only to 150% of control by day 5. The only significant change in bFGF expression in either the oxidative or glycolytic muscles was a small increase (129% of control) at 21 days in SOL. This study demonstrates that the magnitude and direction of change in VEGF protein expression depend on VEGF subtype, muscle fiber type, and duration of ischemia. These findings suggest that strategies in therapeutic angiogenesis may need to differ depending on muscle fiber type.     

Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells

Background

Bisphosphonates are a class of pharmacologic compounds that are commonly used to treat postmenopausal osteoporosis and malignant osteolytic processes. Studies have shown that bone marrow-derived endothelial progenitor cells (EPCs) play a significant role in postnatal neovascularization. Whether the nitrogen-containing bisphosphonate zoledronate inhibits ischemia-induced neovascularization by modulating EPC functions remains unclear.

Methodology/Principal Findings

Unilateral hindlimb ischemia was surgically induced in wild-type mice after 2 weeks of treatment with vehicle or zoledronate (low-dose: 30 μg/kg; high-dose: 100 μg/kg). Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio was significantly lower in wild-type mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in controls 4 weeks after ischemic surgery (control vs. low-dose vs. high-dose: 87±7% vs. *61±18% vs. **49±17%, *p<0.01, **p<0.005 compared to control). Capillary densities were also significantly lower in mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in control mice. Flow cytometry analysis showed impaired mobilization of EPC-like cells (Sca-1⁺/Flk-1⁺) after surgical induction of ischemia in mice treated with zoledronate but normal levels of mobilization in mice treated with vehicle. In addition, ischemic tissue from mice that received zoledronate treatment exhibited significantly lower levels of the active form of MMP-9, lower levels of VEGF, and lower levels of phosphorylated eNOS and phosphorylated Akt than ischemic tissue from mice that received vehicle. Results of the in vitro studies showed that incubation with zoledronate inhibited the viability, migration, and tube-forming capacities of EPC.

Conclusions/Significance

Zoledronate inhibited ischemia-induced neovascularization by impairing EPC mobilization and angiogenic functions. These findings suggest that administration of zoledronate should be withheld in patients with ischemic events such as acute limb ischemia.     

Zinc supplementation results in improved therapeutic potential of bone marrow-derived mesenchymal stromal cells in a mouse ischemic limb model

Background aimsWe wanted to determine whether zinc supplementation can inhibit bone marrow-derived mesenchymal stromal cell (MSC) apoptosis and enhance their tissue regenerative potential a in mouse ischemic hindlimb model.MethodsRat bone marrow cells were cultured and the resulting MSC were passaged for 3–7 generations. The proliferation and apoptosis of MSC was examined by 3-[4,5-dimethyl-2-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis. The activation of protein kinases B (Akt) was determined by Western blots. Vascular endothelial growth factor (VEGF) levels were examined by enzyme-linked immunosorbent assay. The mouse hindlimb ischemic model was established by ligating the right femoral artery. Mice received MSC, zinc-treated MSC or vehicle. The blood flow was assessed by laser Doppler imaging. The survival rate of donor cells was quantified by real-time polymerase chain reaction for the sex-determining region of the Y-chromosome (Sry). Angiogenesis was assessed by histochemical staining and immunofluoresence staining.ResultsSupplementation with physiologic amounts of zinc caused a marked attenuation of cell apoptosis, enhanced cell viabilities, increased VEGF release and up-regulated Akt activation. Zinc-treated MSC delivered into ischemic hindlimbs resulted in significant improvements in limb blood perfusion by increased implanted MSC survival and stimulated angiogenesis.ConclusionsThis study demonstrates the potential of zinc supplement to enhance survival of engrafted MSC and ameliorate their tissue regenerative potential in a mouse ischemic hindlimb model.     

Interleukin-10 promotes pathological angiogenesis by regulating macrophage response to hypoxia during development

Aberrant angiogenesis in the eye is the most common cause of blindness. The current study examined the role of interleukin-10 (IL-10) in ischemia-induced pathological angiogenesis called neovascularization during postnatal development. IL-10 deficiency resulted in significantly reduced pathological retinal angiogenesis. In contrast to the choroicapillaris where IL-10 interferes with macrophage influx, IL-10 did not prevent anti-angiogenic macrophages from migrating to the retina in response to hypoxia. Instead, IL-10 promoted retinal angiogenesis by altering macrophage angiogenic function, as macrophages from wild-type mice demonstrated increased vascular endothelial growth factor (VEGF) and nitric oxide (NO) compared to IL-10 deficient macrophages. IL-10 appears to directly affect macrophage responsiveness to hypoxia, as macrophages responded to hypoxia with increased levels of IL-10 and STAT3 phosphorylation as opposed to IL-10 deficient macrophages. Also, IL-10 deficient macrophages inhibited the proliferation of vascular endothelial cells in response to hypoxia while wild-type macrophages failed to do so. These findings suggest that hypoxia guides macrophage behavior to a pro-angiogenic phenotype via IL-10 activated pathways.     

Deficiency in endothelial nitric oxide synthase impairs myocardial angiogenesis

We recently demonstrated that mice deficient in endothelial nitric oxide (NO) synthase (eNOS) have congenital septal defects and postnatal heart failure. However, the mechanisms by which eNOS affects heart development are not clear. We hypothesized that deficiency in eNOS impairs myocardial angiogenesis. Myocardial capillary densities were measured morphometrically in neonatal mouse hearts. In vitro tube formation on Matrigel was investigated in cardiac endothelial cells. In vivo myocardial angiogenesis was performed by implanting Matrigel in the left ventricular myocardium. Myocardial capillary densities and VEGF mRNA expression were decreased in neonatal eNOS(-/-) compared with neonatal wild-type mice (P < 0.01). Furthermore, in vitro tube formation from cardiac endothelial cells and in vivo myocardial angiogenesis were attenuated in eNOS(-/-) compared with wild-type mice (P < 0.01). In vitro tube formation was inhibited by N(G)-nitro-l-arginine methyl ester in wild-type mice and restored by a NO donor, diethylenetriamine-NO, in eNOS(-/-) mice (P < 0.05). In conclusion, deficiency in eNOS decreases VEGF expression and impairs myocardial angiogenesis and capillary development. Decreased myocardial angiogenesis may contribute to cardiac abnormalities during heart development in eNOS(-/-) mice.     

Protein profiling and angiogenic effect of hypoxia-cultured human umbilical cord blood-derived mesenchymal stem cells in hindlimb ischemia

The aim of the present study was to investigate protein profiles of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions, and evaluate oxygenation effects on angiogenesis in an ischemic hindlimb mouse model using a modified ischemic scoring system. Hypoxic conditions did not change the expression of phenotypic markers and increased adipogenesis and chondrogenesis. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β RII, and vascular endothelial growth factor (VEGF) were upregulated in the conditioned medium of hypoxic hUCB-MSCs, which are commonly related to angiogenesis and proliferation of biological processes by Gene Ontology. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, significant enrichment of the phosphorylation of abelson murine leukemia viral oncogene homolog 1 (ABL1) (Phospho-Tyr204) and B-cell lymphoma-extra large (BCL-XL) (Phospho-Thr47) as anti-apoptotic pathways was observed in hypoxic hUCB-MSCs. Furthermore, hypoxic conditions induced proliferation and migration, and reduced apoptosis of hUCB-MSCs in vitro. Based on the results of protein antibody array, we evaluated the angiogenic effects of injecting normoxic or hypoxic hUCB-MSCs (1 × 10⁶) into the ischemic hindlimb muscles of mice. Ischemic scores and capillary generation were significantly greater in the hypoxic hUCB-MSC injection group than in the normoxic hUCB-MSC group. Our findings demonstrate that culturing hUCB-MSCs in hypoxic conditions not only significantly enriches phosphorylation in the anti-apoptosis pathway and enhances the secretion of several angiogenic proteins from cells, but also alleviates ischemic injury of hindlimb of mice.     

Effects of PEMF on microcirculation and angiogenesis in a model of acute hindlimb ischemia in diabetic rats

Hindlimb ischemia is a major complication of diabetic patients due to poor neovascularization. Therapy with pulsed electromagnetic fields (PEMF) can promote angiogenesis in ischemic lesions. However, the efficacy and therapeutic mechanisms of PEMF in diabetes‐related hindlimb ischemia are unclear. Sprague–Dawley rats were injected with streptozocin to induce diabetes, and 10 weeks later diabetic rats were subjected to surgical induction of acute hindlimb ischemia. The rats were randomized and treated with PEMF, and the blood perfusion of individual rats was determined longitudinally by laser Doppler perfusion imaging (LDPI). The neovascular density was examined using immunofluorescent analysis of CD31 expression and alkaline phosphatase (AP) staining. The levels of VEGF, VEGFR, FGF‐2, and FGFR1 expression, and ERK 1/2 and P38 phosphorylation in the muscles were characterized using enzyme‐linked immunosorbent assay (ELISA) and Western blot assays. The values of LDPI in the PEMF‐treated rats at 14 and 28 days post surgery were significantly greater than those in the controls, accompanied by significantly elevated levels of anti‐CD31 and AP staining. The relative levels of FGF‐2 and FGFR1, but not VEGF and VEGFR expression, and ERK1/2, but not P38 phosphorylation, in the muscles of the PEMF‐treated rats were significantly higher than those in the controls. Our data indicated that PEMF enhanced acute hindlimb ischemia‐related perfusion and angiogenesis, associated with up‐regulating FGF‐2 expression and activating the ERK1/2 pathway in diabetic rats. Therefore, PEMF may be valuable for the treatment of diabetic patients with ischemic injury. Bioelectromagnetics 34:180–188, 2013. © 2012 Wiley Periodicals, Inc.     

Acetylbritannilactone Modulates MicroRNA-155-Mediated Inflammatory Response in Ischemic Cerebral Tissues

Inflammatory responses play a critical role in ischemic brain injury. MicroRNA-155 (miR-155) induces the expression of inflammatory cytokines, and acetylbritannilactone (ABL) exerts potent antiinflammatory actions by inhibiting expression of inflammation-related genes. However, the functions of miR-155 and the actual relationship between ABL and miR-155 in ischemia-induced cerebral inflammation remain unclear. In this study, cerebral ischemia of wild-type (WT) and miR-155^−/− mice was induced by permanent middle cerebral artery occlusion (MCAO). pAd-miR-155 was injected into the lateral cerebral ventricle 24 h before MCAO to induce miR-155 overexpression. MCAO mice and oxygen-glucose deprivation (OGD)-treated BV2 cells were used to examine the effects of ABL and miR-155 overexpression or deletion on the expression of proinflammatory cytokines. We demonstrated that ABL treatment significantly reduced neurological deficits and cerebral infarct volume by inhibiting tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression in ischemic cerebral tissue and OGD-treated BV2 cells. Mechanistic studies suggested that the observed decrease in TNF-α and IL-1β expression was attributable to the ABL-induced suppression of the expression of nuclear factor-kappa B (NF-κB) and Toll-like receptor 4 (TLR4). We further found that miR-155 promoted TNF-α and IL-1β expression by upregulating TLR4 and downregulating the expression of suppressor of cytokine signaling 1 (SOCS1) and myeloid differentiation primary response gene 88 (MyD88), while ABL exerted an inhibitory effect on miR-155-mediated gene expression. In conclusion, miR-155 mediates inflammatory responses in ischemic cerebral tissue by modulating TLR4/MyD88 and SOCS1 expression, and ABL exerts its antiinflammatory action by suppressing miR-155 expression, suggesting a novel miR-155-based therapy for ischemic stroke.