Shuxuetong injection and its peptides enhance angiogenesis after hindlimb ischemia by activating the MYPT1/LIMK1/Cofilin pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxuetong (SXT) injection is formulated by leech and earthworm, has been widely used in the treatment of thrombotic cardiovascular and cerebrovascular diseases with remarkable clinical efficacy. AIM OF THE STUDY: The purpose of this study is to investigate the protective mechanism of SXT injection on the mice model of hindlimb ischemia, and to evaluate the angiogenic effects of SXT injection and its main active substances. MATERIALS AND METHODS: Hindlimb ischemia was induced by left femoral artery ligation. After operation, the mice were injected with saline, 10 mg/kg/d cilostazol, 37.5 mg/kg/d SXT injection, 75 mg/kg/d SXT injection and 150 mg/kg/d SXT injection via tail vein for 4 weeks. Ischemia severity was assessed using laser Doppler perfusion imaging system. Tissue recovery and capillary density were evaluated by histological and immunofluorescent staining. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF-BB) expression were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. Human umbilical vein endothelial cells (HUVECs) proliferation was measured using a BrdU kit and the viability of HUVECs was performed by MTT assay. Migration of HUVECs was performed by the wound healing method and a modified transwell assay. Capillary tube formation by HUVECs was examined by using Matrigel assay. Western blotting was used to detect the expressions of p-Cofilin, p-MYPT1, and p-LIMK1. RESULTS: SXT injection treatment significantly restored the blood flow and reduced tissue injury in mouse gastrocnemius muscle. SXT injection treatment increased capillary density and promoted angiogenesis in hindlimb ischemia. Moreover, SXT injection enhanced the expression of VEGF-A and PDGF-BB at both mRNA and protein levels in ischemic tissue of mice. SXT injection and its main active peptides dramatically increased the migration and capillary tube formation of HUVECs. SXT injection and its peptides enhanced protein expressions of the phosphorylation of MYPT1, Cofilin, and LIMK1. DSYVGDEAQSKR, YNELRVAPEEHP, and IQFLPEGSPVTM may act as the active components of SXT injection. CONCLUSION: SXT injection promoted angiogenesis and improved function recovery in hindlimb ischemia mice by regulation of VEGF-A/PDGF-BB. Moreover, SXT injection and its active peptides induced cell migration and tube formation in HUVECs through activating the MYPT1/LIMK1/Cofilin pathway. This study provided experimental basis for SXT injection in the treatment of ischemic diseases and revealed the effective substance of SXT injection in regulating angiogenesis, providing better evidence for the clinical application of SXT injection.

Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3ß/NRF2 Pathway in Diabetic Hindlimb Ischemia.

BACKGROUND: Peripheral arterial disease (PAD) is a typical disease of atherosclerosis, most commonly influencing the lower extremities. In patients with PAD, revascularization remains a preferred treatment strategy. Buyang Huanwu decoction (BHD) is a popular Chinese herbal prescription which has showed effects of cardiovascular protection through conducting antioxidant, antiapoptotic, and anti-inflammatory effects. Here, we intend to study the effect of BHD on promoting revascularization via the Akt/GSK3ß/NRF2 pathway in diabetic hindlimb ischemia (HLI) model of mice. MATERIALS AND METHODS: All db/db mice (n = 60) were randomly divided into 6 groups by table of random number. (1) Sham group (N = 10): 7-0 suture thread passed through the underneath of the femoral artery and vein without occlusion. The remaining 5 groups were treated differently on the basis of the HLI (the femoral artery and vein from the inguinal ligament to the knee joint were transected and the vascular stump was ligated with 7-0 silk sutures) model: (2) HLI+NS group (N = 15): 0.2 ml NS was gavaged daily for 3 days before modeling and 14 days after occlusion; (3) HLI+BHD group (N = 15): 0.2 ml BHD (20 g/kg/day) was gavaged daily for 3 days before modeling and 14 days after occlusion; (4) HLI+BHD+sh-NC group (N = 8): local injection of adenovirus vector carrying the nonsense shRNA (Ad-GFP) in the hindlimbs of mice before treatment; (5) HLI+BHD+sh-NRF2 group (N = 8): knockdown of NRF2 in the hindlimbs of mice by local intramuscular injection of adenovirus vector carrying NRF2 shRNA (Ad-NRF2-shRNA) before treatment; and (6) HLI+BHD+LY294002 group (N = 4): intravenous injection of LY294002 (1.5 mg/kg) once a day for 14 days on the basis of the HLI+BHD group. Laser Doppler examination, vascular cast, and immunofluorescence staining were applied to detect the revascularization of lower limbs in mice. Western blot analysis was used to detect the expression of vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor- (TNF-) α, heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone-1 (NQO-1), catalase (CAT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated protein kinase B (p-AKT), and phosphorylated glycogen synthase kinase-3 beta (p-GSK3ß). HE staining was used to assess the level of muscle tissue damage and inflammation in the lower extremities. Local multipoint injection of Ad-NRF2-shRNA was used to knock down NRF2, and qPCR was applied to detect the mRNA level of NRF2. The blood glucose, triglyceride, cholesterol, MDA, and SOD levels of mice were tested using corresponding kits. The SPSS 20.0 software and GraphPad Prism 6.05 were used to do all statistics. Values of P < 0.05 were considered as statistically significant. Results and Conclusions. BHD could enhance the revascularization of lower limbs in HLI mice, while BHD has no effect on blood glucose and lipid level in db/db mice (P > 0.05). BHD could elevate the protein expression of VEGF, HO-1, NQO-1, and CAT (P < 0.05) and decrease the expression of IL-1ß, IL-6, and TNF-α (P < 0.05) in HLI mice. Meanwhile, BHD could activate NRF2 and promote the phosphorylation of AKT/GSK3ß during revascularization (P < 0.05). In contrast, knockdown of NRF2 impaired the protective effects of BHD on HLI (P < 0.05). LY294002 inhibited the upregulation of NRF2 activated by BHD through inhibiting the phosphorylation of the AKT/GSK3ß pathway (P < 0.05). The present study demonstrated that BHD could promote revascularization on db/db mice with HLI through targeting antioxidation, anti-inflammation, and angiogenesis via the AKT/GSK3ß/NRF2 pathway.

Shexiang Baoxin Pill Attenuates Ischemic Injury by Promoting Angiogenesis by Activation of Aldehyde Dehydrogenase 2.

ABSTRACT: Promoting angiogenesis is a critical treatment strategy for ischemic cardiovascular diseases. Shexiang Baoxin Pill (SBP), a traditional Chinese medicine, has been reported to be capable of relieving angina and improve heart function by promoting angiogenesis. The aim of this study was to determine the role of mitochondrial aldehyde dehydrogenase 2 (ALDH2) in SBP-induced angiogenesis. Left femoral artery ligation was performed in wild-type mice (WT) and ALDH2 knockout mice, which were administrated with SBP (20 mg/kg/d) or equal volume saline per day by gastric gavage for 2 weeks. Perfusion recovery, angiogenesis in chronic hind limb ischemia, was significantly improved in the WT + SBP group than in the WT group. However, these beneficial effects were absent in ALDH2 knockout mice. In vitro, hypoxia impaired the ability of proliferation, migration and tube formation, sprouting angiogenesis, and promoted apoptosis in cardiovascular microvascular endothelial cells, whereas the hypoxia damage was restored by SBP. The protective effect of SBP was remarkably weakened by ALDH2 knockdown. Furthermore, SBP suppressed hypoxia-induced ALDH2/protein kinase B (AKT)/mammalian target of rapamycin pathways. In conclusion, this study demonstrated that SBP protected lower limb from ischemia injury through the ALDH2-dependent pathway. The protective mechanism of SBP in cardiovascular microvascular endothelial cells was partly mediated through ALDH2/AKT/mammalian target of rapamycin pathways.

Salviae miltiorrhizae radix and puerariae lobatae radix herbal formula improves circulation, vascularization and gait function in a peripheral arterial disease rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: DG is a herbal formula, containing the root of Salvia miltiorrhiza Bunge (Danshen) and the root of Pueraria lobate (Willd.) Ohwi (Gegen), has a history of usage in China for cardiovascular protection and anti-atherosclerosis. AIM OF THE STUDY: The present study aims to determine the beneficial effect of DG on the hind-limb ischemia rat model which mimics peripheral arterial disease (PAD) and its vasodilative effect on isolated femoral artery. MATERIALS AND METHODS: The vasodilatory effects were assessed by contractile responses to DG in the isolated femoral artery and its underlying mechanisms were evaluated by the involvement of endothelium, potassium channel and calcium channel. For hind-limb ischemia study, treatment outcomes were assessed by evaluating hind-limb blood flow, functional limb recovery, muscle histology and angiogenesis. RESULTS: Our results demonstrated positive dose-dependent vasodilatory response to DG via an endothelium-independent mechanism that involved inwardly rectifying K+ channels and Ca2+ channels. We also demonstrated significant improvement in blood perfusion and micro-vessel density in the ischemic limb and positive effects in functional limb recovery. CONCLUSION: In conclusion, our study supported the potential use of DG as a novel treatment for symptomatic PAD.

Circulatory Rejuvenated EPCs Derived from PAOD Patients Treated by CD34+ Cells and Hyperbaric Oxygen Therapy Salvaged the Nude Mouse Limb against Critical Ischemia.

This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice (n = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood prior to CD34+ cell and HBO treatment (EPCPr-T) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood after CD34+ cell and HBO treatment (EPCAf-T) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 (p < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all p < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all p < 0.0010). The number of small vessels and endothelial cell surface markers (CD31+/vWF+) in the CLI area displayed an identical pattern of INBF (all p < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all p < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.

Serum metabolomic profiles reveal the impact of BuZangTongLuo formula on metabolic pathways in diabetic mice with hindlimb ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: BuZangTongLuo Formula (BZTLF) was the decoction of eight traditional Chinese medicines including Astragalus membranaceus, Dioscorea opposita, Salvia miltiorrhiza, Scrophularia ningpoensis, Ophiopogon japonicus, Panax ginseng, Fritillariae cirrhosae and Whitmania pigra. This formula has been used as an effective remedy for treatment of diabetic ischemia clinically. AIM OF THE STUDY: In previous study, we have reported the therapeutic effect of BZTLF on diabetic vascular dysfunction. However, it remains obscure about the role of metabolic pathways in BZTLF-initiated improvement on hindlimb ischemia. MATERIALS AND METHODS: Diabetic mice with hindlimb ischemia were orally administrated with BZTLF by gavage. The serum samples were prepared for untargeted metabolomic analysis by ultra-performance liquid chromatography-mass spectrometer. The metabolic network was built by integrating metabolite data with the Gene Expression Omnibus (GEO) dataset (GSE3313). Further, quantitative PCR was used to confirm the key target genes. RESULTS: BZTLF treatment remarkably led to the reversal of changed metabolite levels in serum of diabetic mice with hindlimb ischemia, which mainly derived from bacteria, plant and signaling molecules. Also, BZTLF reshaped the metabolic pathways, especially those responsible for metabolism of lipid, gluthanine and tryptophan. In addition, BZTLF led to the reduction of lysophosphatidic acids (LPAs) and increment of triglycerides (TGs) conjugation with non-saturated fatty acids in serum. BZTLF significantly restored the down-regulation of vascular endothelial growth factor receptor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) or the up-regulation of interleukin 4-induced 1 (IL4I1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1) at mRNA level, which were key regulatory genes located in metabolic pathways of glutamate and tryptophan. CONCLUSIONS: BZTLF improved hindlimb ischemia in diabetic mice by the positive regulation of metabolome changes in serum.

BuZangTongLuo decoction improved hindlimb ischemia by activating angiogenesis and regulating gut microbiota in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Seven traditional medicinal plants (including Astragalus membranaceus, Dioscorea hemsleyi, Salvia miltiorrhiza, Scrophularia ningpoensis, Ophiopogon japonicus, Panax ginseng and Fritillariae cirrhosae) and one insect leech (Whitmania pigra Whitman) were combined into BuZangTongLuo formula (BZTLF) under the guidance of traditional Chinese medicine. BZTLF is potentially effective against diabetic vascular complications. AIM OF THE STUDY: Previous studies failed to clarify the molecular mechanism through which BZTLF suppressed diabetic ischemia. In this study, we aimed to explore whether BZTLF treatment could prevent the occurrence of type 2 diabetic (T2D) hindlimb ischemia in mice. Further, we investigated the regulatory effect of BZTLF on angiogenesis-related VEGF signaling pathway and gut microbiota dysfunction in diabetic ischemia mice. MATERIALS AND METHODS: C57BL/6J mice fed with high-fat diet (HFD) received STZ injection and femoral artery ligation to build T2D diabetic hindlimb ischemia model. Mice were gavaged with BZTLF (5 g [raw materials]/kg/d) or with metformin plus atorvastatin for three weeks. Laser doppler imaging system was utilized for the visualization of blood flow. Histochemistry analysis was performed for microvascular vessel staining. Western blot was applied to detect the protein changes of signaling molecules responsible for VEGF pathway. Finally, 16S rDNA gene sequencing was conducted for analysis of gut microbiota structure. RESULTS: BZTLF treatment remarkably restored blood flow and capillary density of diabetic hindlimb ischemia. And the protein changes of VEGF signaling molecules were reversed in BZTLF-treated diabetic ischemia mice, including the decreased VEGF and HIF-1α, and the increased NO, eNOS and p-ERK1/2. The gut microbiota analysis suggests that BZTLF treatment increased the abundances of several beneficial bacteria (Akkermansia, Bifidobacterium and Bacteroides), while decreased the populations of some harmful bacteria(Blautia, Weissella, Escherichia Shigella and Kurthia). By using Spearman's correlation analysis, these changed gut flora were positively/negatively correlated with VEGF signaling pathway or glycometabolic parameters. CONCLUSION: BZTLF displayed beneficial effects on diabetic hindlimb ischemia by reshaping the gut microbiota structure and stunning the VEGF/HIF-1α pathway.

Effect of Xuefu Zhuyu Capsule () on Angiogenesis in Hindlimb Ischemic Rats.

OBJECTIVE: To investigate the effect and mechanism of Xuefu Zhuyu Capsule (, XZC) on pro-angiogenesis in the hindlimb ischemic model rats. METHODS: A total of 100 Sprague Dawley rats were randomly divided into a model group, a regular-dose XZC group (0.48 g•kg-1•d-1) and a high-dose XZC group (0.96 g•kg-1•d-1) using random number table method. The model of hindlimb ischemic rats were made through femoral artery embolization with Bletilla microsphere agent. XZC were given on the first day after embolization surgery and lasted 5 days. Finally 72 models were obtained with 12 in each group for each time point. The lower ischemic limb was amputated on the third day after embolization surgery. Histopathological characters and the number of blood vessels of granulation tissues were observed at 36 and 48 h after amputation, respectively. The main genes were obtained from microarray analysis and were validated using real-time quantitative polymerase chain reaction. RESULTS: The vascular number of granulation tissues at both 36 and 48 h were characterized by new and fresh vessels. The number of angiogenesis in the high-dose XZC group at 36 and 48 h was greater compared with that in the regular-dose XZC and model groups (P<0.01), and high-dose XZC at 36 h increased more vessels than that at 48 h (P<0.01). Consequently, granulation tissues from the high-dose XZC group at 36 h were chosen for microarray analysis. In all, 2,085 differentially expressed genes (DEGs) were detected and 25 DEGs were determined to be directly related to angiogenesis. Four biological process terms were found including angiogenesis, regulation of angiogenesis, positive regulation of angiogenesis, and positive regulation of vascular endothelial growth factor receptor signaling pathway (P<0.05). Microarray analysis also showed 49 pathways including 11 pathways related to angiogenesis. CONCLUSION: XZC promoted angiogenesis moderately and the mechanism involved multiple DEGs and multiple pathways.

Sodium Tanshinone IIA sulfonate improves post-ischemic angiogenesis in hyperglycemia.

BACKGROUND: Diabetes 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 RESULTS: Unilateral 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. CONCLUSION: These findings demonstrate STS improves angiogenesis via inhibiting miR-133a expression and increasing GCH-1 protein levels in experimental PAD with diabetes.

Vitamin D intervention does not improve vascular regeneration in diet-induced obese male mice with peripheral ischemia.

Vitamin D appears to either promote or inhibit neovascularization in a disease context-dependent manner. The effects of vitamin D, alone or in combination with niacin, on endothelial cell (EC) angiogenic function and on revascularization in obese animals with peripheral ischemia are unknown. Here, we report that supplementation of high palmitate medium with vitamin D, niacin or both vitamins increased EC tube formation, which relies primarily on cell migration, and also maintained tube stability over time. Transcriptomic analyses revealed that both vitamins increased stress response and anti-inflammatory gene expression. However, vitamin D decreased cell cycle gene expression and inhibited proliferation, while niacin induced stable expression of miR-126-3p and -5p and maintained cell proliferation in high palmitate. To assess vascular regeneration, diet-induced obese mice received vitamin D, niacin or both vitamins following hind limb ischemic injury. Niacin, but not vitamin D or combined treatment, improved recovery of hind limb use. Histology of tibialis anterior sections revealed no improvements in revascularization, regeneration, inflammation or fibrosis with vitamin D or combined treatment. In summary, although both vitamin D and niacin increased angiogenic function of EC cultures in high fat, only niacin improved recovery of hind limb use following ischemic injury in obese mice. It is possible that inhibition of cell proliferation by vitamin D in high-fat conditions limits vascular regeneration and recovery from peripheral ischemia in obesity.

Combination of cells-based therapy with apelin-13 and hyperbaric oxygen efficiently promote neovascularization in ischemic animal model.

OBJECTIVE: Critical lower-limb ischemia (CLLI) is characterized by high morbidity and mortality. The aim of this study was to explore the effectiveness of the combination of cell therapy with apelin-13 and hyperbaric oxygen in CLLI animal model. MATERIALS AND METHODS: The experimental ischemic rats were divided into five groups, including negative control, bone marrow derived mononuclear cells (BM-MNCs), apelin-13, hyperbaric oxygen treatment (HBOT) and apelin-13 with HBOT group. Each group was composed of 10 rats. Endothelial progenitor cells (EPCs) derived from bone marrow were transplanted into the ischemia rat model. After 3 weeks of transplantation, the formation of new vessels was evaluated by examining cluster of differentiation (CD)31, CD34 and vascular endothelial growth factor receptor 2 (VEGFR-2) expressions as well as a direct vision of vessels by hematoxylin and eosin (HE) staining and immunohistochemistry. RESULTS: Compared with the negative control group, both angiogenic factors expressions and the number of new vessels increased notably by the transplantation of BM-MNCs in the ischemic models. Apelin-13 or HBOT alone improved the efficacy within limit while the combination of the three elements remarkably promoted the neovascularization in ischemic limbs. CONCLUSIONS: BM-MNC induced angiogenesis in the ischemic limbs and was considered an effective resource for cell therapy. The preliminary data of this study showed that the combination of cell therapy with apelin-13 and HBOT improved the efficacy of angiogenesis.

Ameliorating effects of Cuscuta chinensis Lamak extract on hind­limb ischemia, and angiogenic­ or inflammatory associated factors in ovariectomized mice.

Cuscuta chinensis Lamak (CCL) has traditionally been used in Korea to treat sexual disorders and skin problems. The aim of the present study was to investigate the effects of CCL extract on surgical injury­induced ischemia in the hind limbs of mice. Specifically, female C57BL/6 mice were ovariectomized, and their hind­limb vessels were ligated with surgical silk (6­0) and excised. CCL (150 or 450 mg/kg/BW) was then administered to the mice for 3 weeks, and the blood flow rate was evaluated using a laser Doppler system at ­7, 0, 7, 14 and 21 days following hind­limb ischemia. The serum expression profiles of angiogenic and inflammatory mediators were measured using an antibody array, and the transcript levels were reverse transcription­quantitative polymerase chain reaction. The rate of hind limb blood flow was normalized to non­ischemic lesions and revealed to be markedly elevated at 14 and 21 days following ischemia when compared with the vehicle group. The density of capillaries in the hind limbs was also significantly increased following treatment with CCL in a dose­dependent manner. In addition, the transcriptional expression of angiogenetic factors were upregulated, whereas that of inflammatory cytokines were downregulated. Finally, vascular endothelial cell migration and tube formation were evaluated in vitro using human umbilical vein endothelial cells (HUVECs) and identified to be significantly increased following treatment with CCL. Overall, the results of the present study indicate that CCL extract exhibits therapeutic potential for the treatment of hind­limb ischemia as it promotes peripheral angiogenic and anti­inflammatory effects in mice.

Magnesium lithospermate B ameliorates microcirculation perfusion in rats by promoting vascular NO production via activating the PI3K/AKT pathway.

Microcirculation morphologically refers to the blood flow in vessels of less than 150 µm in diameter, including arterioles, capillaries and venules, which provides nutrients and removes metabolic byproducts within tissues. Microcirculation dysfunction is involved in the pathological progress of many diseases, such as obesity, hypertension, and insulin resistance. In this study we investigated the effects of magnesium lithospermate B (MLB), an active compound of the traditional Chinese medicine Slavia miltiorrhiza, on the microcirculation dysfunction in rats and the underlying molecular mechanisms. The effects of MLB on microcirculation were assessed in vivo by measuring the hindlimb blood perfusion in dextran-induced microcirculation dysfunction rats and mesentery blood flow in anesthetized rats. We demonstrated that administration of MLB restored the impaired rat hindlimb blood flow and promoted the mesenteric micoperfusion in vivo. We further revealed in these two animal models that MLB treatment significantly increased the production of total nitrite in vascular tissues (mesentery, aorta, and heart), which was confirmed in human microvascular endothelial cells (HMEC-1) treated with MLB in vitro. Moreover, we showed that MLB treatment significantly increased the phosphorylation of endothelium nitric oxide synthase (eNOS) via inducing AKT phosphorylation in vivo and in vitro. Co-administration of the eNOS inhibitor L-NAME (20 mg/kg) abolished the protective effects of MLB against dextran-induced microcirculation dysfunction in rats, whereas pretreatment with PI3K inhibitor LY294002 (10 µM) prevented eNOS activation in MLB-treated HMEC-1 cells. Our results suggest that MLB can restore the microcirculation dysfunction via activating eNOS, and in turn enhancing the vascular nitric oxide production, which is medicated by MLB-caused activation of the PI3K/AKT pathway.

A long-term treatment with taurine prevents cardiac dysfunction in mdx mice.

Taurine is an amino acid abundantly present in heart and skeletal muscle. Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to skeletal muscle wasting and heart failure. An altered taurine metabolism has been described in dystrophic animals and short-term taurine administration exerts promising amelioration of early muscular alterations in the mdx mouse model of DMD. To reinforce the therapeutic and nutraceutical taurine potential in DMD, we evaluated the effects of a long-term treatment on cardiac and skeletal muscle function of mdx mice in a later disease stage. Taurine was administered in drinking water (1 g/kg/day) to wt and mdx mice for 6 months, starting at 6 months of age. Ultrasonography evaluation of heart and hind limb was performed, in parallel with in vivo and ex vivo functional tests and biochemical, histological and gene expression analyses. 12-month-old mdx mice showed a significant worsening of left ventricular function parameters (shortening fraction, ejection fraction, stroke volume), which were significantly counteracted by the taurine treatment. In parallel, histologic signs of damage were reduced by taurine along with the expression of proinflammatory myocardial IL-6. Interestingly, no effects were observed on hind limb volume and percentage of vascularization or on in vivo and ex vivo muscle functional parameters, suggesting a tissue-specific action of taurine in relation to the disease phase. A trend toward increase in taurine was found in heart and quadriceps from treated animals, paralleled by a slight decrease in mdx mice plasma. Our study provides evidences that taurine can prevent late heart dysfunction in mdx mice, further corroborating the interest on this amino acid toward clinical trials.

Naoxintong restores collateral blood flow in a murine model of hindlimb ischemia through PPARδ-dependent mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Naoxintong (NXT) is a compound preparation that is widely used in patients with cardiovascular and cerebrovascular diseases. AIM OF STUDY: The aim of this study is to investigate the protective mechanism of NXT on the mice model of peripheral vascular disease (PAD). MATERIALS AND METHODS: In the study, hindlimb ischemia was induced by ligation of femoral artery on the right leg of mice. After surgery, the mice were administrated with saline solution, 10 mg/kg/d simvastatin and 700 mg/kg/d NXT for 4 weeks. The blood flow perfusion was measured by laser Doppler perfusion imaging system. Histological and immunofluorescent staining was used to determine muscle recovery, capillary density, tissue vascular endothelial growth factor (VEGF), phosphorylated-Akt (p-Akt) and phosphorylated-endothelial nitric oxide synthase (p-eNOS) expression. Terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) was performed to detect the apoptosis of myocytes in hindlimb. The autophagy-associated gene expression and peroxisome proliferator-activated receptors (PPARs) expression were measured by Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Western blotting was performed to detect the expressions of light-chain 3 (LC3), VEGF, p-Akt, p-eNOS and PPARs. The EMSA experiment was performed to figure out whether PPARδ could directly bind to the predicted PPRE motif of VEGF. RESULTS: NXT treatment significantly accelerated perfusion recovery and reduced tissue injury in mice muscle. Apoptosis and autophagy were decreased within the ischemic muscle of NXT-treated mice. Quantification of vessels in hindlimb muscles provided evidences that NXT promoted angiogenesis in peripheral ischemia. In addition, results from western blotting and immunofluorescent staining suggested NXT induced angiogenesis via VEGF/Akt/eNOS signaling pathway. More interestingly, NXT specifically increased the expression of PPARδ in both mRNA and protein levels. EMSA results showed that PPARδ associated with PPRE site of VEGF promoter, suggesting that NXT-induced VEGF expression is mediated, at least in part, by PPARδ. CONCLUSION: In conclusion, the present study implicated that the restoration of hindlimb blood perfusion and recovery of limb functions were improved in NXT-treated mice with significant improvement of angiogenesis mediated by PPARδ-VEGF-Akt-eNOS axis as well as attenuation of autophagy and apoptosis. These results expand knowledge about the beneficial effects of NXT in angiogenesis and blood flow recovery. It might provide insight into the PPARδ regulating neovascularization in hindlimb ischemia and identify NXT as a potent new compound used for the treatment of peripheral vascular disease.

Ginsenoside Rg1 protects against hind-limb ischemia reperfusion induced lung injury via NF-κB/COX-2 signaling pathway.

BACKGROUND/AIMS: Ginsenoside Rg1 is regarded as the primary bioactive ingredient in Panax notoginseng that has been well recognized for its protective effects against ischemia/reperfusion (IR) injury. However, the mechanisms still remain elusive. Our study aims to investigate the effects of Rg1 against lung injury induced by hind-limb IR in rats. METHODS: Twenty-four Sprague Dawley rats were randomly submitted to sham operation (SM group), hind-limb IR (IR group), hind-limb IR + Rg1 (Rg1 group), and hind-limb IR + Pro-DTC group (PD group). All the rats except those in SM group were subjected to 3 h of ischemia followed by 6 h of reperfusion, and extra intravenous Rg1 and pyrrolidine dithiocarbamate (Pro-DTC), a selective inhibitor of nuclear factor kappa B (NF-κB), was administered intravenously before ischemia in the Rg1 and PD group, respectively. The activities of myeloperoxidase (MPO), superoxide dismutase (SOD) and catalase (CAT), as well as protein expressions of NF-κB p65 and cyclooxygenases-2 (COX-2) in lung tissue, and thromboxane B2 (TXB2) and 6-keto-ProstaglandinF1α (6-keto-PGF1α) levels in bronchoalveolar lavage (BAL) fluid were detected. Morphological changes, index of quantitative assessment of histologic lung injury (IQA), apoptosis index (AI) and lung Wet/Dry ratio were also evaluated. RESULTS: The levels of Wet/Dry ratio, IQA, AI, activities of MPO and 6-keto-PGF1α/TXB2 ratio were increased, and NF-κB p65 and COX-2 protein expression were upregulated, while SOD and CAT levels were decreased in lung tissue in IR group as compared with SM group (p < 0.05), all the alterations could be significantly reversed by Rg1 or Pro-DTC pretreatment (p < 0.05). And Rg1 and Pro-DTC also significantly attenuated the pulmonary histological abnormalities induced by IR. CONCLUSION: Ginsenoside Rg1 potentially attenuated lung injury induced by hind-limb IR by regulating NF-κB/COX-2 signaling pathway.

Low molecular weight fucoidan ameliorates hindlimb ischemic injury in type 2 diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Low molecular weight fucoidan (LMWF), extracted from Laminaria japonica Areschoug, is a traditional Chinese medicine, commonly used to alleviate edema, particularly for feet with numbness and pain. AIM OF THE STUDY: Diabetic mellitus (DM) patients are at high risk of developing peripheral arterial disease (PAD). Individuals with DM and PAD co-morbidity have a much higher risk of critical limb ischemia. LMWF showed several beneficial effects, such as anti-inflammation, anti-thrombosis, and enhancing revascularization. Therefore, we hypothesized that LMWF might be beneficial to diabetes-induced PAD, and investigated the therapeutic potential of LMWF on diabetic PAD rats. MATERIALS AND METHODS: Type 2 diabetic Goto-Kakizaki (GK) rats were made PAD by injection of sodium laurate into femoral artery. LMWF (20, 40 or 80mg/kg/day) or cilostazol (100mg/kg/day) were given to diabetic PAD rats for 4 weeks, respectively. The effects of LMWF on foot ulceration and claudication, plantar blood flow, collateral vessel formation, endothelium morphology, gastrocnemius injury, platelet aggregation, vessel vasodilation, and the expressions of inflammation factors, VEGF, eNOS, and nitric oxide were measured. RESULTS: We found that LMWF markedly ameliorated foot ulceration and claudication, and improved the plantar perfusion by reversing hyperreactive platelet aggregation, ameliorating endothelium-dependent vasodilation and revascularization on diabetic PAD rats. In addition, upregulation of several inflammatory factors, such as ICAM-1 and IL-1ß in the gastrocnemius muscles of ischemic hindlimb were suppressed by LMWF administration. And eNOS phosphorylation at Ser1177 and NO production were significantly enhanced in LMWF-treated diabetic PAD rats. CONCLUSIONS: Taken together, our findings demonstrated that LMWF exhibits therapeutic effect on hindlimb ischemia in type 2 diabetic rats likely through ameliorating endothelium eNOS dysfunction and enhancing revascularization, thus, providing a potential supplementary non-invasive treatment for diabetes-induced PAD.

Carthamus tinctorius L. extract improves hemodynamic and vascular alterations in a rat model of renovascular hypertension through Ang II-AT1R-NADPH oxidase pathway.

Carthamus tinctorius L. (CT) is widely used in Asian countries as a beverage and in folk medicine. The effects of CT extract on hemodynamics, vascular remodeling, the renin-angiotensin system (RAS) and oxidative stress in the two-kidney, one clip (2K-1C) hypertensive rat model were investigated. Renovascular hypertension was induced in male Sprague-Dawley rats and were treated with CT extract (500mg/kg/day) or captopril (5mg/kg/day) or vehicle for four weeks. CT extract or captopril reduced blood pressure, hindlimb vascular resistance, and increased hindlimb blood flow in 2K-1C hypertensive rats (p<0.05). Increases in aortic wall thickness, cross-sectional area and collagen deposition in 2K-1C rats were alleviated with CT extract or captopril treatment (p<0.05). CT extract or captopril suppressed RAS activation, including elevated serum ACE activity, and plasma Ang II level and up-regulated aortic AT1R protein expression in 2K-1C rats (p<0.05). Furthermore, CT extract or captopril reduced vascular superoxide production, aortic NADPH oxidase subunit gp91phox expression and increased plasma nitric oxide metabolite levels in 2K-1C rats (p<0.05). These findings suggest that CT extract ameliorated hemodynamic alteration and vascular remodeling in 2K-1C hypertensive rats. Possible mechanisms may involve RAS inhibitor effects and potent antioxidant activity.

Curcumin induces therapeutic angiogenesis in a diabetic mouse hindlimb ischemia model via modulating the function of endothelial progenitor cells.

BACKGROUND: Neovascularization is impaired in diabetes mellitus, which leads to the development of peripheral arterial disease and is mainly attributed to the dysfunction of endothelial progenitor cells (EPCs). Previous studies proved the promotional effect of curcumin on neovascularization in wound healing of diabetes. Thus, we hypothesize that curcumin could promote neovascularization at sites of hindlimb ischemia in diabetes and might take effect via modulating the function of EPCs. METHODS: Streptozotocin-induced type 1 diabetic mice and nondiabetic mice both received unilateral hindlimb ischemic surgery. Curcumin was then administrated to the mice by lavage for 14 days consecutively. Laser Doppler perfusion imaging was conducted to demonstrate the blood flow reperfusion. Capillary density was measured in the ischemic gastrocnemius muscle. In addition, angiogenesis, migration, proliferation abilities, and senescence were determined in EPCs isolated from diabetic and nondiabetic mice. Quantitative PCR was then used to determine the mRNA expression of vascular endothelial growth factor (VEGF) and angiopoetin-1 (Ang-1) in EPCs. RESULTS: Curcumin application to type 1 diabetic mice significantly improved blood reperfusion and increased the capillary density in ischemic hindlimbs. The in-vitro study also revealed that the angiogenesis, migration, and proliferation abilities of EPCs and the number of senescent EPCs were reversed by curcumin application. Quantitative PCR confirmed the overexpression of VEGF-A and Ang-1 in EPCs after curcumin treatment. CONCLUSION: Curcumin could enhance neovascularization via promoting the function of EPCs in a diabetic mouse hindlimb ischemia model.

Adipose-derived stem cell spheroid treated with low-level light irradiation accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND AIMS: We investigated whether low-level light irradiation (LLLI) before adipose-derived stromal cells (ASCs) spheroid transplantation improved hind-limb functional recovery by stimulation of angiogenesis. METHODS: The spheroid, composed of ASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor and basic fibroblast growth factor. From immunochemical staining analysis, the spheroid of ASCs included CD31+, KDR+ and CD34+, whereas monolayer-cultured ASCs were negative for these markers. To evaluate the therapeutic effect of the ASC spheroid treated with LLLI in vivo, phosphate-buffered saline, monolayer ASCs, LLLI-monolayer ASCs, spheroid ASCs and LLLI-spheroid ASCs were transplanted into a hind-limb ischemia model. RESULTS: The LLLI-spheroid ASCs transplanted into the hind-limb ischemia differentiated into endothelial cells and remained differentiated. Transplantation of LLLI-spheroid ASCs into the hind-limb ischemia significantly elevated the density of vascular formations through angiogenic factors released by the ASCs and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of LLLI-spheroid ASCs significantly improved functional recovery compared with ASC or spheroid ASC transplantation and PBS treatment. CONCLUSIONS: These findings suggest that transplantation of ASC spheroid treated with LLLI may be an effective stem cell therapy for the treatment of hind-limb ischemia and peripheral vascular disease.