Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease

The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P < 0.001) and group 1 (16.9 +/- 1.1%, P < 0.001). Immunostaining for CD31 showed significantly increased capillary density in group 3 (14.88 +/- 0.9) compared with group 2 (8.5 +/- 0.49, P < 0.001) and group 1 (5.69 +/- 0.3, P < 0.001). Improved blood flow (ml/min./g) was achieved in animal group 3 (0.173 +/- 0.04) as compared with animal group 2 (0.122 +/- 0.016; P= 0.047) and group 1 (0.062 +/- 0.012; P < 0.001). In conclusion, CD liposome mediated VEGF(165) gene transfer with SkMs effectively induced neovascularization in the ischaemic hind limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.     

Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability: efficacy and mechanism

Distal skin ischemic necrosis is a common complication in skin flap surgery. The pathogenesis of skin flap ischemic necrosis is unclear, and there is no clinical treatment available. Here, we used the 4 x 10 cm rat dorsal skin flap model to test our hypothesis that subcutaneous injection of vascular endothelial growth factor 165 (VEGF165) in skin flaps at the time of surgery is effective in augmentation of skin flap viability, which is associated with an increase in nitric oxide (NO) production, and the mechanism involves 1) an increase in skin flap blood flow in the early stage after surgery and 2) enhanced angiogenesis subsequently to sustain increased skin flap blood flow and viability. We observed that subcutaneous injection of VEGF165 in skin flaps at the time of surgery increased skin flap viability in a dose-dependent manner. Subcutaneous injection of VEGF165 at the dose of 2 microg/flap increased skin flap viability by 28% (P < 0.05; n = 8). Over 80% of this effect was blocked by intramuscular injection of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine (13 mg/kg) 45 min before surgery (P < 0.05; n = 8). The VEGF165 treatment also increased skin flap blood flow (2.68 +/- 0.63 ml x min(-1) x 100 g(-1)) compared with the control (1.26 +/- 0.10 ml x min(-1) x 100 g(-1); P < 0.05, n = 6) assessed 6 h postoperatively. There was no change in skin flap capillary density at this time point. VEGF165-induced increase in capillary density (32.2 +/- 1.1 capillaries/mm2; P < 0.05, n = 7) compared with control (24.6 +/- 1.4 capillaries/mm2) was seen 7 days postoperatively. There was also evidence to indicate that VEGF165-induced NO production in skin flaps was stimulated by activation of NOS activity followed by upregulation of NOS protein expression. These observations support our hypothesis and for the first time provide an important insight into the mechanism of acute local VEGF165 protein therapy in mitigation of skin flap ischemic necrosis.     

Efficacy of combination gene therapy with multiple growth factor cDNAs to enhance skin flap survival in a rat model

The objective of this study was to investigate the efficacy of combination gene therapy with multiple angiogenic growth factor cDNAs to enhance survival of ischemic skin flaps in a rat model. Sixty Sprague-Dawley rats were divided into six groups. Varying combinations of VEGF165, PDGF-B, and bFGF-plasmids were injected to prefabricate the flaps. Random skin flaps were raised on the dorsal aspect of rats following prefabrication with growth factor cDNAs. Flap viability was determined by measurement of percentage area of survival. The efficacy of gene therapy was evaluated by flap survival and neovascularization of representative histologic sections stained immunohistologically. The VEGF165 plus bFGF cDNAs enhanced the viability of the flap and neovascularization most effectively; the flap survival area was 64.3 +/- 8.7% after transfer of these two growth factor genes. Addition of PDGF-B cDNA is deleterious to the effects of combined VEGF165 and bFGF, leading to a significant decrease in flap viability (44.9 +/- 2.7%). Viability of the flaps with combined VEGF165 and bFGF cDNA transfer was significantly greater than that of the flaps with VEGF165 transfer alone (57.6 +/- 5.2%) or sham plasmid control (52.3 +/- 5.0%). Combined transfer of VEGF165 and bFGF cDNA is the most effective combination of multiple growth factor genes to improve flap viability in this model. Simultaneous transfer of three growth factor genes (VEGF165, PDGF-B, and bFGF) is deleterious to flap survival, at least for the ratio of lipofectin:transgene employed.     

Combined transfer of human VEGF165 and HGF genes renders potent angiogenic effect in ischemic skeletal muscle

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of "single-gene" administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo. Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.     

Vasodilator effect and mechanism of action of vascular endothelial growth factor in skin vasculature

Various laboratories have reported that local subcutaneous or subdermal injection of VEGF(165) at the time of surgery effectively attenuated ischemic necrosis in rat skin flaps, but the mechanism was not studied and enhanced angiogenesis was implicated. In the present study, we used the clinically relevant isolated perfused 6 x 16-cm pig buttock skin flap model to 1) test our hypothesis that VEGF(165) is a potent vasodilator and acute VEGF(165) treatment increases skin perfusion; and 2) investigate the mechanism of VEGF(165)-induced skin vasorelaxation. We observed that VEGF(165) (5 x 10(-16)-5 x 10(-11) M) elicited a concentration-dependent decrease in perfusion pressure (i.e., vasorelaxation) in skin flaps preconstricted with a submaximal concentration of norepinephrine (NE), endothelin-1, or U-46619. The VEGF(165)-induced skin vasorelaxation was confirmed using a dermofluorometry technique for assessment of skin perfusion. The vasorelaxation potency of VEGF(165) in NE-preconstricted skin flaps (pD(2) = 13.57 +/- 0.31) was higher (P < 0.05) than that of acetylcholine (pD(2) = 7.08 +/- 0.24). Human placental factor, a specific VEGF receptor-1 agonist, did not elicit any vasorelaxation effect. However, a specific antibody to VEGF receptor-2 (1 microg/ml) or a specific VEGF receptor-2 inhibitor (5 x 10(-6) M SU-1498) blocked the vasorelaxation effect of VEGF(165) in NE-preconstricted skin flaps. These observations indicate that the potent vasorelaxation effect of VEGF(165) in the skin vasculature is initiated by the activation of VEGF receptor-2. Furthermore, using pharmacological probes, we observed that the postreceptor signaling pathways of VEGF(165)-induced skin vasorelaxation involved activation of phospholipase C and protein kinase C, an increase in inositol 1,4,5-trisphosphate activity, release of the intra-cellular Ca(2+) store, and synthesis/release of endothelial nitric oxide, which predominantly triggered the effector mechanism of VEGF(165)-induced vasorelaxation. This information provides, for the first time, an important insight into the mechanism of VEGF(165) protein or gene therapy in the prevention/treatment of ischemia in skin flap surgery and skin ischemic diseases.     

The influence of diet and carnitine supplementation on plasma carnitine, cholesterol and triglyceride in WHHL (Watanabe-heritable hyperlipidemic), Netherland dwarf and New Zealand rabbits (Oryctolagus cuniculus)

1. Plasma carnitine levels in the spontaneously (endogenously) hyperlipidemic Watanabe (WHHL) rabbit are approximately 2-fold higher (P less than 0.001) than in normal rabbits of the New Zealand (NZ) or Netherland Dwarf (NDw) breeds. 2. Plasma carnitine levels in WHHL (44 +/- 3 nmol/ml) can be approximated in NZ and NDw which are rendered exogenously hyperlipidemic by supplementation of the stock chow diet with cholesterol and peanut oil. 3. The induction of endogenous hyperlipidemia in NZ by feeding a sucrose casein rich diet results in a biphasic response of plasma carnitine (elevation followed by normalization). 4. Plasma carnitine in WHHL is readily elevated by supplemental L-carnitine and the elevation is associated with a reduction in plasma triglyceride which shows differences in individual response time; plasma cholesterol is unaffected by supplemental L-carnitine.     

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.     

Reduced lung endothelial angiotensin-converting enzyme activity in Watanabe hyperlipidemic rabbits in vivo

We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White (n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [(3)H]benzoyl-Phe-Ala-Pro (BPAP) and the 5'-nucleotidase (NCT) substrate [(14)C]AMP were estimated, and the first-order reaction parameter A(max)/K(m), where A(max) is the product of enzyme mass and the catalytic rate constant and K(m) is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and A(max)/K(m) were reduced in WHHL (1.69 +/- 0.16 vs. 2.9 +/- 0.44 and 599 +/- 69 vs. 987 +/- 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP K(m) and A(max) values were estimated separately under mixed-order reaction conditions. No differences in K(m) values were found (9.79 +/- 1 vs. 9.9 +/- 1.31microM), whereas WHHL rabbit A(max) was significantly decreased (5.29 +/- 0.88 vs. 7. 93 +/- 0.8 micromol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.     

Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells

Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Here, the impact of erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. During the in vitro differentiation, vascular endothelial growth factor (VEGF) secretion by ECFCs was observed from day 3 (burst-forming unit erythroid cells) to day 10 (erythroblasts). ECFCs from day 5 to day 7 (colony-forming unit erythroid cells) showed the highest VEGF productivity, and day 6 ECFCs were used for the experiments. ECFCs contained larger amounts of VEGF and fibroblast growth factor-2 (FGF-2) than peripheral blood mononuclear cells (PBMNCs). In tubule formation assays with human umbilical vein endothelial cells, ECFCs stimulated 1.5-fold more capillary growth than PBMNCs, and this effect was suppressed by antibodies against VEGF and FGF-2. Using an immunodeficient hindlimb ischemia model and laser-Doppler imaging, we evaluated the limb salvage rate and blood perfusion after intramuscular implantation of ECFCs. ECFC implantation increased both the salvage rate (38% vs. 0%, P < 0.05) and the blood perfusion (82.8% vs. 65.6%, P < 0.01). In addition, ECFCs implantation also significantly increased capillaries with recruitment of vascular smooth muscle cells and the capillary density was 1.6-fold higher than in the control group. Continuous production of human VEGF from ECFCs in the skeletal muscle was confirmed at least 7 days after the implantation. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis.     

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.     

Arterial fatty acid-binding protein activity associated with dietarily-induced and spontaneously occurring atherosclerosis in the rabbit (Oryctolagus cuniculus)

1. Adult WHHL rabbits, or New Zealand rabbits fed either a stock chow diet or a high cholesterol diet were evaluated to assess the relationship between the development of aortic atherosclerosis and arterial FABP activity. 2. Aortic FABP activity was significantly (P less than 0.05) lower in atherosclerotic New Zealand aortas (0.039 +/- 0.008 nmol palmitoyl CoA bound/mg soluble prot) which had developed macroscopic lesions on 80% of the aortic surface as compared to lesion-free New Zealand aortas (0.053 +/- 0.002 nmol palmitoyl CoA bound/mg soluble prot). 3. In spontaneously hyperlipidemic rabbit (WHHL) aortas, FABP activity (0.023 +/- 0.004 nmol palmitoyl CoA bound/mg soluble prot) was significantly lower (P less than 0.05) than in either the normal or atherosclerotic New Zealand aortas. 4. To our knowledge, this study is the first to report a change in arterial FABP with the atherogenic process.     

MicroRNA let‐7g possesses a therapeutic potential for peripheral artery disease

Peripheral artery disease (PAD) is a manifestation of systemic atherosclerosis and conveys a significant health burden globally. Critical limb ischaemia encompasses the most severe consequence of PAD. Our previous studies indicate that microRNA let‐7g prevents atherosclerosis and improves endothelial functions. This study aimed to investigate whether and how let‐7g therapy may improve blood flow to ischaemic limbs. The present study shows that let‐7g has multiple pro‐angiogenic effects on mouse ischaemic limb model and could be a potential therapeutic agent for PAD. Mice receiving intramuscular injection of let‐7g had more neovascularization, better local perfusion and increased recruitment of endothelial progenitor cells after hindlimb ischaemia. The therapeutic effects of let‐7g's on angiogenesis are mediated by multiple regulatory machinery. First, let‐7g increased expression of vascular endothelial growth factor‐A (VEGF‐A) and VEGF receptor‐2 (VEGFR‐2) through targeting their upstream regulators HIF‐3α and TP53. In addition, let‐7g affected the splicing factor SC35 which subsequently enhanced the alternative splicing of VEGF‐A from the anti‐angiogenic isoform VEGF‐A_165b towards the pro‐angiogenic isoform VEGF‐A_164a. The pleiotropic effects of let‐7g on angiogenesis imply that let‐7g may possess a therapeutic potential in ischaemic diseases.     

Partial ileal bypass reduces the production rate of low density lipoproteins in Watanabe heritable hyperlipidemic rabbits

Partial ileal bypass surgery in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits resulted in a decrease of low density lipoproteins (LDL)-cholesterol from 14.2 +/- 2.4 to 7.0 +/- 1.2 mmol/l. To investigate the effect of partial ileal bypass on receptor-mediated and receptor-independent LDL catabolism, turnover studies were performed of radiolabeled native LDL and chemically modified LDL (methyl-LDL) in WHHL rabbits after partial ileal bypass, in WHHL control rabbits, and in New Zealand White ("normal") rabbits. The plasma LDL pool in WHHL control rabbits was increased 10-fold. The receptor-mediated LDL clearance was essentially zero in WHHL rabbits, both in controls and after ileal bypass surgery; the fractional catabolic rates for total LDL were equal in both WHHL groups and were also similar to that for methyl-LDL in the normal rabbits. Seventy percent of the total LDL clearance in the normal rabbits occurred via the LDL receptor pathway. In the animals with a partial ileal bypass, the plasma LDL-protein pool was appreciably lower than in WHHL controls (41.6 +/- 5.7 vs 73.4 +/- 9.9 mg/kg, P less than 0.02). The absolute catabolic rate was almost 50% lower in the PIB group (21.4 +/- 2.0 vs 40.0 +/- 7.5 mg X kg-1 X day-1, P less than 0.02). These results indicate that the decrease of LDL after partial ileal bypass surgery in WHHL rabbits is the result of a reduced production rate of LDL.     

Overexpression of lipoprotein lipase in transgenic Watanabe heritable hyperlipidemic rabbits improves hyperlipidemia and obesity

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of the triglyceride-rich lipoproteins and plays a critical role in lipoprotein and free fatty acid metabolism. Genetic manipulation of LPL may be beneficial in the treatment of hypertriglyceridemias, but it is unknown whether increased LPL activity may be effective in lowering plasma cholesterol and improving insulin resistance in familial hypercholesterolemic patients. To test the hypothesis that stimulation of LPL expression may be used as an adjunctive therapy for treatment of homozygous familial hypercholesterolemia, we have generated transgenic (Tg) Watanabe heritable hyperlipidemic (WHHL) rabbits that overexpress the human LPL transgene and compared their plasma lipid levels, glucose metabolism, and body fat accumulation with those of non-Tg WHHL rabbits. Overexpression of LPL dramatically ameliorated hypertriglyceridemia in Tg WHHL rabbits. Furthermore, increased LPL activity in male Tg WHHL rabbits also corrected hypercholesterolemia (544 +/- 52 in non-Tg versus 227 +/- 29 mg/dl in Tg, p < 0.01) and reduced body fat accumulation by 61% (323 +/- 27 in non-Tg versus 125 +/- 21ginTg, p < 0.01), suggesting that LPL plays an important role in mediating plasma cholesterol homeostasis and adipose accumulation. In addition, overexpression of LPL significantly suppressed high fat diet-induced obesity and insulin resistance in Tg WHHL rabbits. These results imply that systemic elevation of LPL expression may be potentially useful for the treatment of hyperlipidemias, obesity, and insulin resistance.     

Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia-reperfusion via nitric oxide mechanism

Caveolin-1 is a protein constituent of cell membranes. The caveolin-1 scaffolding region (residues 82-101) is a known inhibitor of protein kinase C. Inhibition of protein kinase C results in maintained nitric oxide (NO) release from the endothelium, which attenuates cardiac dysfunction after ischemia-reperfusion (I/R). Therefore, we hypothesized that the caveolin-1 scaffolding region of the molecule, termed caveolin-1 peptide, might attenuate postischemia polymorphonuclear neutrophil (PMN)-induced cardiac dysfunction. We examined the effects of caveolin-1 peptide in isolated ischemic (20 min) and reperfused (45 min) rat hearts reperfused with PMNs. Caveolin-1 peptide (165 or 330 microg) given intravenously 1 h before I/R significantly attenuated postischemic PMN-induced cardiac dysfunction, as exemplified by left ventricular developed pressure (LVDP) (P < 0.01) and the maximal rate of developed pressure (+dP/dt(max)) (P < 0.01), compared with I/R hearts obtained from rats given 0.9% NaCl. In addition, caveolin-1 peptide significantly reduced cardiac PMN infiltration from 195 +/- 5 PMNs/mm2 in untreated hearts to 103 +/- 5 and 60 +/- 5 PMNs/mm2 in hearts from 165 and 330 microg caveolin-1 peptide-treated rats, respectively (P < 0.01). PMN adherence to the rat coronary vasculature was also significantly reduced in rats given either 165 or 330 microg caveolin-1 peptide compared with rats given 0.9% NaCl (P < 0.01). Moreover, caveolin-1 peptide-treated rat aortas exhibited a 2.2-fold greater basal release of NO than vehicle-treated aortas (P < 0.01), and this was inhibited by NG-nitro-L-arginine methyl ester. These results provide evidence that caveolin-1 peptide significantly attenuated PMN-induced post-I/R cardiac contractile dysfunction in the isolated perfused rat heart, probably via enhanced release of endothelium-derived NO.     

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.     

Exposure to ambient particles accelerates monocyte release from bone marrow in atherosclerotic rabbits

Exposure to air pollution [particulate matter, particles <10 microm (PM(10))] causes a systemic inflammatory response that includes stimulation of the bone marrow (BM) and progression of atherosclerosis. Monocytes are known to play a key role in atherogenesis by migration into subendothelial lesions where they appear as foam cells. The present study was designed to quantify the BM monocyte response in Watanabe heritable hyperlipidemic (WHHL) rabbits after PM(10) exposure. WHHL rabbits were given twice weekly intrapharyngeal instillations of 5 mg of PM(10) for 4 wk to a total of 40 mg and compared with control WHHL or New Zealand White (NZW) rabbits. The thymidine analog 5'-bromo-2'-deoxyuridine was used to label dividing cells in the BM and a monoclonal antibody to identify monocytes in peripheral blood. The transit time of monocytes through the BM was faster in WHHL than in NZW rabbits (30.4 +/- 1.9 h vs. 35.2 +/- 0.9 h, WHHL vs. NZW; P < 0.05). PM(10) instillation exposure increased circulating band cell counts, caused rapid release of monocytes from the BM, and further shortened their transit time through the BM to 23.2 +/- 1.6 h (P < 0.05). The percentage of alveolar macrophages containing particles in the lung correlated with the BM transit time of monocytes (r(2) = 0.45, P <0.05). We conclude that atherosclerosis increases the release of monocytes from the BM, and PM(10) exposure accelerates this process in relation to the amount of particles phagocytosed by alveolar macrophages.     

Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats

Background

Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy.

Methods and Results

Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed.

Conclusions

These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.     

Combination of simvastatin administration and EPC transplantation enhances angiogenesis and protects against apoptosis for hindlimb ischemia

The aim of this present study is to investigate the impacts of combinatorial simvastatin administration and endothelial progenitor cell (EPC) transplantation on therapeutic angiogenesis in an athymic nude mouse model of hind limb ischemia. Athymic nude mice were divided into four groups (n = 10/group): vehicle administration plus PBS injection (control), simvastatin administration plus PBS injection (simvastatin), vehicle administration plus EPC transplantation (EPC), and simvastatin administration plus EPC transplantation (combination). The combination therapy had the greatest laser Doppler blood perfusion imager (LDPI) index and capillary density among the four groups. Importantly, this combination therapy significantly reduced apoptosis of ischemic skeletal muscle cells in part through downregulation of Bax and upregulation of Bcl-2 compared with the other groups. Moreover, the combination therapy exhibited the highest efficacy of increasing the ratio of phospho-Akt to Akt among the four groups. Taken together, the simvastatin and EPC combination therapy promotes powerful angiogenesis in hindlimb ischemia. The combination therapy not only inhibites apoptosis of ischemic skeletal muscle cells partially via downregulation of Bax and upregulation of Bcl-2, but also activates Akt phosphorylation significantly. These efficacies may be mediated by the angiogenic potency of simvastatin, EPCs, and by the beneficial effects of simvastatin on transplanted EPCs as well.