Rat Model Of Hindlimb Ischemia Research Articles

Kallistatin is a new inhibitor of angiogenesis and tumor growth

AbstractKallistatin is a unique serine proteinase inhibitor (serpin) and a heparin-binding protein. It has been localized in vascular smooth muscle cells and endothelial cells of human blood vessels, suggesting that kallistatin may be involved in the regulation of vascular function. Our previous study showed that kallistatin plays a role in neointima hyperplasia. In this study, we investigated the potential role of kallistatin in angiogenesis in vitro and in vivo. Purified human kallistatin significantly inhibited vascular endothelial growth factor (VEGF)– or basic fibroblast growth factor (bFGF)–induced proliferation, migration, and adhesion of cultured endothelial cells. Kallistatin attenuated VEGF- or bFGF-induced capillary density and hemoglobin content in subcutaneously implanted Matrigel plugs in mice. To further investigate the role of kallistatin in angiogenesis, we prepared adenovirus carrying the human kallistatin cDNA (Ad.HKBP) and evaluated the effect of kallistatin gene delivery on spontaneous angiogenesis in a rat model of hind-limb ischemia. Local kallistatin gene delivery significantly reduced capillary formation and regional blood perfusion recovery in the ischemic hind limb after removal of the femoral artery. Furthermore, a single intratumoral injection of Ad.HKBP into pre-established human breast tumor xenografts grown in athymic mice resulted in significant inhibition of tumor growth. CD31 immunostaining of tumor sections showed a decreased number of blood vessels in the kallistatin-treated group as compared to the control. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and tumor growth.

Evaluation of tissue perfusion in a rat model of hind-limb muscle ischemia using dynamic contrast-enhanced magnetic resonance imaging.

To evaluate the feasibility of using dynamic contrast-enhanced magnetic resonance imaging (MRI) for assessment of muscle perfusion in a rat model of hind-limb ischemia. The acute alteration and chronic recovery in muscle perfusion and perfusion reserve after femoral artery ligation were quantified using the maximum Gd-DTPA uptake rate obtained by a T(1)-weighted gradient-recalled echo sequence. Radionuclide-labeled microsphere blood flow measurements were performed for comparison with the MR perfusion measurement on a separate set of animals. After femoral artery ligation, a significant reduction in resting muscle perfusion was only observed at 1 hour post-ligation during the 28-day follow-up period. Muscle perfusion reserve was severely diminished following the ligation. Despite significant recovery over time, perfusion reserve to the ligated limb reached only 63% of the perfusion capacity in the unaffected limb by 42 days post ligation. A strong correlation (r = 0.86) between MR perfusion and microsphere blood flow measurements was observed for evaluation of relative changes in muscle perfusion. Dynamic contrast-enhanced MRI with Gd-DTPA is useful to assess time-dependent changes in muscle perfusion and perfusion reserve in this hind-limb ischemia model.

Angiogenesis Induced by the Injection of Peripheral Leukocytes and Platelets

Background. Peripheral leukocytes and platelets (LAPs) contain many kinds of cells with the ability to secrete several growth factors and cytokines. We attempted to induce therapeutic angiogenesis by injecting self-LAPs into a rat ischemic hindlimb model.Materials and methods. Supernatants from cultured LAPs were used for the endothelial cell (EC) proliferation assay, and LAPs were used in a cornea model to evaluate angiogenic potency. LAPs were injected directly into the male Dark Agouti rat ischemic hindlimb model, after which a microangiogram was done and the capillary/muscle fiber ratio was examined histologically. ELISA revealed the levels of contributing growth factors and cytokines present in the ischemic muscles.Results. The EC proliferation assay showed that the supernatants of LAPs accelerated proliferation and that the LAPs induced angiogenesis in the cornea model. The microangiograms and histological evaluation revealed that angiogenesis was induced more effectively in the rats injected with LAPs (LAP group) than in the those injected with phosphate-buffered saline (PBS group). The levels of basic fibroblast growth factor (bFGF) in the ischemia, PBS, and LAP groups were significantly increased compared to those in the sham group. The level of interleukin-1β (IL-1β) in the LAP group was significantly more elevated than in the other groups.Conclusions. The injection of self-LAPs induced angiogenesis in a rat ischemic hindlimb model. Ischemia caused an elevation in the level of bFGF and also in IL-1β derived from LAPs, which contributed to angiogenesis. This is a novel, yet simple and safe method of inducing therapeutic angiogenesis.

AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model.

Clinical trials on therapeutic angiogenesis using vascular endothelial growth factor (VEGF) are ongoing, however the benefits of these therapies are still controversial. To establish a more efficient gene transfer method for ischemic diseases, we investigated the therapeutic potential of adeno-associated virus (AAV)-mediated VEGF gene transfer. We produced VEGF(165)-expressing AAV vectors (AAV-VEGF). HEK-293 cells were transduced with AAV-VEGF in vitro and VEGF expression and secretion were examined. We used a rat ischemic hindlimb model and AAV-VEGF was administered intramuscularly into the ischemic limb. Gene expression was evaluated by RT-PCR and ELISA. Six weeks after gene transfer, we measured the blood flow of limb vessels and the skin temperature of limbs. Histochemical examination was performed to illustrate capillary growth. Western blotting and ELISA revealed VEGF protein expression and secretion from AAV-VEGF-transduced HEK-293 cells. VEGF mRNA and protein expression was consistently observed in the injected muscle at least 10 weeks after the injection, while no VEGF mRNA could be detected at remote organs. The mean blood flow in AAV-VEGF-transduced ischemic limbs was significantly higher than in AAV-LacZ-transduced limbs. Capillary density was significantly higher in AAV-VEGF-injected tissues than in AAV-LacZ-injected tissues. This study demonstrates that (1) AAV-mediated VEGF gene transfer into rat skeletal muscles is efficient and stable without ectopic expression, and (2) AAV-mediated VEGF gene transfer stimulates angiogenesis and thereby improves blood flow in a rat hindlimb ischemia model. These findings suggest that AAV-mediated VEGF gene transfer may be useful for treatment of ischemic diseases.

The induction of angiogenesis by the implantation of autologous bone marrow cells: A novel and simple therapeutic method

Background. Bone marrow contains many kinds of primitive cells that could differentiate to endothelial cells and secrete several growth factors. In the current study, we attempted to induce therapeutic angiogenesis by implanting autologous bone marrow cells (BMCs) and using a rat ischemic hind limb model. Methods. BMCs were prepared by removing red blood cells. A rat ischemic hind limb model was made by the ligation of the left femoral artery and its branches. BMCs were injected into 7 points of the ischemic muscles. To assess angiogenesis, a microangiogram, laser Doppler, and histologic evaluation were performed after the surgical procedure. Results. A microangiogram and histologic evaluation showed that angiogenesis was significantly induced in the ischemic hind limb by the implantation of BMCs. Laser Doppler imaging analysis showed that blood flow was significantly increased after implantation of BMCs. Some implanted BMCs were stained positively with CD31 and vascular endothelial-cadherin (VE-cadherin), which might have been incorporated into the vasculature. The condition of ischemia caused an elevation in the level of basic fibroblast growth factor in the ischemic muscle and also in interleukin-1β derived from the implanted BMCs, which might contribute to angiogenesis. Conclusion. These findings indicate that autologous bone marrow implantation may be a novel and simple method for inducing therapeutic angiogenesis. (Surgery 2001;130:44-54.)

Autologous Bone Marrow Implantation Induced Angiogenesis and Improved Deteriorated Exercise Capacity in a Rat Ischemic Hindlimb Model

Background. Bone marrow possesses endothelial progenitor cells that secrete several growth factors and can contribute to the formation of new capillaries. In the present study, we investigated the extent of angiogenesis induced by implantation of autologous bone marrow cells (BMCs) in a rat ischemic hindlimb model and studied whether the increased collateral vessels can improve deteriorated physical function.Materials and methods. Ischemic hindlimb was created by ligation of the femoral artery and its branches in Dark Agouti (DA) rats. BMCs (1 × 107) were injected percutaneously at six points into the gastrocnemius muscle. To assess angiogenesis, histologic evaluation and microangiography were performed at 2 weeks postligation. Severity of the ischemic insult was evaluated by measuring blood flow in the adductor and gastrocnemius muscles using nonradioactive colored microspheres and by determining the femoral arteriovenous oxygen difference (AVDO2) at 2 weeks postligation. Running time on a motor-driven treadmill was used to represent exercise capacity.Results. The histologic evaluation and microangiogram showed that the implanted BMCs induce angiogenesis. Blood flow to the adductor muscle on the treated side in the bone marrow cell implantation (BMI) group was significantly restored to 77.3 ± 19.3% of that of the normally perfused limb in comparison to that in control groups (P < 0.05). AVDO2 in the BMI group significantly decreased when compared with AVDO2 in control groups. Rats in the BMI group ran approximately 1.5 times longer than rats in control groups at 2 and 4 weeks postligation (P < 0.01).Conclusions. Implantation of autologous BMCs induced angiogenesis and improved deteriorated exercise capacity in our rat ischemic hindlimb model.

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models: preclinical study for treatment of peripheral arterial disease.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells, and acts as a survival factor against endothelial cell death. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. We have previously reported that intra-arterial administration of recombinant HGF induced angiogenesis in a rabbit hindlimb ischemia model. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy, due to its disadvantages. Initially, we examined the transfection of 'naked' human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imaging, accompanied by the detection of human HGF protein. A significant increase in capillary density was found in rats transfected with human HGF as compared with control vector, in a dose-dependent manner (P < 0.01). Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid once on day 10 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (P < 0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-transfected animals. In addition, a significant increase in blood flow was assessed by a Doppler flow wire and the ratio in blood pressure of the ischemic limb to the normal limb was observed in rabbits transfected with HGF plasmid as compared with rabbits transfected with control vector (P < 0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hindlimb models, as potential therapy for peripheral arterial disease.

Efficient Expression of the Vascular Endothelial Growth Factor Gene In Vitro and In Vivo, Using an Adeno-associated Virus Vector

Vascular endothelial growth factor (VEGF) has proven to be one of the most effective growth factors for therapeutic angiogenesis. The biological efficacy of the adeno-associated virus (AAV) vector has recently been demonstrated in muscle tissues, including the heart. Apart from these promising insights into VEGF and the AAV vector, studies on VEGF gene transfer using the AAV vector have been limited. Here, we evaluate AAV-mediated VEGF gene transfer, both in vitro andin vivo , using the AAV-mVEGF vector that contains cDNA for murine VEGF120within an HCMV-driven expression cassette. Transient transfection of AAV-mVEGF plasmid significantly increased mVEGF expression in 293T cells. The secreted VEGF in the conditioned medium had strong biological activity, as confirmed by the Miles» vascular permeability assay. Transduction of 293T and HeLa cells with AAV-mVEGF stock of high titer, that is essentially adenovirus-free, showed significantly increased mVEGF expression above that of AAV-eGFP-transduced cells. When human umbilical vein endothelial cells were transduced, a higher level of mVEGF expression, together with higher cell counts, was observed compared to AAV-eGFP-transduced cells. In vivo transduction of mouse tibialis anterior muscle resulted in an increased level of mVEGF expression, and higher capillary-to-myofibre ratio, 8 weeks post-transduction. In a rat hindlimb ischemia model, regional blood flow, as well as the capillary-to-myofibre ratio, was significantly increased at 4 weeks post-transduction. These findings demonstrate the efficient delivery of the VEGF gene using an AAV vector, which has implications for angiogenic gene therapy in ischemic diseases.

Novel roles of kallistatin, a specific tissue kallikrein inhibitor, in vascular remodeling.

We have purified, cloned and characterized kallistatin, a tissue kallikrein-binding protein (KBP) in humans and rodents. Kallistatin is a unique serine proteinase inhibitor (serpin) with Phe-Phe residues at the P2 and P1 positions. Structural and functional analysis of kallistatin by site-directed mutagenesis and protein engineering indicate that wild-type kallistatin is selective for tissue kallikrein. Kallistatin is expressed and localized in endothelial and smooth muscle cells of blood vessels and has multiple roles in vascular function independent of the tissue kallikrein-kinin system. First, kallistatin induces vasorelaxation of isolated aortic rings and reduces renal perfusion pressure in isolated rat kidneys. Transgenic mice overexpressing rat kallistatin are hypotensive, and adenovirus-mediated gene delivery of human kallistatin attenuates blood pressure rise in spontaneously hypertensive rats. Second, kallistatin stimulates the proliferation and migration of vascular smooth muscle cells in vitro and neointima formation in balloon-injured rat arteries. Third, kallistatin inhibits the proliferation, migration and adhesion of endothelial cells in vitro and angiogenesis in the rat model of hindlimb ischemia. These results demonstrate novel roles of kallistatin in blood pressure regulation and vascular remodeling.

Hyperhomocysteinemia impairs angiogenesis in response to hindlimb ischemia.

Hyperhomocysteinemia (HH) is an independent risk factor for atherosclerosis, including peripheral arterial occlusive disease (PAOD). Because angiogenesis and collateral vessel formation are important self-salvage mechanisms for ischemic PAOD, we examined whether HH modulates angiogenesis in vivo in a rat model of hindlimb ischemia. Rats were divided into 3 groups: the control group was given tap water, the HH group was given water containing L-methionine (1 g x kg(-1) x d(-1)), and the HH+L-arg group was given water containing methionine (1 g x kg(-1) x d(-1)) and l-arginine (2.25 vol%). At day 14 of the dietary modifications, the left femoral artery and vein were excised, and the extent of angiogenesis and collateral vessels in the ischemic limb were examined for 4 weeks. Plasma homocysteine levels significantly increased (P:<0.001), and plasma and tissue contents of nitrite+nitrate as well as tissue cGMP levels significantly decreased in the HH group compared with the control group (P:<0.01). Laser Doppler blood flowmetry (LDBF) revealed a significant decrease in the ischemic/normal limb LDBF ratio in the HH group at days 7, 14, 21, and 28 (P:<0.01 versus control). Angiography revealed a significant decrease in the angiographic score in the HH group at day 14 (P:<0.001 versus control). Immunohistochemistry of ischemic tissue sections showed a significant reduction in the capillary density in the HH group (P:<0. 001 versus control). Oral l-arginine supplementation in rats with HH (HH+L-arg) restored the decreased plasma and tissue nitrite+nitrate and cGMP contents (P:<0.05) as well as angiogenesis, as assessed by LDBF (P:<0.05 versus HH), angiographic score (P:<0.01 versus HH), and capillary density (P:<0.001 versus HH). In summary, HH impaired ischemia-induced angiogenesis and collateral vessel formation in a rat model of hindlimb ischemia in vivo. The mechanism of the HH-induced impairment of angiogenesis might be mediated in part by a reduced bioactivity of endogenous NO in the HH state.

Pre-Clinical Studies of Therapeutic Angiogenesis Induced by Human Hepatocyte Growth Factor (HGF) Gene in Rat and Rabbit Hind Limb Ischemia Models

P72 A novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy. Initially, we examined the transfection of “naked” human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imager. Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (C100% vs H 245%, P

Pre-Clinical Studies of Therapeutic Angiogenesis Induced by Human Hepatocyte Growth Factor (HGF) Gene in Rat and Rabbit Hind Limb Ischemia Models

P72 A novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy. Initially, we examined the transfection of “naked” human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imager. Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (C100% vs H 245%, P&lt;0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hind limb models, as potential therapy for peripheral arterial disease.

Kallistatin Inhibits Endothelial Cell Proliferation, Migration and Adhesion in Vitro and Angiogenesis in the Ischemic Hindlimb of Rats

P76 Kallistatin is a serine proteinase inhibitor (serpin) which has multifunctions including regulation of tissue kallikrein activity, blood pressure, inflammation and neointima hyperplasia. In this study, we investigated the potential role of kallistatin in vascular biology by studying its effects on the proliferation, migration and adhesion of cultured primary human endothelial cells in vitro, and angiogenesis in the ischemic hindlimb of rats. Purified kallistatin significantly inhibits cultured endothelial cell proliferation, migration and adhesion induced by VEGF or bFGF. To further investigate the role of kallistatin in vascular growth in vivo, we prepared adenovirus carrying the human kallistatin gene under the control of the cytomegalovirus promoter/enhancer (Ad.CMV-cHKBP). Expression of recombinant human kallistatin in HEK 293 cells transfected with Ad.CMV-cHKBP was identified by a specific ELISA. The effect of adenovirus-mediated kallistatin gene delivery on angiogenesis was evaluated in a rat model of hindlimb ischemia. Adenovirus carrying the human kallistatin or green fluorescent protein (GFP) gene were injected locally into the ischemic adductor at the time of surgery. Histological and morphometric analysis at 14 days post injection showed that adenovirus-mediated kallistatin gene delivery significantly reduced capillary density in the ischemic muscle as compared to that of control rats injected with GFP. The anti-angiogenic effect of kallistatin was associated with reduced regional blood flow in the ischemic hindlimb measured by microsphere assays. Expression of human kallistatin was identified in the injected muscle and immunoreactive human kallistatin levels were measured in the muscle and in the circulation of rats following kallistatin gene delivery. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and in vascular remodeling.

Kallistatin Inhibits Endothelial Cell Proliferation, Migration and Adhesion in Vitro and Angiogenesis in the Ischemic Hindlimb of Rats

P76 Kallistatin is a serine proteinase inhibitor (serpin) which has multifunctions including regulation of tissue kallikrein activity, blood pressure, inflammation and neointima hyperplasia. In this study, we investigated the potential role of kallistatin in vascular biology by studying its effects on the proliferation, migration and adhesion of cultured primary human endothelial cells in vitro, and angiogenesis in the ischemic hindlimb of rats. Purified kallistatin significantly inhibits cultured endothelial cell proliferation, migration and adhesion induced by VEGF or bFGF. To further investigate the role of kallistatin in vascular growth in vivo, we prepared adenovirus carrying the human kallistatin gene under the control of the cytomegalovirus promoter/enhancer (Ad.CMV-cHKBP). Expression of recombinant human kallistatin in HEK 293 cells transfected with Ad.CMV-cHKBP was identified by a specific ELISA. The effect of adenovirus-mediated kallistatin gene delivery on angiogenesis was evaluated in a rat model of hindlimb ischemia. Adenovirus carrying the human kallistatin or green fluorescent protein (GFP) gene were injected locally into the ischemic adductor at the time of surgery. Histological and morphometric analysis at 14 days post injection showed that adenovirus-mediated kallistatin gene delivery significantly reduced capillary density in the ischemic muscle as compared to that of control rats injected with GFP. The anti-angiogenic effect of kallistatin was associated with reduced regional blood flow in the ischemic hindlimb measured by microsphere assays. Expression of human kallistatin was identified in the injected muscle and immunoreactive human kallistatin levels were measured in the muscle and in the circulation of rats following kallistatin gene delivery. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and in vascular remodeling.

Protective effect of TA-993, a novel therapeutic agent for peripheral circulatory insufficiency, on skeletal muscle fatigue in a rat model of hindlimb ischemia.

TA-993 (cis-(-)-2-(4-methylphenyl)-3-acetoxy-2,3-dihydro-5-(2-dimethylaminoethy l)-8-methyl1,5-benzothiazepine-4(5H)-one maleate), a new 1,5-benzothiazepine derivative, has a selective increasing action on limb blood flow in addition to an antiplatelet action. In this report we studied the effect of TA-993 on a time dependent decrease in developed tension of electrically-induced contraction of tibialis anterior muscle in a rat model of peripheral circulatory insufficiency induced by occlusion of abdominal aorta. In our preparation, the developed tension decreased by 20-30% in a sham-operated group and 30-40% in an abdominal aorta-occluded group at the end of the experimental period of 60 min. Intraduodenal administration (i.d.) of TA-993 (10 mg/kg) to the abdominal aorta-occluded rats ameliorated the decrease in developed tension to the level of the sham-operated group. Moreover, TA-993 at 10 mg/kg, i.d. significantly increased femoral arterial blood flow supplied through collateral circulation and decreased the whole blood viscosity in this model. These results suggest that TA-993 improves dysfunction of skeletal muscle contraction due to peripheral circulating insufficiency through an increase in collateral blood flow and an improvement of red blood cell deformability.

Protective Effect of TA-993, a Novel Therapeutic Agent for Peripheral Circulatory Insufficiency, on Skeletal Muscle Fatigue in a Rat Model of Hindlimb Ischemia

TA-993 (cis-(–)-2-(4-methylphenyl)-3-acetoxy-2,3-dihydro-5-(2-dimethylaminoethyl)-8-methyll,5-benzothiazepine-4(5H)-one maleate), a new 1,5-benzothiazepine derivative, has a selective increasing action on limb blood flow in addition to an antiplatelet action. In this report we studied the effect of TA-993 on a time dependent decrease in developed tension of electrically-induced contraction of tibialis anterior muscle in a rat model of peripheral circulatory insufficiency induced by occlusion of abdominal aorta. In our preparation, the developed tension decreased by 20–30% in a sham-operated group and 30–40% in an abdominal aorta-occluded group at the end of the experimental period of 60 min. Intraduodenal administration (i.d.) of TA-993 (10 mg/kg) to the abdominal aorta-occluded rats ameliorated the decrease in developed tension to the level of the sham-operated group. Moreover, TA-993 at 10 mg/kg, i.d. significantly increased femoral arterial blood flow supplied through collateral circulation and decreased the whole blood viscosity in this model. These results suggest that TA-993 improves dysfunction of skeletal muscle contraction due to peripheral circulating insufficiency through an increase in collateral blood flow and an improvement of red blood cell deformability.

Establishment of a simple and practical procedure applicable to therapeutic angiogenesis.

Therapeutic angiogenesis is thought to be beneficial for serious ischemic diseases. This investigation was designed to establish a simple and practical procedure applicable to therapeutic angiogenesis. When cultured skeletal muscle cells were electrically stimulated at a voltage that did not cause their contraction, vascular endothelial growth factor (VEGF) mRNA was augmented at an optimal-frequency stimulation. This increase of VEGF mRNA was derived primarily from transcriptional activation. Electrical stimulation increased the secretion of VEGF protein into the medium. This conditioned medium then augmented the growth of endothelial cells. The effect of electrical stimulation was further confirmed in a rat model of hindlimb ischemia. The tibialis anterior muscle in the ischemic limb was electrically stimulated. The frequency of stimulation was 50 Hz and strength was 0.1 V, which was far below the threshold for muscle contraction. After a 5-day stimulation, there was a significant increase in blood flow within the muscle. Immunohistochemical analysis revealed that VEGF protein was synthesized and capillary density was significantly increased in the stimulated muscle. Rats tolerated this procedure very well, and there was no muscle contraction, muscle injury, or restriction in movement. We propose this procedure as a simple and practical method of therapeutic angiogenesis.

Endothelium-dependent relaxation of collateral microvessels after intramuscular gene transfer of vascular endothelial growth factor in a rat model of hindlimb ischemia.

Recent investigations have demonstrated the ability of vascular endothelial growth factor (VEGF) to augment the development of collateral arteries in vivo. In vitro studies have suggested that the use of VEGF also improves the endothelium-dependent relaxation of collaterals at the microvascular level. The purpose of this study was to determine in vivo the extent to which vasomotor responses of collateral microvessels are altered after VEGF treatment. Ischemia was induced in the hindlimb of 35 rats by excision of the femoral artery. Immediately thereafter, 400 microg of a plasmid encoding VEGF or ss-galactosidase (control) was transfected into limb muscles. Four weeks later, synchrotron radiation microangiography, with a spatial resolution of 30 microm, was performed to document the reactivity of collateral microvessels. Administration of the endothelium-dependent vasodilator acetylcholine failed to induce dilation of collateral microvessels in control animals. By contrast, profound dilation of collaterals was observed after acetylcholine in VEGF-treated animals. This response was evident in vessels with a linear appearance but not in those with an undulating appearance. The resulting blood flow in the ischemic limb after administration of acetylcholine in the control animals was only 64.6+/-17.0% of that of the contralateral normal limb, whereas blood flow was augmented to 106.1+/-8.4% in VEGF-treated animals (P<0.05). These results demonstrate in vivo that the use of VEGF restores impaired vasomotor responses in some types of collateral microvessels, which may help to provide a basis for understanding the microcirculation after therapeutic angiogenesis with VEGF.

Use of Synchrotron Radiation Microangiography to Assess Endothelium-dependent Relaxation of Microvessels in a Rat Model of Hindlimb Ischemia

Use of Synchrotron Radiation Microangiography to Assess Endothelium-dependent Relaxation of Microvessels in a Rat Model of Hindlimb Ischemia

Use of synchrotron radiation microangiography to assess endothelium-dependent relaxation of microvessels in a rat model of hindlimb ischemia

Use of synchrotron radiation microangiography to assess endothelium-dependent relaxation of microvessels in a rat model of hindlimb ischemia