Administration Of Vascular Endothelial Growth Factor Research Articles

Comparison of vascular endothelial growth factor and fibroblast growth factor-2 in a swine model of endothelial dysfunction☆☆☆

Growth factor based angiogenesis, with or without cell therapy, is a promising therapeutic modality for patients with coronary artery disease. We compared the relative efficacies of surgically delivered vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in a swine model of hypercholesterolemia-induced endothelial dysfunction which captures many of the pathophysiologic abnormalities of human coronary disease. Yucatan mini-swine (20-30 kg), fed a high cholesterol diet (total 20 weeks), underwent circumflex ameroid placement to create chronic myocardial ischemia, followed three weeks later by perivascular administration of VEGF (2 microg; n=6), FGF-2 (100 microg; n=6), or placebo (n=7) in the ischemic territory. Normocholesterolemic animals (n=7) served as controls. Four weeks later, endothelial function, collateral-dependent perfusion, as well as myocardial protein and mRNA levels of angiogenic mediators were assessed. Endothelial dysfunction was observed in all hypercholesterolemic animals as impaired microvessel relaxation in response to adenosine diphosphate and VEGF. VEGF administration improved baseline-adjusted collateral-dependent perfusion at rest (-0.03+/-0.05 vs -0.12+/-0.04, VEGF vs placebo, p=0.09), but FGF-2 delivery caused a significantly greater improvement in perfusion compared to either group (+0.15+/-0.03, p<0.05 vs HC-placebo and HC-VEGF) at rest. Molecular analysis revealed increased eNOS expression (135%+/-8%, p=0.03 vs placebo) in all growth factor treated animals and increased expression of FGF-2 receptor, FGFR1 (65+/-26%, p=0.04 vs placebo), in FGF-2 treated animals. No significant changes were demonstrated in other angiogenic mediators including Akt, Syndecan-4. In the setting of hypercholesterolemic endothelial dysfunction, FGF-2 is more effective than VEGF at enhancing collateral-dependent perfusion and thus, may be a better candidate than VEGF for angiogenic therapy in patients with end-stage CAD.

Vascular Endothelial Growth Factor Attenuates Nω-Nitro-L-Arginine Methyl Ester-Induced Preeclampsia-Like Manifestations in Rats

Objectives. To verify the hypothesis that vascular endothelial growth factor (VEGF) attenuates Nω-Nitro-L-arginine Methyl Ester (L-NAME)-induced preeclampsia-like manifestations in rats. Study design. Forty pregnant Wistar rats were randomly divided into four groups: control, preeclampsia model, VEGF treatment, and VEGF prophylactic. On day 5 of gestation, L-NAME was injected subcutaneously in rats of the preeclampsia model, VEGF treatment, and VEGF prophylactic groups. VEGF was given after the occurrence of hypertension and proteinuria in the VEGF treatment group and from day 5 in the VEGF prophylactic group. Blood pressure was monitored and urine protein was assayed. Blood platelet was counted, and serum nitric oxide metabolites, endothelin-1, 6-keto-PGF-1α, and TXB2 were determined. Results. Blood pressure increased significantly on day 8 of gestation in the preeclampsia model and VEGF treatment groups compared with control (p < 0.05 for both) and remained elevated through the pregnancy in the preeclampsia model group. Blood pressure was significantly decreased after the administration of VEGF in the VEGF treatment group (p < 0.05). There was no significant difference in blood pressure between the VEGF prophylactic group and control (p > 0.05). Urine protein, platelet count, serum nitric oxide metabolites, endothelin-1, 6-keto-PGF-1α, and TXB2 were significantly different between control and the preeclampsia model group (p < 0.05), but not between control and the VEGF treatment or VEGF prophylactic groups (p > 0.05 for all). Conclusion. VEGF attenuates L-NAME-induced preeclampsia-like manifestations in rats, suggesting the important role of VEGF in preeclampsia and providing a potential strategy for the prevention and treatment of preeclampsia.

Intracerebral VEGF injection highly upregulates AQP4 mRNA and protein in the perivascular space and glia limitans externa

Stroke is the third leading cause of death and the leading cause of adult disability in the industrialized nations. One of the consequences of stroke is blood–brain barrier (BBB) leakage and subsequent edema, which is one of the causes of mortality in this pathology. Aquaporin-4 (AQP4) is the most abundant water channel in the brain. Studies in AQP4 knock-out mice have shown a prominent role of this water channel in edema development and resolution after ischemia. Here we have studied changes in AQP4 mRNA and protein expression in response to vascular endothelial growth factor (VEGF), a potent angiogenic factor. VEGF administration highly upregulated AQP4 mRNA and protein in the ventral midbrain. Perfusion of the animals with FITC-albumin prior to sacrifice demonstrated localization of AQP4 protein in close proximity to the VEGF-induced new blood vessels. Expression levels of AQP4 mRNA were maximum 7 days after VEGF injection whereas our previous report showed that BBB leakage is resolved at this time point. Therefore, we speculate a positive role of AQP4 in edema resolution, which may partially explain the previously reported beneficial effects of delayed VEGF administration in ischemic rats. Our results provide new insights into the molecular changes in the edematous brain and may help in future therapeutical directions.

VEGF modulation of retinal pigment epithelium resistance

Fluid accumulation into the subretinal space and the development of macular edema is a common condition in age-related macular degeneration, diabetic retinopathy, and following ocular surgery, or injury. Vascular endothelial growth factor (VEGF) and other cytokines have been implicated in the disruption of retinal pigment epithelium (RPE) barrier function and a reduction in the regulated removal of subretinal fluid; however, the cellular and molecular events linking these agents to the disruption of barrier function have not been established. In the current study, cultures of ARPE-19 and primary porcine retinal pigment epithelium (RPE) cells were utilized to investigate the effects of the VEGF-induced modifications to the barrier properties of the RPE. The barrier function was determined by transepithelial resistance (TER) measurements and morphology of the RPE monolayers. In both ARPE-19 and primary porcine RPE cells the administration of VEGF produced a significant drop in TER, and this response was only observed following apical administration. Maximum reduction in TER was reached 5 h post VEGF administration. These responses were concentration-dependent with an EC 50 of 502 pg/mL in ARPE-19 cells and 251 pg/mL in primary porcine cells. In both ARPE-19 and primary RPE cells, the response to VEGF was blocked by pretreatment with the relatively selective VEGF-R2 antagonists, SU5416 or ZM323881, or the protein tyrosine kinase inhibitor, genistein. Administration of the relatively selective VEGF-R2 agonist, VEGF-E, also reduced TER in a concentration-dependent manner (EC 50 of 474 pg/mL), while VEGF-R1 agonist, placental growth factor (PlGF), did not significantly alter the TER. Immunolocalization studies demonstrated that confluent monolayers exhibited continuous cell-to-cell ZO-1 protein contacts and apical localization of the VEGF-R2 receptors. These data provide evidence that the VEGF-induced breakdown of RPE barrier function is mediated by the activation of apically-oriented VEGF-R2 receptors. Thus, VEGF-mediated increases in RPE permeability are initiated by a rise in intraocular levels of VEGF.

Vascular endothelial growth factor does not improve liver regeneration and survival after 90% subtotal liver resection

Vascular endothelial growth factor (VEGF) has been shown to stimulate liver regeneration after 70% partial hepatectomy (PH). It is unclear, however, whether exogenous administration of VEGF can also be used to improve liver regeneration and survival after 90% subtotal liver resection. The aim of this study was to determine the effect of exogenous and endogenous VEGF after 90% subtotal hepatectomy (SH). Rats were subjected to 90% SH and treated with VEGF, anti-VEGF or NaCl. Postoperatively (3 h - 5 days) liver body weight ratio (LBR), hepatocyte proliferation and biochemical markers were assessed. ELISA was performed to measure protein levels for VEGF. Gene expression was determined by customized cDNA arrays and quantitative RT-PCR. Administration of VEGF did not enhance LBR or hepatic proliferation, or reduce the serum parameters. VEGF levels were the highest in VEGF-treated animals. The overall survival after 90% SH reached 78% in VEGF-treated animals, but did not differ significantly from that of anti-VEGF or NaCl-treated animals (74% and 75%, respectively). Gene expression analysis showed a modulation of anti-apoptotic and cell cycle control genes that was independent of VEGF. In contrast to PH, liver regeneration and survival after SH cannot be modulated by VEGF. This indicates that the relevant mechanisms that stimulate liver regeneration after hepatectomy at least partially depend upon the extent of liver resection.

In Vitro and In Vivo Effects of Deoxyribonucleic Acid–Based Coatings Funtionalized with Vascular Endothelial Growth Factor

Vascularization is important in wound healing and essential for tissue ingrowth into porous tissue-engineering matrices. Furthermore, peri-implant tissue vascularization is known to be important for the functionality of subcutaneously implanted biosensors (e.g., glucose sensors). As a first exploration of the use of deoxyribonucleic acid (DNA)-based coatings for the optimization of biosensor functionality, this study focused on the effect of DNA-based coatings functionalized with vascular endothelial growth factor (VEGF) on in vitro endothelial cell behavior and vascularization of the peri-implant tissue in vivo. To that end, DNA-based coatings consisting of poly-D-lysine and DNA were functionalized with different amounts of VEGF (25 and 250 ng) and compared to non-coated controls and non-functionalized DNA-based coatings. The results demonstrated the superiority of VEGF-functionalized DNA-based coatings in increasing endothelial cell proliferation and migration in vitro over non-coated controls and non-functionalized DNA-based coatings. In vivo, a significant increase in vascularization of the peri-implant area was observed for VEGF-functionalized DNA-based coatings. Because no dosage-dependent effects were observed, future experiments should focus on optimizing VEGF concentration for this purpose. Additionally, the administration of VEGF in combination with other (pro-angiogenic) factors should be considered.

Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells

Hypoxia has been shown to stimulate the expression of vascular endothelial growth factor (VEGF), which is a major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF promotes angiogenesis in the testis. However, the effect of VEGF on the steroidogenesis of testosterone and the cell proliferation in Leydig cells is unclear. To assess the effects and the action mechanisms of hypoxia, a mouse TM3 Leydig cell line was employed in the present study. The Leydig cells were incubated in an incubator chamber (95% N2-5% CO2) for 1-24 h. The cultured media were collected and assayed by testosterone RIA and VEGF enzyme immunoassay. 3-(4,50-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide assay was used to detect the proliferation of Leydig cells. The present results showed that the proliferation of Leydig cells was enhanced significantly by hypoxia. The basal VEGF release was increased, and the response of VEGF production to human chorionic gonadotropin (hCG) was also enhanced in hypoxic condition. During hypoxia, administration of hCG or VEGF stimulated proliferation of Leydig cells, but the stimulatory effect was abolished by the administration of anti-VEGF antibody. Higher doses of VEGF stimulated testosterone release in a dose-dependent manner. Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on testosterone release. These data suggest that hypoxia stimulates cell proliferation and testosterone release in Leydig cells via an increase of VEGF production.

Pulmonary effects of antenatal intratracheal VEGF administration in preterm fetal rabbits

IN PRETERM FETAL RABBITS ANNE DEBEER, JACQUES JANI, FREDERIK DE BUCK, LAURENCO SBRAGIA, GUNNAR NAULAERS, PETER CARMELIET, JAN DEPREST, Catholic University Leuven, Centre for Surgical Technologies, Leuven, Belgium, Catholic University Leuven, Center of Transgene Technology and Gene Therapy, Leuven, Belgium OBJECTIVE: Vascular endothelial growth factor (VEGF) is critical for angio& vasculogenesis and pneumocyte type II cell differentiation. We aimed to assess functional pulmonary effects of antenatal intratracheal administration of VEGF in preterm rabbits. STUDY DESIGN: At 26d gestational age (GA) ovarian-end fetuses underwent tracheal occlusion (TO) to avoid leakage of instilled 30 mg recombinant rat VEGF164 in 70 mL vehicle(TO/VEGF; n=7) or 70 mL normal saline (TO/ placebo; n=8). Internal controls were weight-matched littermates (n=9). Fetuses were harvested at GA=d28 (term=31d) and ventilated with a Scireq Flexivent ventilator. Lung mechanics (total lung capacity (TLC mL/kg), dynamic resistance (R cmH2O.s/mL) and dynamic compliance (C mL/cmH2O/ kg) were measured during 16 min at 4 min intervals. RESULTS: TLC was significantly lower in TO/placebo than TO/VEGF and controls. R was higher C resp. lower in TO/placebo than controls (all time points) and TO/VEGF (R:0, 4, 8 min; C:4, 8, 12 min). CONCLUSION: In preterm rabbits, antenatal TO has an adverse effect on pulmonary mechanics nearly completely countered by in utero administered intratracheal VEGF.

High-dose atorvastatin is associated with impaired myocardial angiogenesis in response to vascular endothelial growth factor in hypercholesterolemic swine

The disappointing results of myocardial angiogenic therapy have been attributed, in part, to endothelial dysfunction present in patients with coronary disease. Statins have established proendothelial properties but seem to have dose-dependent effects on angiogenesis. We investigated the functional and molecular effects of high-dose atorvastatin on vascular endothelial growth factor-induced myocardial angiogenesis in hypercholesterolemic swine. Yucatan miniswine (20-30 kg) were fed either a normal (ND group, n = 8) or high-cholesterol diet, with (HC-ATOR group, n = 8) or without (HC group, n = 8) atorvastatin (3 mg x kg(-1) x d(-1)), for 13 weeks. Chronic ischemia was induced by ameroid constrictor placement around the circumflex artery, followed 3 weeks later by perivascular vascular endothelial growth factor administration (2 microg over 4 weeks) with a sustained release osmotic pump. Microvessel relaxation responses, myocardial perfusion, and myocardial expression of angiogenic mediators were assessed 4 weeks later. Hypercholesterolemic swine demonstrated impaired microvessel relaxation to vascular endothelial growth factor (P < .01 vs ND group) and adenosine diphosphate (P < .001 vs ND group), which was normalized in the HC-ATOR group. After perivascular vascular endothelial growth factor administration, collateral-dependent myocardial perfusion was significantly increased in the ND group but decreased in both the HC and HC-ATOR groups (both P < .01 vs the ND group). The animals in the HC-ATOR group demonstrated increased myocardial expression of the antiangiogenic protein endostatin and increased Akt phosphorylation without significant changes in Akt and endothelial nitric oxide synthase expression. Atorvastatin treatment reverses hypercholesterolemia-induced endothelial dysfunction without appreciable improvements in collateral-dependent myocardial perfusion in response to vascular endothelial growth factor treatment. Increased myocardial endostatin expression and chronic Akt activation, associated with atorvastatin therapy, might account for the lack of improvement in the angiogenic response to vascular endothelial growth factor therapy.

Participation of adrenomedullin and its relation with vascular endothelial growth factor in androgen regulation of prostatic blood flow in vivo

Objectives We had previously reported that androgen-regulated prostatic blood flow and vascular endothelial growth factor (VEGF) were involved in the signal transduction pathway. Adrenomedullin (ADM) is a multifunctional regulatory peptide with mitogenic and angiogenic capabilities that are regulated by androgen. ADM is abundantly expressed in the prostate. We focused on ADM and evaluated its participation and relation with VEGF in androgen prostatic blood flow regulation using a castrated rat model. Methods We examined the effect of locally injected dihydrotestosterone (DHT) and ADM, and the co-administration of DHT with an ADM receptor antagonist (ADM 22-52) on prostatic blood flow. Furthermore, prostatic blood flow was evaluated after ADM and VEGF administration with each other’s antagonist, VEGF neutralizing antibody and ADM 22-52, respectively. Changes in the mRNA expression levels of ADM in the prostate after castration and successive androgen stimulation were also evaluated. Results The administration of ADM promptly increased prostatic blood flow in a dose-dependent manner within 30 minutes. The DHT-induced increase in prostatic blood flow was completely abolished by co-administration with anti-ADM. Anti-ADM inhibited the VEGF-induced prostatic blood flow elevation, but a VEGF neutralizing antibody did not affect the ADM-mediated blood flow elevation. Furthermore, upregulation of the ADM gene induced by DHT was inhibited by co-administration with a VEGF-neutralizing antibody. Conclusions These results have clearly demonstrated the direct regulation of prostatic blood flow by ADM and its involvement in androgenic prostatic blood flow regulation. Furthermore, ADM was estimated to be a downstream mediator of VEGF action in the signal transduction pathway.

Impact of exogenous VEGF administration after subtotal liver resection

Aims: Recent data suggest that vascular endothelial growth factor (VEGF) can improve liver regeneration after partial hepatectomy. It is unclear, whether exogenous administration of VEGF can also be used to ameliorate impaired liver regeneration and improve overall survival after massive (90%) resection of the liver.

Exogenous Pro-Angiogenic Stimuli Cannot Prevent Physiologic Vessel Regression

In healing wounds, rising levels of vascular endothelial growth factor (VEGF) induce a period of robust angiogenesis. The levels of pro-angiogenic factors in the wound begin to decline just before a period of vascular regression, suggesting that these mediators are necessary to sustain vessel density. The purpose of this study was to determine if the maintenance of pro-angiogenic stimuli in the wound would prevent physiological vessel regression. A standard subcutaneous sponge wound model was modified by the addition of a mini-osmotic pump, allowing manipulation of the wound milieu by the addition of exogenous growth factors. After initial characterization of this model, exogenous VEGF (10 microg/mL), FGF (10 microg/mL), PDGF (10 microg/mL), or VEGF (10 microg/mL) plus FGF (10 microg/mL) were delivered to wounds and blood vessel density analyzed by immunohistochemistry. VEGF administration resulted in a transient increase in wound vessel density (P < 0.05). None of the pro-angiogenic growth factors (VEGF, FGF, PDGF, VEGF/FGF) were able to prevent vascular regression (P = NS). These findings suggest that the anti-angiogenic signals that mediate physiological vascular regression in wounds are strongly dominant over pro-angiogenic stimuli during the later phases of wound healing. Clinical manipulation of anti-angiogenic signals in addition to the currently used pro-angiogenic targets may be needed to achieve therapeutic modulation of blood vessel density.

Vascular endothelial growth factor administration does not improve microvascular disease in the salt-dependent phase of post-angiotensin II hypertension

Renal microvascular injury and tubulointerstitial inflammation may provide a potential mechanism for the development of salt-sensitive hypertension. Therefore, we hypothesized that vascular endothelial growth factor (VEGF) administration would prevent the development of salt-sensitive hypertension induced by ANG II. Infusion of ANG II in rats for 2 wk led to an elevation in blood pressure and an increase in blood urea nitrogen. Prominent tubular injury, focal areas of peritubular capillary loss accompanied by a decrease in urinary nitrites, thickening of the afferent arteriole, and an elevation in systemic and renal VEGF protein levels also occurred. In separate studies, animals were infused with ANG II and then placed on a low-salt diet for 1 wk. At this point, the animals were paired on the basis of weight and blood pressure and treated with either VEGF(121) or vehicle subcutaneously for 8 wk while being fed a high-salt diet. During the treatment period, a spontaneous improvement in many parameters, including both renal function and healing of the peritubular capillaries, occurred to the same degree in both vehicle- and VEGF(121)-treated rats. VEGF(121) significantly reduced blood pressure and accelerated the recovery of tubular injury. In contrast, vehicle-treated rats demonstrated a persistent increase in afferent arteriolar media-to-lumen ratio, which was further enhanced in rats treated with VEGF(121). Therefore, VEGF therapy has only limited benefits on the healing of renal lesions in the salt-dependent phase of post-ANG II-mediated hypertension.

Improvement of Venous Flap Survival by Application of Vascular Endothelial Growth Factor in a Rat Model

Expression of vascular endothelial growth factor (VEGF) in the venous flap and the effect of exogenous VEGF on survival of the venous flap were studied in rats. A 4- x 4-cm groin type 2 venous skin flap was used in the study. In part 1, biopsies were taken from the flap at 0, 6, 12, 24, and 48 hours after the flaps were raised. VEGF gene expression was measured. In part 2, exogenous VEGF (1 microg/mL) was injected subdermally into the flaps in 10 rats before the flaps were replaced. Flaps that received a saline injection were used as the control. Skin paddle survival was measured on postoperative day 7. The results showed that VEGF expression was significantly increased at 24 and 48 hours after venous flap elevation (P < 0.05). Injection of exogenous VEGF to the flap significantly improved survival of the flap (73% of the flap) when compared with the control, which had a 39% mean percent survival (P < 0.05). We conclude that VEGF expression was increased in the venous flap. Administration of exogenous VEGF significantly improved survival of the venous flap.

A potent angiogenic factor, vascular endothelial growth factor, improves the survival of the on-going acute hepatic failure in rats

Although it has been shown that a simultaneous administration of the vascular endothelial growth factor (VEGF); a potent angiogenic factor, could improve the overall survival of chemically induced acute hepatic failure (AHF) in rats, it has not been elucidated yet whether this salvage effect can be observed in the on-going AHF or not. For future clinical application, we examined the effect of VEGF on the on-going AHF. A combination of d-galactosamine (Gal-N) and lipopolysaccharide (LPS) was administered to induce AHF in rats. The survival rate and several indices were compared with or without VEGF treatment at 12 and 24h after the intoxication. Even after the establishment of severe liver injury, the overall survival and the serum ALT elevation were significantly improved by treatment with VEGF. The proliferation of the hepatocytes and sinusoidal endothelial cells (SEC) was also stimulated by VEGF. Furthermore, VEGF prevented the destruction of the architecture of the hepatic sinusoids. Since VEGF significantly improved the survival of the on-going AHF, the exogenous VEGF administration may represent a feasible new therapeutic strategy for AHF in the future.

Transmyocardial revascularization and vascular endothelial growth factor administration enhance effect of cell transplantation for myocardial infarct repair in rats

To test the hypothesis that transmyocardial revascularization and vascular endothelial growth factor (VEGF) administration would enhance cell transplantation effect for myocardial infarct. After ligation of left anterior descending coronary artery, the recipient rats were treated with transmyocardial revascularization. Ten minutes after transmyocardial revascularization, recombinant murine VEGF165 was injected into the clot of the drilling channel and the myocardium bordering the channel. Two weeks after transmyocardial revascularization, differentiated cells from embryo stem cells were injected into the infarcted myocardium and vascular endothelial growth factor was injected again in the same dose. Four weeks after the differentiated cells were grafted, cardiac function was assessed by hemodynamic measurements. Capillary density and infarct size in the infarct region were measured with a previous experimental method. Graft histology and morphology were also evaluated. Four weeks after the differentiated cells were grafted, myocardial infarct rats treated with transmyocardial revascularization and vascular endothelial growth factor showed a significantly higher cardiac function in hemodynamic measurements (P<0.01) than other control groups. A significant increase in capillary density and reduction in infarct size were observed in the infarct hearts of the myocardial infarct rats under combination therapy (P<0.01). Before differentiated cell transplantation, transmyocardial revascularization and vascular endothelial growth factor administration caused an angiogenesis effect to enhance neovascularization. It may be superior in attenuating the progression of cardiac dysfunction in the rat model compared with the myocardial infarct rat transplanted cell alone.

New Model of Liver Regeneration Induced Through Use of Vascular Endothelial Growth Factor

Introduction Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen. The objective of this study was to verify the proregenerative effects of VEGF in an experimental model of acute liver failure. Materials and methods Sixty four rats that underwent intraperitoneal injection of carbon tetrachloride (CCl 4) were randomly divided into two groups: group B animals received intravenous injection of VEGF 164 1 hour following CCl 4 poisoning. Group A hosts were untreated. To obtain daily liver function tests (LFTs) and histological samples, on each day up to 8 days we sacrificed four rats in each group. Results The laboratory examinations showed notable alteration of LFTs in group A, while group B revealed only slight changes. The histological examination showed greater liver damage in group A compared with group B. Conclusion Our results suggest that administration of exogenous VEGF protects the liver from CCl 4-induced acute hepatic failure. Further studies are underway to assess whether exogenous VEGF is effective in other liver injuries.

Contraindications of VEGF-based therapeutic angiogenesis: Effects on macrophage density and histology of normal and ischemic brains

Therapeutic angiogenesis by vascular endothelial growth factor (VEGF) is advocated as a promising treatment strategy for brain ischemic stroke. However, data in the literature demonstrating the benefit of therapeutic angiogenesis are contradictory. In this paper, we describe the effects of non-angiogenic and angiogenic doses of VEGF 165 on macrophage density and histology of normal and ischemic brains of adult rats. VEGF 165 was administered intra-arterially for 7 days following temporary occlusion of the middle cerebral artery. In contrast to ischemic brains treated with non-angiogenic doses of VEGF 165 which showed preserved neuropil and reduced numbers of macrophages, ischemic brains treated by an angiogenic dose showed phagocytized neuropil and high macrophage density. Though neither non-angiogenic nor angiogenic doses caused macrophage infiltration in normal brains, damage of the brain matrix occurred with the angiogenic dose. These results suggest an angiogenic dose of VEGF 165 injures the nervous tissue rather than promote recovery. Angiogenesis by VEGF monotherapy for ischemic stroke should be viewed with caution, or avoided. Since our data show intravascular administration of VEGF 165 does not cause macrophage inflammation, in contrast to reports in the literature whereby VEGF 165 was applied directly to the brain, our findings also indicate the relationships between VEGF, angiogenesis, and macrophage inflammation are governed by the route VEGF is administered to the brain.

Intramuscular Administration of Vascular Endothelial Growth Factor Augmenting Collateral Circulation in a Rat Hindlimb Replantation Model

This study evaluated the effect of exogenous vascular endothelial growth factor (VEGF) on augmentation of collateral circulation in a rat hindlimb replantation model. Twenty-eight rats, divided into four groups, underwent right hindlimb replantation. In experimental Group 1, the rats received daily intramuscular VEGF injections (1 microg/kg). In Group 2, the rats received 25 microg/kg VEGF injections. In Group 3, the rats received 50 microg/kg VEGF injections. In Group 4, the control group, rats received saline injections. Seven days after replantation, the repaired femoral vessels were excised. Seven days after excision of the femoral vessels, toe necrosis ratios of the replanted hindlimbs were grossly examined, and calf blood pressures were measured. Angiography was performed and muscle tissue was biopsied for histology. The results showed that administration of VEGF significantly decreased toe necrosis and improved calf blood pressure ratio, angiographic score, and capillary density in the replanted hindlimbs that underwent late vessel excision. The effect of VEGF was significantly dose-dependent. The authors conclude that VEGF can augment angiogenesis and collateral vessel formation in replanted limbs, and improve survival in replanted limbs with potential late arterial insufficiency.

Effects of VEGF and nitric oxide synthase inhibition on blood–brain barrier disruption in the ischemic and non-ischemic cerebral cortex

Objectives: This study was performed to compare the effects of exogenous vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) inhibition on blood–brain barrier (BBB) disruption in the ischemic cortex (IC) and non-ischemic contralateral cortex (CC) during the early stage of focal cerebral ischemia in rats.Methods: A middle cerebral artery (MCA) was occluded after a craniotomy in each rat under isoflurane anesthesia. Two more craniotomies were performed over the contralateral non-ischemic hemisphere to expose cerebral cortex. For the control rats, the normal saline patches were applied to all three craniotomy holes (control group). To inhibit NOS, NG-nitro-L-arginine-methyl ester (L-NAME) (10 mg/kg) was administered i.v. 20 minutes after MCA occlusion (L-NAME group). In another group, VEGF (10−10 M) was topically applied 30 minutes after MCA occlusion on the IC as well as one of the holes of the contralateral cortex (VEGF group). To investigate the effects of the combination of VEGF and L-NAME, both L-NAME and VEGF were administered as described above (L-NAME+ VEGF group). The transfer coefficient (Ki) of 14C-α-aminoisobutyric acid and the volume of 3H-dextran (70000 Da) distribution were determined to measure the degree of BBB disruption at 1 hour after MCA occlusion.Results: In the control group, Ki of the IC was significantly higher than the contralateral cortex (CC) (p<0.005). VEGF application increased the Ki of the IC further when compared with the control group (+51%, p<0.05%). L-NAME administration produced no significant decrease in the Ki of the IC when compared with the control group. With L-NAME+ VEGF administration, the Ki of the IC became significantly lower than that of the VEGF alone (−38%, p<0.005). Thus, L-NAME produced a much greater decrease in the Ki of the IC in the VEGF treated than the control animals (p<0.05). In the non-IC, VEGF, L-NAME, or their combination did not affect BBB disruption. The volume of dextran distribution followed a similar pattern to Ki.Discussion: Our data suggest that even in the early stage of focal cerebral ischemia, the degree of BBB disruption in response to the exogenous VEGF is much greater in the ischemic than in the non-IC and that the mechanism of the increase of BBB disruption by VEGF in the IC involves the NOS pathway.