Determination of the effectiveness of Dorema ammoniacum gum on wound healing: an experimental study.

OBJECTIVE: For a long time, natural compounds have been used to accelerate wound healing. In this study, the topical effects of ammoniacum gum extract on wound healing were investigated in white male rats. METHOD: Following skin wound induction in aseptic conditions, 48 Wistar rats were divided into six equal groups; phenytoin cream 1% (standard), untreated (control), Eucerin (control), and 5%, 10% and 20% ointments of Dorema ammoniacum gum extract (treatment groups). All experimental groups received topical drugs daily for 14 days. The percentage of wound healing, hydroxyproline content, histological parameters, and growth factors (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-α) were measured in experimental groups. RESULTS: The areas of the wounds in the treatment groups were significantly decreased compared with the wound areas of control groups at 5, 7 and 10 days after wounding. On the 12th day, the wounds in the treatment groups were completely healed. Hydroxyproline contents were significantly increased in the treatment groups compared with the control groups (p<0.001). In histological evaluation, the re-epithelialisation, increasing thickness of the epithelial layer, granulation tissue and neovascularisation parameters in the treatment groups showed significant increases compared with the control groups. Also, serum levels of TGF-ß, PDGF, EGF and VEGF in the treatment groups were significantly increased compared to the control groups. CONCLUSION: The topical application of ammoniacum gum extract significantly increases the percentage of wound healing in rats and reduces the time of wound closure.

VEGF receptor antagonist Cyclo-VEGI reduces inflammatory reactivity and vascular leakiness and is neuroprotective against acute excitotoxic striatal insult.

BACKGROUND: Excitotoxic brain insult is associated with extensive neuronal damage but could also cause inflammatory reactivity and vascular remodeling. The effects of the vascular endothelial growth factor (VEGF) inhibitor, Cyclo-VEGI on expression of VEGF, microgliosis and astrogliosis, blood-brain barrier (BBB) integrity and neuronal viability have been studied following intra-striatal injection of the excitotoxin, quinolinic acid (QUIN). The purpose of this study was to examine VEGF-dependent inflammatory responses in excitotoxin-injected brain and their dependence on pharmacological antagonism of VEGF receptors. METHODS: Single and double immunofluorescence staining of cellular (microglia, astrocyte, neuron) responses and dye and protein infiltration of blood-brain barrier have been applied in the absence, and presence, of pharmacological modulation using a VEGF receptor antagonist, Cyclo-VEGI. Dunn-Bonferroni statistical analysis was used to measure for significance between animal groups. RESULTS: Detailed analysis, at a single time point of 1 d post-QUIN injection, showed excitotoxin-injected striatum to exhibit marked increases in microgliosis (ED1 marker), astrogliosis (GFAP marker) and VEGF expression, compared with PBS injection. Single and double immunostaining demonstrated significant effects of Cyclo-VEGI treatment of QUIN-injected striatum to inhibit microgliosis (by 38%), ED1/VEGF (by 42%) and VEGF striatal immunoreactivity (by 43%); astrogliosis and GFAP/VEGF were not significantly altered with Cyclo-VEGI treatment. Leakiness of BBB was indicated by infiltration of Evans blue dye and plasma protein fibrinogen into QUIN-injected striatum with barrier permeability restored by 62% (Evans blue permeability) and 49% (fibrinogen permeability) with Cyclo-VEGI application. QUIN-induced toxicity was demonstrated with loss of striatal neurons (NeuN marker) and increased neuronal damage (Fluoro-Jade marker) with significant neuroprotection conferred by Cyclo-VEGI treatment (33% increase in NeuN and 38% decrease in Fluoro-Jade). CONCLUSION: An antagonist for VEGF receptor-mediated signaling, Cyclo-VEGI, has shown efficacy in a broad spectrum of activity against striatal excitotoxic insult including inhibition of microgliosis, reduction in leakiness of BBB and parenchymal infiltration of plasma fibrinogen and in conferring significant protection for striatal neurons. Antagonism of VEGF-mediated activity, possibly targeting VEGF receptors on reactive microglia, is suggested as a neuroprotective mechanism against inflammatory reactivity and a novel strategy to attenuate acute excitotoxic damage.

Chemotherapy and antiangiogenesis--drug-specific, dose-related effects.

Dose-response effects of fluorouracil, paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide and etoposide on VEGF165/164-mediated angiogenesis using the rat mesenteric-window angiogenesis assay are reported. VEGF is a pivotal pro-angiogenic factor in most tumors. Microvessel spatial extension, density, pattern formation and segment length were assessed quantitatively and objectively. A single i.v. injection of each drug was given at a low, intermediate or high dose, 7 days before sacrifice. All the drugs elicited significant responses in terms of one or more measured variables. Only paclitaxel, doxorubicin and cyclophosphamide significantly suppressed the overall angiogenic response (p < or = 0.0001, p < or = 0.0002 and p < or = 0.05, respectively), however. Taking toxicity into account, paclitaxel was more potent in inhibition of angiogenesis than the other agents. No clear correlation was found between drug half-life, the degree of toxic effects (in terms ofbody weight changes) and the antiangiogenic effect. The antiangiogenic effects were distinctly drug specific.

Malignant mesothelioma growth inhibition by agents that target the VEGF and VEGF-C autocrine loops.

Malignant mesothelioma (MM) is a locally aggressive tumor that originates from the mesothelial cells of the pleural and sometimes peritoneal surface. Conventional treatments for MM, consisting of chemotherapy or surgery give little survival benefit to patients, who generally die within 1 year of diagnosis. Hence, there is an urgent need for the development of alternative therapies. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for MM. The closely related molecule, VEGF-C, is also implicated in malignant mesothelioma growth. VEGF-C and its cognate receptor VEGFR-3 are co-expressed in mesothelioma cell lines. A functional VEGF-C autocrine growth loop was demonstrated in mesothelioma cells by targeting VEGF-C expression and binding to VEGFR-3. The ability of novel agents that reduce the levels of VEGF and VEGF-C to inhibit mesothelioma cell growth in vitro was assessed. Antisense oligonucleotide (ODN) complementary to VEGF that inhibited VEGF and VEGF-C expression simultaneously specifically inhibited mesothelioma cell growth. Similarly, antibodies to VEGF receptor (VEGFR-2) and VEGF-C receptor (VEGFR-3) were synergistic in inhibiting mesothelioma cell growth. In addition, a diphtheria toxin-VEGF fusion protein (DT-VEGF), which is toxic to cells that express VEGF receptors was very effective in inhibiting mesothelioma cell growth in vitro. These results indicate that targeting VEGF and VEGF-C simultaneously may be an effective therapeutic approach for malignant mesothelioma.

Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors.

Decalcified freeze-dried bone allograft (DFDBA), believed to serve as a matrix for new bone growth and to contain various bone-inducing growth factors, is currently used to regenerate periodontal defects and to restore and maintain dental alveolar ridges. Growth factors within DFDBA are extracted during the demineralization process, thus rendering the allograft incapable of spontaneous osteogenesis; however, exogenous growth factor addition to DFDBA may enhance the osteogenic capacity of native osteoblasts. This study's purpose is to evaluate murine osteoblast proliferation in the presence of various exogenous soluble growth factors as measured by fluorescence units. Osteoblasts harvested from mouse pup calvaria were cultured with 2% residual calcium-DFDBA and supplemented by one of the following growth factors or combinations of these factors: transforming growth factor-beta (TGF-beta), insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), fibroblast growth factors basic (bFGF), or vascular endothelial growth factors (VEGF). Osteoblast proliferation rates indicate that the in vitro supplementation of 2% residual calcium-DFDBA with the combination of IGF and TGF-beta, IGF and PDGF, and PDGF and TGF-beta significantly (P < or = .05) enhance murine osteoblast activity and proliferation at 7 days compared with the control containing no exogenous growth factors.

Possible involvement of the vascular endothelial growth factor-Flt-1-focal adhesion kinase pathway in chemotaxis and the cell proliferation of osteoclast precursor cells in arthritic joints.

Vascular endothelial growth factor (VEGF) plays a crucial role in the pathogenesis of inflammatory joint disease, including angiogenesis and synovitis. Rheumatoid arthritis is a chronic inflammatory disease characterized by progressive synovitis and subsequent bone destruction mediated by osteoclasts (OCs). In this study, we investigate the effects of VEGF on OC precursor cells (pOCs) using Raw cells and adjuvant-induced arthritis in rats. OCs and pOCs in the arthritic joints express VEGF and VEGF receptor type I (Flt-1). Raw cells also express Flt-1, and VEGF treatment stimulated chemotaxis, cell proliferation, the association of Flt-1 with focal adhesion kinase (FAK), and the tyrosine phosphorylation of FAK in Raw cells. The tyrosine phosphorylation of FAK was also observed in pOCs in the arthritic joints of adjuvant-induced arthritis. Adenovirus-mediated expression of FAK-related nonkinase in Raw cells inhibited the effects of VEGF in a dominant negative manner. Furthermore, intra-articular injection of the FAK-related nonkinase virus suppressed the recruitment of pOCs and bone destruction. Our results suggest the possible involvement of the VEGF-Flt-1-FAK pathway in inflammatory disease-induced joint destruction.

Intracavernosal injection of vascular endothelial growth factor induces nitric oxide synthase isoforms.

OBJECTIVE: To identify genes that are affected by vascular endothelial growth factor (VEGF), as an intracavernosal injection with VEGF improved the recovery of erectile function in a rat model of arteriogenic impotence, specifically examining the three nitric oxide synthase (NOS) genes, nNOS, eNOS, and iNOS. MATERIALS AND METHODS: Male rats had their pudendal arteries ligated or underwent a sham operation. They were then treated by an intracavernosal injection with 4 microg of VEGF in phosphate-buffered saline (PBS) or PBS alone. At 6 and 24 h after treatment electrostimulation was applied to the cavernosal nerve and the intracorporal pressure measured. The erectile tissue was then harvested for RNA isolation and cryo-sectioning. The isolated RNA was used for microarray and reverse transcription-polymerase chain reaction (RT-PCR) analyses, and the tissue sections for immunohistochemical analysis. RESULTS: Microarray analysis detected nNOS, eNOS and iNOS at very low expression levels in PBS-treated rats; expression levels were higher for eNOS and iNOS in all VEGF-treated rats. These results were further confirmed by RT-PCR analysis. Immunohistochemical analysis identified the cavernosal endothelium and smooth muscle as the tissue types where eNOS and iNOS were up-regulated, respectively. CONCLUSIONS: This is the first report of the induction of both eNOS and iNOS in the penis after intracavernosal VEGF. These events may help support a significant recovery of erectile function after interrupting the blood supply to the penis.

The effect of vascular endothelial growth factor on a rat model of traumatic arteriogenic erectile dysfunction.

PURPOSE: We tested the hypothesis that intracavernous injection of vascular endothelial growth factor (VEGF) can restore erectile function in a rat model of traumatic arteriogenic erectile dysfunction. MATERIALS AND METHODS: Exploration of bilateral internal iliac arteries was performed in 50, 3-month-old male rats. A total of 44 rats underwent bilateral ligation of the internal iliac arteries and 6 that underwent exploration only served as the sham operated group. Minutes later intracavernous injection of phosphate buffered saline (PBS) plus bovine serum albumin in 16 rats, 2 microg. VEGF plus PBS plus BSA in 12 and 4 microg. VEGF plus PBS plus BSA in 16 was performed. At weeks 1, 2 and 6 about a third of the rats in each group underwent electrostimulation of the cavernous nerves to assess erectile function and were then sacrificed. Penile tissues were collected for histochemical and electron microscopy examinations. RESULTS: No impairment of erectile function was noted in sham operated rats. Immediately after arterial ligation all rats showed little or no erectile response to neurostimulation. In PBS treated rats modest recovery of erectile function was noted at week 6. Significant recovery of erectile function was noted in VEGF treated rats at weeks 1 and 2 in the 4 microg. group only and at week 6 in the 2 and 4 microg. groups. Neuronal nitric oxide synthase staining showed a reduction in neuronal nitric oxide synthase positive nerve fibers in the dorsal or intracavernous nerves at week 1. Moderate recovery of neuronal nitric oxide synthase positive nerve fibers was noted in the 2 and 4microg. VEGF treated groups but not in the PBS treated group. Electron microscopy revealed no pathological change in sham operated rats. In dorsal nerves the atrophy of myelinated and nonmyelinated nerve fibers was noted in ligated plus PBS treated rats. Partial recovery was observed in VEGF treated rats. Scattered atrophic smooth muscle cells were seen in PBS and occasionally in VEGF treated rats but not in the sham operated group. The most dramatic findings in VEGF treated rats were hypertrophy and hyperplasia of the endothelial cells, especially those lining the small capillaries. CONCLUSIONS: Ligation of bilateral internal iliac arteries produced a reliable animal model of traumatic arteriogenic erectile dysfunction. Intracavernous injection of VEGF minutes after arterial ligation facilitated the recovery of erectile function.

An update on angiogenesis therapy.

Angiogenesis is a novel approach for the therapy of various ischemia-related pathophysiologic conditions. Proangiogenic growth factors have shown promising results in preclinical studies using protein- and gene-based therapies. However, their success in clinical trials is hindered by the lack of an optimal delivery strategy that would provide sustained and localized levels of the growth factors in the diseased tissue. Targeted delivery of proangiogenic agents is expected to demonstrate therapeutic efficacy of growth factors at relatively lower doses, without the risk of systemic toxicity in terms of unwanted angiogenesis. To achieve the above objectives, various drug delivery systems are under investigation. This review describes the basic mechanism of action of growth factors, their current status in preclinical and clinical studies, and the issue of drug delivery.

Matrix metalloproteinase 9, apoptosis, and vascular morphology in early arthritis.

OBJECTIVE: To examine matrix metalloproteinase 9 (MMP-9) in the synovial fluid (SF) and synovial membrane (SM) in relation to vascular endothelial cell (EC) apoptosis, vascular endothelial growth factor (VEGF), and SM vascular pattern. METHODS: Thirty-four patients underwent needle arthroscopy of the knee joint; 12 had early rheumatoid arthritis (RA), 12 had early psoriatic arthritis (PsA), and 10 had osteoarthritis (OA). The early RA and early PsA patients were matched for disease activity. SF levels of MMP-9 and VEGF were measured by an enzyme-linked immunosorbent assay, and EC apoptosis was measured by TUNEL assay. MMP-9 expression was examined in SM by immunohistochemistry. Synovial tissue explants were stimulated with VEGF, and MMP-9 levels were measured in the supernatants. The synovial vascular pattern was recorded. RESULTS: SF MMP-9 levels were significantly higher in early PsA patients than in early RA patients; OA patients had minimal levels. MMP-9 levels correlated with blood vessel morphology and SF VEGF levels. MMP-9 expression was greater in early PsA SM than in early RA SM, but the difference was not significant. In contrast however, EC apoptosis was greater in early RA SM than in early PsA SM. MMP-9 levels increased 2-fold and 9-fold, respectively, in SM explant culture supernatants on day 7 in response to stimulation with 25 ng/ml and 50 ng/ml of VEGF. CONCLUSION: SF MMP-9 levels correlate with the pattern of SM neovascularization and SF VEGF levels in early inflammatory arthritis, and VEGF increases MMP-9 production by SM. Endothelial cell apoptosis, however, appears to be more prevalent in early RA. This combination of factors may explain the pattern of differential angiogenesis in these arthritides.

Inhibition of inflammatory corneal angiogenesis by TNP-470.

PURPOSE: To determine the efficacy of the angiogenic inhibitor TNP-470 on inflammatory corneal neovascularization. Topical and systemic delivery of the drug were investigated in a murine model as well as inhibition of endothelial cell proliferation in vitro and in vivo. METHODS: The effect of TNP-470 on VEGF- and bFGF-stimulated bovine capillary endothelial (BCE) cell proliferation was evaluated in vitro. Corneal neovascularization was induced in vivo by mechanical debridement of the corneal and limbal epithelium with 0.15 M NaOH on C57BL6 mice. TNP-470 was administered systemically at 30 mg/kg body weight (BW) every other day or topically three times daily in a concentration of 5 ng/ml dissolved in methylcellulose. Vessel length was investigated on day 7. VEGF protein content in murine corneas was analyzed by ELISA on days 2, 4, and 7 of treatment. A modified bromouridine (BrdU) ELISA was used to quantify endothelial cell proliferation. RESULTS: TNP-470 exerted a dose-dependent inhibition of bFGF- and VEGF-induced endothelial cell proliferation in vitro. Both systemic and topical application of TNP-470 led to a significant reduction of inflammatory corneal neovascularization (P < 1 x 10(-5)). BrdU labeling showed that TNP-470 inhibited endothelial cell proliferation. VEGF protein levels were reduced by systemic TNP-470 treatment. CONCLUSIONS: These results suggest that TNP-470 reduces inflammatory corneal angiogenesis by directly inhibiting endothelial cell proliferation. Topical and systemic treatment with TNP-470 reduces VEGF levels that are responsible for vessel growth during the neovascularization process.

Healing of a free tracheal autograft is enhanced by topical vascular endothelial growth factor in an experimental rabbit model.

OBJECTIVE: In 1996, we introduced the free tracheal autograft technique for repair of congenital tracheal stenosis from complete tracheal rings in infants and children. Sources of possible concern with this procedure include the potential for autograft ischemia, patch dehiscence, and recurrent stenosis. Vascular endothelial growth factor is a potent angiogenic inducer (particularly in the setting of ischemia, hypoxia, or both) and is postulated to promote tissue healing. The purpose of this study was to test the hypothesis that pretreatment of tracheal autografts with topical vascular endothelial growth factor would enhance tracheal healing. METHODS: In a rabbit model of tracheal reconstruction (n = 32), an elliptically shaped portion of the anterior tracheal wall was excised. The excised portion of trachea was one third of the tracheal circumference and 2 cm in length (6 tracheal rings). This portion of trachea (the autograft) was soaked in either vascular endothelial growth factor (5 microg/mL, n = 16) or normal saline solution (n = 16) for 15 minutes before being reimplanted in the resultant tracheal opening. Animals were killed and autografts were examined at 2 weeks, 1 month, and 2 months postoperatively for gross and microscopic characteristics. RESULTS: By 2 weeks, and progressing through 1 and 2 months, autografts treated with vascular endothelial growth factor, as compared with control autografts, had reduced luminal stenosis, submucosal fibrosis, and inflammatory infiltrate (P <.05). The autografts tended to become malaligned in control animals, whereas the tracheal architecture was preserved in rabbits treated with vascular endothelial growth factor. Microvascular vessel density was significantly greater in all vascular endothelial growth factor groups (P <.05) at all time intervals. CONCLUSIONS: Topical treatment of free tracheal autografts with vascular endothelial growth factor in a rabbit tracheal reconstruction model enhanced healing, as evidenced by accelerated autograft revascularization, reduced submucosal fibrosis and inflammation, and preservation of the normal tracheal architecture. Topical vascular endothelial growth factor may improve future results of tracheal reconstruction.

Extensive in vivo angiogenesis following controlled release of human vascular endothelial cell growth factor: implications for tissue engineering and wound healing.

Vascular endothelial cell growth factor (VEGF) has strong stimulating effects on vascularization. Though very potent, VEGF is rapidly degraded due to its short half-life and when administrated by uncontrolled and nonspecific methods; however, its systemic administration in large doses can cause harmful side effects. Controlled release technology would allow delivering desired levels of bioactive VEGF within extended periods and permit examination of the in vivo effects of the compound in a broader way. The objective of this study was to determine the in vitro release behavior of VEGF from calcium alginate microspheres and the potency of this controlled release system in promoting localized neovascularization at the subcutaneous site of the rat model. In vitro release of human VEGF165 (2 and 4 microg/cm3 microsphere) was studied for 3 weeks under static conditions at 25 degrees C, and daily hormone release was measured using a competitive enzyme immunoassay. Following an uncontrolled release within the first 4 days, a quite constant zero-order VEGF release of 50 to 90 and 70 to 120 ng/day was achieved from 2 and 4 microg/cm3 polymer loaded microspheres respectively. In vivo angiogenesis was studied for a period of 8 weeks and evaluated using immunoperoidase staining and histopathological measurements. In vivo studies with rats (n = 24) showed a considerable level of capillary network formation at the epigastric groin fascia of VEGF microsphere-implanted rats starting from the first week. The most extensive neovascularization was observed in the group with 3 week postimplanted 4 microg VEGF containing microspheres; this level of vascularization was quite similar after 8 weeks. While the control group showed no evidence of angiogenesis, the difference in VEGF-induced neovascularization is statistically significant (p < 0.03). Immunostaining of the specimens showed a strong relationship between the release of human VEGF and neovascularization. The controlled VEGF release system described here promotes vigorous angiogenesis and has applicability for tissue engineering and wound healing studies.

Vascular endothelial growth factor-B-deficient mice display an atrial conduction defect.

BACKGROUND: Vascular endothelial growth factors (VEGFs) and their receptors are essential regulators of vasculogenesis and angiogenesis in both embryos and adults. One of the factors with a still unknown physiological function is VEGF-B, which is expressed in many tissues, including the heart. METHODS AND RESULTS: Mice carrying a targeted deletion in the VEGF-B gene were developed. In VEGF-B(-/-) animals, no gross abnormalities were observed in organs that normally show high expression of VEGF-B, such as the heart, muscle, and kidney. Analysis of heart function by ECG showed that adult VEGF-B(-/-) mice have an atrial conduction abnormality characterized by a prolonged PQ interval. VEGF- or basic fibroblast growth factor-induced corneal angiogenesis was similar in normal and VEGF-B(-/-) mice. CONCLUSIONS: VEGF-B seems to be required for normal heart function in adult animals but is not required for proper development of the cardiovascular system either during development or for angiogenesis in adults.

Inhibitory effects of tetrandrine and related synthetic compounds on angiogenesis in streptozotocin-diabetic rodents.

Structure-activity relationships of tetrandrine, isolated from a Kampo medicine, Stephania tetrandrae S. MOORE (root), and related synthetic compounds, were investigated in in vitro fetal bovine serum (FBS)-stimulated angiogenesis of cultured choroids in streptozotocin-diabetic Wistar rats, and air-pouch granuloma angiogenesis in vivo in diabetic mice. Tetrandrine, KS-1-1 (6,7-dimethoxy-1-[[4-[5-(6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroiso quinolinyl)methyl-2-methoxy]phenoxy]benzyl]-2-methyl-1,2,3,4-tetrahyd roisoquinoline), and KS-1-4 (6,7-dimethoxy-1-[[4-[4-(6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroiso quinolinyl)methyl]phenoxy]benzyl]-2-methyl-1,2,3,4-tetrahydroisoquino line), potently inhibited choroidal angiogenesis and air-pouch granuloma angiogenesis in the diabetic state. Their inhibitory effects on diabetic choroids were greater than those on normal choroids. Among these compounds, KS-1-4 inhibited only diabetic angiogenesis. These compounds significantly inhibited FBS-stimulated tube formation in vascular endothelial cells from normal rats. Tetrandrine and KS-1-4, but not KS-1-1, inhibited vascular endothelial growth factor- and platelet-derived growth factor-BB-stimulated angiogenesis in normal choroids. The bis[tetrahydroisoquinoline] moiety, connected by oxy-bis[phenylenemethylene] and 2,2'-dimethyl groups in tetrandrine, contributes to the inhibition of diabetic choroidal angiogenesis. KS-1-4 may be a candidate for anti-choroidopathy and retinopathy drugs in the diabetic state.

Hypoxia augments cytokine (transforming growth factor-beta (TGF-beta) and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts.

Vascular endothelial growth factor (VEGF) is abundant in synovium and synovial fluids, where it probably contributes to vascular permeability and angiogenesis in arthritic joints. To investigate the probable sources of VEGF in synovium, we compared the ability of several cytokines (TGF-beta, platelet-derived growth factor (PDGF), IL-1, tumour necrosis factor (TNF), basic fibroblast growth factor (bFGF) that are associated with arthritis and angiogenesis, to stimulate secretion of VEGF protein by human synovial fibroblasts. TGF-beta was the strongest inducer of VEGF secretion; six times more VEGF was secreted when cells were stimulated by TGF-beta than when stimulated by PDGF or IL-1 for 24 h. TNF-alpha and bFGF did not stimulate any secretion of VEGF. The stimulatory effects of TGF-beta and IL-1 on VEGF secretion were additive. Hypoxic culture alone also stimulated VEGF secretion, but more importantly, hypoxic culture conditions doubled the rate of VEGF secretion stimulated by the cytokines TGF-beta and IL-1. When dermal and synovial fibroblasts were stimulated identically by hypoxia and cytokines (TGF-beta and IL-1), synovial fibroblasts secreted four times more VEGF than did dermal fibroblasts. Thus in rheumatoid arthritis, the capacity of synovial fibroblasts in the hypoxic environment to secrete large amounts of VEGF in response to cytokines such as TGF-beta probably contributes significantly to angiogenesis in the synovium.

Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.

We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.