Carbon Dots Conjugated with Vascular Endothelial Growth Factor for Protein Tracking in Angiogenic Therapy.

One of the challenges of using growth factors for tissue regeneration is to monitor their biodistributions and delivery to injured tissues for minimally invasive detection. In the present study, tracking of human vascular endothelial growth factor (VEGF) was achieved by chemically linking it to photoluminescent carbon dots (CDs). Carbon dots were synthesized by the hydrothermal method and, subsequently, conjugated with VEGF using carbodiimide coupling. ELISA and western blot analysis revealed that VEGF-conjugated CDs preserve the binding affinity of VEGF to its antibodies. We also show that VEGF-conjugated CDs maintain the functionality of VEGF for tube formation and cell migration. The VEGF-conjugated CDs were also used for in vitro imaging of human umbilical vein endothelial cells. The results of this work suggest that cell-penetrating VEGF-conjugated CDs can be used for growth factor protein tracking in therapeutic and tissue engineering applications.

Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.

VEGF-induced neovascularization plays a pivotal role in corneal neovascularization (CoNV). The current study investigated the potential effect of ginsenoside Rh2 (GRh2) on neovascularization. In HUVECs, pretreatment with GRh2 largely attenuated VEGF-induced cell proliferation, migration, and vessel-like tube formation in vitro. At the molecular level, GRh2 disrupted VEGF-induced VEGF receptor 2 (VEGFR2)-Grb-2-associated binder 1 (Gab1) association in HUVECs, causing inactivation of downstream AKT and ERK signaling. Gab1 knockdown (by targeted short hairpin RNA) similarly inhibited HUVEC proliferation and migration. Notably, GRh2 was ineffective against VEGF in Gab1-silenced HUVECs. In a mouse cornea alkali burn model, GRh2 eyedrops inhibited alkali-induced neovascularization and inflammatory cell infiltrations in the cornea. Furthermore, alkali-induced corneal expression of mRNAs/long noncoding RNAs in cornea were largely attenuated by GRh2. Overall, GRh2 inhibits VEGF-induced angiogenic effect via inhibiting VEGFR2-Gab1 signaling in vitro. It also alleviates angiogenic and inflammatory responses in alkali burn-treated mouse corneas.-Zhang, X.-P., Li, K.-R., Yu, Q., Yao, M.-D., Ge, H.-M., Li, X.-M., Jiang, Q., Yao, J., Cao, C. Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.

Systemic biopolymer-delivered vascular endothelial growth factor promotes therapeutic angiogenesis in experimental renovascular disease.

We recently developed a therapeutic biopolymer composed of an elastin-like polypeptide (ELP) fused to vascular endothelial growth factor (VEGF) and showed long-term renoprotective effects in experimental renovascular disease after a single intra-renal administration. Here, we sought to determine the specificity, safety, efficacy, and mechanisms of renoprotection of ELP-VEGF after systemic therapy in renovascular disease. We tested whether kidney selectivity of the ELP carrier would reduce off-target binding of VEGF in other organs. In vivo bio-distribution after systemic administration of ELP-VEGF in swine was determined in kidneys, liver, spleen, and heart. Stenotic-kidney renal blood flow and glomerular filtration rate were quantified in vivo using multi-detector computed tomography (CT) after six weeks of renovascular disease, then treated with a single intravenous dose of ELP-VEGF or placebo and observed for four weeks. CT studies were then repeated and the pigs euthanized. Ex vivo studies quantified renal microvascular density (micro-CT) and fibrosis. Kidneys, liver, spleen, and heart were excised to quantify the expression of angiogenic mediators and markers of progenitor cells. ELP-VEGF accumulated predominantly in the kidney and stimulated renal blood flow, glomerular filtration rate, improved cortical microvascular density, and renal fibrosis, and was accompanied by enhanced renal expression of VEGF, downstream mediators of VEGF signaling, and markers of progenitor cells compared to placebo. Expression of angiogenic factors in liver, spleen, and heart were not different compared to placebo-control. Thus, ELP efficiently directs VEGF to the kidney after systemic administration and induces long-term renoprotection without off-target effects, supporting the feasibility and safety of renal therapeutic angiogenesis via systemic administration of a novel kidney-specific bioengineered compound.

Injecting vascular endothelial growth factor into the temporomandibular joint induces osteoarthritis in mice.

It is unclear whether vascular endothelial growth factor (VEGF) can initiate osteoarthritis (OA) in the temporomandibular joint (TMJ). In this study we evaluated the effects of intra-articular injection of exogenous VEGF in the TMJ in mice on the early stage. Forty-eight male Sprague-Dawley mice were equally divided into 3 groups. In the vegf group, the mice received an injection of VEGF solution (50 µL) in the TMJ once a week over a period of 4 weeks. In the sham group, the mice received an injection of saline (50 µL). The control group did not receive any injection. Four mice from each group were sacrificed at 1, 2, 4, and 8 weeks. Gradual prominent cartilage degeneration was observed in the vegf group. Additionally, this group showed higher expressions of metalloproteinase (MMP)-9, MMP-13, receptor activator of nuclear factor-kappa-B ligand (RANKL), and a higher number of apoptotic chondrocytes and VEGF receptor 2 (VEGFR2)-positive chondrocytes. Micro-computed tomography (CT) revealed prominent subchondral bone resorption in the vegf group, with a high number of osteoclasts in the subchondral bone. In vitro study demonstrated that VEGF can promote osteoclast differentiation. In conclusion, our study found that VEGF can initiate TMJ OA by destroying cartilage and subchondral bone.

Vaccination with a mutated variant of human Vascular Endothelial Growth Factor (VEGF) blocks VEGF-induced retinal neovascularization in a rabbit experimental model.

Vascular Endothelial Growth Factor (VEGF) is a key driver of the neovascularization and vascular permeability that leads to the loss of visual acuity of eye diseases like wet age-related macular degeneration, diabetic macular edema, and retinopathy of premature. Among the several anti-VEGF therapies under investigation for the treatment of neovascular eye diseases, our group has developed the vaccine candidate CIGB-247-V that uses a mutated form of human VEGF as antigen. In this work we evaluated if the vaccine could prevent or attenuate VEGF-induced retinal neovascularization in the course of a rabbit eye neovascularization model, based on direct intravitreal injection of human VEGF. Our experimental findings have shown that anti-VEGF IgG antibodies induced by the vaccine were available in the retina blood circulation, and could neutralize in situ the neovascularization effect of VEGF. CIGB-247-V vaccination proved to effectively reduce retinal neovascularization caused by intravitreal VEGF injection. Altogether, these results open the way for human studies of the vaccine in neovascular eye syndromes, and inform on the potential mechanisms involved in its effect.

Polychromatic angiography for the assessment of VEGF-induced BRB dysfunction in the rabbit retina.

PURPOSE: To determine the utility of polychromatic angiography (PCA) in the assessment of VEGF-induced blood retinal barrier (BRB) dysfunction in rabbits. METHODS: Twenty-six eyes of 24 Dutch Belted rabbits were injected intravitreally with 1.25 µg (group A, n = 5), 10 µg (group C, n = 7), or 4 µg (group B, n = 6; group D, n = 4; and group E, n = 4) of VEGF on day 0. Groups D and E were also injected intravitreally with 1.25 µg and 12.5 µg bevacizumab, respectively, on day 2. On days 0, 2, 4, 7, 11, and 14, PCA was performed using a contrast agent mixture composed of fluorescein sodium, indocyanine green, PCM102, and PCM107 and imaged with a modified fundus camera. PCA scores were based on detected leaking fluorophores. RESULTS: On day 7, there was a statistically significant difference between PCA scores of group A (0.6 ± 0.89) and both groups B (2.67 ± 1.37, P = 0.0154) and C (3.33 ± 0.52, P = 0.00085). There was also a statistically significant difference between groups B and E (PCA score 0.75 ± 0.96, P = 0.032) on day 7. On day 11, there was statistically significant difference between group C (1.80 ± 1.1) and both groups A (0, P = 0.021) and B (0.33 ± 0.52, P = 0.037). CONCLUSIONS: A differential response to both increasing VEGF dose and administration of bevacizumab could be discerned using the PCA. PCA allowed stratification of VEGF-induced BRB dysfunction and inhibitory effects of bevacizumab therapy in the rabbit retina.

A novel ex vivo murine retina angiogenesis (EMRA) assay.

Pathological retinal angiogenesis results from the imbalance of pro-angiogenic and anti-angiogenic factors. In particular, vascular endothelial growth factor (VEGF) plays a pivotal role in retinal neovascularization and various therapeutic VEGF blockers have evolved over time. Nevertheless, new retinal angiogenesis models are crucial for investigating anti-angiogenic therapies and bringing them to patients. Here, we developed a novel ex vivo murine retina angiogenesis (EMRA) assay in which endothelial sprouts originate from mature and quiescent retinal vessels. In this model, retina fragments from adult mice are embedded in a three-dimensional fibrin gel in the presence of human recombinant VEGF. Starting from the 3rd-4th day of incubation, endothelial cell sprouts invading the fibrin gel can be observed under an inverted microscope and measured at different time points thereafter. The effect of VEGF is dose-dependent, maximal stimulation being observed at day 7 for retina fragments stimulated with 25-75 ng/ml of the growth factor. To assess whether the EMRA assay is suitable for testing the activity of anti-angiogenic compounds, retina fragments were incubated with VEGF in the presence of the neutralizing anti-VEGF antibodies bevacizumab and ranibizumab. The results demonstrate that both antibodies inhibit VEGF activity in a dose-dependent manner. In conclusion, the EMRA assay represents a new ex vivo model of retinal neovascularization suitable for the rapid screening of novel anti-angiogenic therapeutics.

VEGF, which is elevated in the CSF of patients with hydrocephalus, causes ventriculomegaly and ependymal changes in rats.

Hydrocephalus is a condition characterized primarily by excessive accumulation of fluid in the ventricles of the brain for which there is currently no effective pharmacological treatment. Surgery, often accompanied by complications, is the only current treatment. Extensive research in our laboratory along with work from others has suggested a link between hydrocephalus and vascular function. We hypothesized that vascular endothelial growth factor (VEGF), the major angiogenic factor, could play a role in the pathogenesis of hydrocephalus. We tested this hypothesis by examining two predictions of such a link: first, that VEGF is present in many cases of clinical hydrocephalus; and second, that exogenous VEGF in an animal model could cause ventricular enlargement and tissue changes associated with hydrocephalus. Our results support the idea that VEGF elevation can potentiate hydrocephalus. The clinical relevance of this work is that anti-angiogenic drugs may be useful in patients with hydrocephalus, either alone or in combination with the currently available surgical treatments.

Assessment of the differences in pharmacokinetics and pharmacodynamics between four distinct formulations of triamcinolone acetonide.

PURPOSE: To compare the durability of Kenalog, Trivaris, Triesence, and compounding pharmacy preservative-free triamcinolone acetonide in pigmented rabbits with syneretic vitreous using direct visualization, pharmacodynamics, and pharmacokinetics. METHODS: Twenty-five Dutch-belted rabbits were used. Pharmacokinetic experiment: Rabbits were intravitreally injected with one of four 4-mg triamcinolone acetonide formulations. Wide-field imaging was serially performed to document residual drug mass. Pharmacodynamics experiment: Four triamcinolone acetonide groups and one control group received intravitreal recombinant human vascular endothelial growth factor 165 every 2 weeks and were followed with fluorescein angiography to assess vascular endothelial growth factor retinal vasculopathy as a measure of residual steroid effect. Particle size of the formulations was measured with Mastersizer 2000. RESULTS: Remaining triamcinolone acetonide mass after 19 weeks: 12,091 ± 2,512 pixels for the Kenalog group, 1,307.36 ± 695.57 for Trivaris, 5577 ± 1477 for Triesence, and 1,535 ± 329 for compounded preservative-free triamcinolone acetonide. Kenalog suppressed recombinant human vascular endothelial growth factor-induced retinopathy more effectively than the other triamcinolone acetonide groups at Week 39, the final time point assessed. Particle size (90th percentile) was 47 µm for Kenalog, 26 µm for Triesence, and 22 µm for both compounded preservative-free triamcinolone acetonide and Trivaris. CONCLUSION: Triamcinolone acetonide formulations do not have the same pharmacokinetics/pharmacodynamics. Kenalog has the longest vitreous visibility and durability. Particle size appears to correlate with efficacy and durability.

Topical pazopanib blocks VEGF-induced vascular leakage and neovascularization in the mouse retina but is ineffective in the rabbit.

PURPOSE: To test the effect of pazopanib, a tyrosine kinase inhibitor that blocks VEGF and platelet-derived growth factor (PDGF) receptors and c-Kit, on vascular leakage and neovascularization (NV) in the retina. METHODS: Pazopanib was tested to determine its effect on VEGF-induced vascular permeability via measurement of [(3)H]mannitol retina to lung (RLLR) and retina to renal leakage ratios (RRLR) and in rho/VEGF mice with subretinal NV. In rabbits, the effect of intravitreal, topical, and systemic pazopanib on VEGF-induced leakage was tested by vitreous fluorophotometry. RESULTS: In mice, oral pazopanib (40 mg/kg twice a day [bid]) reduced RLLR (0.84 to 0.58, P = 0.0014) and RRLR (0.55 to 0.30, P = 0.0018) in VEGF-injected eyes. After intraocular injection of VEGF into both eyes, topical pazopanib (10 mg/mL three times a day [tid] for 14 days) reduced RLLR (0.85 vs. 0.56, P = 0.001), RRLR (0.44 vs. 0.28, P = 0.0075), and immunoreactive albumin in the retina compared to values in fellow eye controls. Treatment of one eye of rho/VEGF mice with 10 mg/mL, but not 5 mg/mL, pazopanib tid reduced the mean area of subretinal NV compared to that in fellow eyes (0.0055 vs. 0.0025 mm(2), P = 0.020). In rabbits, intravitreal pazopanib suppressed VEGF-induced fluorescein leakage, but topical (10 mg/mL four times a day [qid] or 12 mg/mL bid) had no significant effect. Systemic administration of pazopanib by osmotic pump with or without 10 mg/mL drops tid also failed to suppress VEGF-induced leakage. CONCLUSIONS: Administration of pazopanib topically or systemically suppressed retinal vascular leakage in mice, but not rabbits. These data suggest differences in the blood-retinal barrier (BRB) of mice and rabbits and indicate that penetration through the outer BRB may be needed for topically administered drugs to exert effects in the retina.

Doxycycline-mediated inhibition of corneal angiogenesis: an MMP-independent mechanism.

PURPOSE: We explored the underlying mechanisms of doxycycline inhibition of corneal angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro in the presence or absence of VEGF, doxycycline, or broad-spectrum matrix metalloproteinase (MMP) inhibitors (1,10-phenanthroline [1PT] and batimastat). HUVEC proliferation was determined using Cell Counting Kit-8. Rats with VEGF-induced corneal neovascularization (CNV) were treated with saline solution, 0.1% doxycycline, 0.1% 1PT, or 50 µM batimastat (n = 7/group). The length and area of CNV were measured on day 7. The activity of MMP-2 and MMP-9 was determined by a fluorogenic peptide substrate. Western blotting and ELISA were used to analyze the expression of phosphorylated eNOS and Akt, and PI3K activity. RESULTS: Our results showed that doxycycline inhibited HUVEC proliferation induced by VEGF in a dose-dependent manner in vitro, and decreased CNV induced by VEGF in vivo in terms of vessel length and area. 1PT and batimastat showed similar MMP inhibitory functions with doxycycline in vitro and in vivo, but they had no effects on HUVEC proliferation, and only partially mimicked the inhibitory properties of doxycycline (∼45%) on angiogenesis induced by VEGF. In addition, although doxycycline is capable of modulating the PI3K/Akt-eNOS pathway in vitro and in vivo in an MMP-independent manner, 1PT and batimastat were not. CONCLUSIONS: The mechanism of doxycycline-mediated inhibition of angiogenesis occurs through MMP inhibitory activity and modulation of the PI3K/Akt-eNOS pathway, an MMP-independent mechanism.

Injection of vascular endothelial growth factor into knee joints induces osteoarthritis in mice.

UNLABELLED: Osteoarthritis (OA) is a common joint disorder affecting circa 2% of the population. OBJECTIVES: It has been suggested that secretion of vascular endothelial growth factor (VEGF) could play a role in the chain of events leading to OA. METHODS: In the present study, healthy mice were injected intra-articularly with VEGF. RESULTS: Shortly after the administration of VEGF, synovial hyperplasia, increased calcification of the articular cartilage and bone sclerosis were observed. Consequently, cartilage degradation characteristic of OA was found. These changes were seen to a lesser degree in the opposite knees of VEGF-injected mice and did not occur in the control mice. CONCLUSIONS: The findings suggest an active role of VEGF in the pathogenesis of OA and render support to a possible role for subchondral bone sclerosis in the pathogenesis of cartilage degradation.

A novel and effective human hepatocyte growth factor kringle 1 domain inhibits ocular neovascularization.

Neovascularization is the critical pathological process and the leading cause of blindness in a variety of clinical conditions. This angiogenesis process is still uncertain. Human hepatocyte growth factor 1 (HGFK1) is derived from the mature form of hepatocyte growth factor, which contains four kringle domains in its α-chain. This study aimed to investigate the antiangiogenic activity of HGFK1 using in vitro and in vivo assays. HGFK1 was added into the DMEM to test the proliferation and migration of human umbilical vascular endothelial cells (HUVEC), and it was intravitreously injected in laser photocoagulation-induced choroidal neovascularization (NV) model, oxygen-induced retinopathy model and rho/VEGF transgenic mice to test its antiangiogenic effect. The results showed that HGFK1 effectively inhibited VEGF-stimulated HUVEC proliferation and migration, and also had anti-NV activity in choroidal NV and retinal NV. It is suggested that HGFK1 has antiangiogenic activity in vitro and in vivo. It may lead to new potential drug discoveries and the development in addition to anti-VEGF therapy in the future clinical anti-angiogenesis treatment.

Bevacizumab attenuates VEGF-induced angiogenesis and vascular malformations in the adult mouse brain.

BACKGROUND AND PURPOSE: Vascular endothelial growth factor (VEGF) expression is elevated in human brain arteriovenous malformations (bAVM). We have developed a bAVM model in the adult mouse by focal Alk1 gene deletion and human VEGF stimulation. We hypothesized that once the abnormal vasculature has been established, tonic VEGF stimulation is necessary to maintain the abnormal phenotype, and VEGF antagonism by bevacizumab (Avastin) would reduce vessel density and attenuate the dysplastic vascular phenotype. METHODS: Angiogenesis and bAVM were induced by injection of adeno-associated viral vector expressing human VEGF alone into the brain of wild-type mice or with adenoviral vector expressing Cre recombinase (Ad-Cre) into Alk1(2f/2f) mice. Six weeks later, bevacizumab or trastuzumab (Herceptin, bevacizumab control) was administered. Vessel density, dysplasia index, vascular cell proliferation and apoptosis, and human IgG were assessed (n=6/group). RESULTS: Compared with trastuzumab (15 mg/kg), administration of 5, 10, and 15 mg/kg of bevacizumab to adeno-associated viral vector expressing human VEGF treated wild-type mice reduced focal vessel density (P<0.05); administration of 5 mg/kg bevacizumab decreased proliferating vascular cells (P=0.04) and increased TUNEL-positive vascular cells (P=0.03). More importantly, bevacizumab (5 mg/kg) treatment reduced both vessel density (P=0.01) and dysplasia index (P=0.02) in our bAVM model. Human IgG was detected in the vessel wall and in the parenchyma in the angiogenic foci of bevacizumab-treated mice. CONCLUSIONS: We provide proof-of-principle that, once abnormal AVM vessels have formed, VEGF antagonism may reduce the number of dysplastic vessels and should be evaluated further as a therapeutic strategy for the human disease.

Actions of bevacizumab and ranibizumab on microvascular retinal endothelial cells: similarities and differences.

BACKGROUND: Retinal endothelial cells are crucially involved in the genesis of diabetic retinopathy which is treated with vascular endothelial growth factor (VEGF) inhibitors. Of these, ranibizumab can completely restore VEGF-induced effects on immortalised bovine retinal endothelial cells (iBREC). In most experiments supporting diabetic retinopathy therapy with bevacizumab, only non-retinal EC or retinal pigment epithelial cells have been used. Also, bevacizumab but not ranibizumab can accumulate in retinal pigment epithelial cells. OBJECTIVE: To investigate the effects of bevacizumab on VEGF-induced changes of iBREC properties and potential uptake and accumulation of both inhibitors. METHODS: Uptake of VEGF inhibitors by iBREC with or without pretreatment with VEGF(165) was visualised by immunofluorescence staining and western blot analyses. Measured transendothelial resistance (TER) of iBREC (±VEGF(165)) showed effects on permeability, indicated also by the western blot-determined tight junction protein claudin-1. The influence of bevacizumab on proliferation and migration of iBREC was studied in the presence and absence of VEGF(165). RESULTS: Bevacizumab strongly inhibited VEGF-stimulated and basal migration, but was less efficient than ranibizumab in inhibiting VEGF-induced proliferation or restoring the VEGF-induced decrease of TER and claudin-1. This ability was completely lost after storage of bevacizumab for 4 weeks at 4°C. Ranibizumab and bevacizumab were detectable in whole cell extracts after treatment for at least 1 h; bevacizumab accumulated during prolonged treatment. Ranibizumab was found in the membrane/organelle fraction, whereas bevacizumab was associated with the cytoskeleton. CONCLUSION: Both inhibitors had similar effects on retinal endothelial cells; however, some differences were recognised. Although barrier properties were not affected by internalised bevacizumab in vitro, potential adverse effects due to accumulation after repetitive intravitreal injections remain to be investigated.

Diaminothiazoles inhibit angiogenesis efficiently by suppressing Akt phosphorylation.

The prevention of neovessel formation or angiogenesis is a recent popular strategy for limiting and curing cancer. Diaminothiazoles are a class of compounds that have been reported to show promise in the treatment of cancer by inhibiting cancer cell proliferation and inducing apoptosis, because of their effects on microtubules and as inhibitors of cyclin-dependent kinases. Many microtubule-targeting agents are being studied for their antiangiogenic activity, and a few have shown promising activity in the treatment of cancer. Here, we report that diaminothiazoles can be highly effective as antiangiogenic agents, as observed in the chick membrane assay. The lead compound, 4-amino-5-benzoyl-2-(4-methoxyphenylamino)thiazole (DAT1), inhibits endothelial cell processes such as invasion, migration, and tubule formation, which require a functional cytoskeleton. DAT1 also decreases the expression of cell adhesion markers. The antiangiogenic activities of DAT1 occur at concentrations that are not cytotoxic to the normal endothelium. Analysis of intracellular signaling pathways shows that DAT1 inhibits Akt phosphorylation, which is actively involved in the angiogenic process. The antiangiogenic properties of diaminothiazoles, in addition to their promising antimitotic and cytotoxic properties in cancer cell lines, give them an extra advantage in the treatment of cancer.

The antiangiogenic effects of integrin alpha5beta1 inhibitor (ATN-161) in vitro and in vivo.

PURPOSE: Integrin α5ß1 is involved in the development of choroidal neovascularization (CNV). Thus, the inhibition of integrin α5ß1 may provide an alternative to the current standard of CNV therapy, which involves inhibition of vascular endothelial growth factor (VEGF) and is not effective in all patients. This study evaluated the antiangiogenic effects of ATN-161, a small peptide inhibitor of integrin α5ß1, in human choroidal endothelial cells (hCECs) and in laser-induced CNV in rats. Furthermore, the utility of spectral-domain optical coherence tomography (SD-OCT) for dynamic observation of the development of CNV in animal models was assessed. METHODS: The antiangiogenic potential of ATN-161 was evaluated in VEFG-stimulated hCECs by MTS proliferation assays, migration assays, and synthetic matrix capillary tube formation assays. To evaluate the antiangiogenic effects of ATN-161 in vivo, CNV was induced in rats by laser photocoagulation. ATN-161, scrambled peptide, or AF564 anti-VEGF antibody, were injected intravitreally immediately after photocoagulation. Eyes were examined by SD-OCT and fluorescein angiography on days 1, 7, and 14 after injection, and the areas of CNV were measured by analysis of choroidal flatmounts at day 14. RESULTS: ATN-161 inhibited VEGF-induced migration and capillary tube formation in hCECs, but did not inhibit proliferation. In vivo, injection of ATN-161 after laser photocoagulation inhibited CNV leakage and neovascularization to an extent similar to AF564. Furthermore, SD-OCT and histologic examinations indicated that ATN-161 significantly decreased the size of laser-induced lesions. CONCLUSIONS: Integrin α5ß1 inhibition by ATN-161 may be a promising alternative therapy for CNV-related angiogenesis. In addition, SD-OCT technology allows excellent visualization of experimentally induced CNV in vivo.

Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of specific ROS in vascular pathology and may be translated into remedies for these ROS-induced abnormalities.

Docosahexaenoic acid improves the nitroso-redox balance and reduces VEGF-mediated angiogenic signaling in microvascular endothelial cells.

PURPOSE: Disturbances to the cellular production of nitric oxide (NO) and superoxide (O(2)(-)) can have deleterious effects on retinal vascular integrity and angiogenic signaling. Dietary agents that could modulate the production of these signaling molecules from their likely enzymatic sources, endothelial nitric oxide synthase (eNOS) and NADPH oxidase, would therefore have a major beneficial effect on retinal vascular disease. The effect of ω-3 polyunsaturated fatty acids (PUFAs) on angiogenic signaling and NO/superoxide production in retinal microvascular endothelial cells (RMECs) was investigated. METHODS: Primary RMECs were treated with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 48 hours. RMEC migration was determined by scratch-wound assay, proliferation by the incorporation of BrdU, and angiogenic sprouting using a three-dimensional model of in vitro angiogenesis. NO production was quantified by Griess assay, and phospho-eNOS accumulation and superoxide were measured using the fluorescent probe dihydroethidine. eNOS localization to caveolin-rich microdomains was determined by Western blot analysis after subfractionation on a linear sucrose gradient. RESULTS: DHA treatment increased nitrite and decreased superoxide production, which correlated with the displacement of eNOS from caveolar subdomains and colocalization with the negative regulator caveolin-1. In addition, both ω-3 PUFAs demonstrated reduced responsiveness to VEGF-stimulated superoxide and nitrite release and significantly impaired endothelial wound healing, proliferation, and angiogenic sprout formation. CONCLUSIONS: DHA improves NO bioavailability, decreases O(2)(-) production, and blunts VEGF-mediated angiogenic signaling. These findings suggest a role for ω-3 PUFAs, particularly DHA, in maintaining vascular integrity while reducing pathologic retinal neovascularization.

SDF-1/CXCR4 contributes to the activation of tip cells and microglia in retinal angiogenesis.

PURPOSE. Although stromal cell-derived factor (SDF)-1 contributes to angiogenesis, its effects on sprouting angiogenesis remain ill defined. The authors investigated how SDF-1 and its receptor, CXCR4, influence neovascular sprouting. METHODS. In vivo retinal vascular development was evaluated and ex vivo retinal angiogenesis induced by vascular endothelial growth factor (VEGF). Time-sequential observation was followed by the quantification of movements in neovascular sprouts and microglia. Real-time PCR was performed for the measurement of mRNA levels. RESULTS. Neutralizing antibodies against SDF-1 or an antagonist of CXCR4, AMD3100, reduced the radius of the vascularized area in retinal vascular development. These inhibitions disturbed the filopodial extensions in tip cells and proliferation in stalk cells, reduced the number of microglia, and decreased the mRNA levels of KDR/Flk-1, UNC5B, and PDGFB, which are abundantly expressed in tip cells. In ex vivo experiments, VEGF induced SDF-1 mRNA expression, and the inhibition of SDF-1/CXCR4 decreased the number of VEGF-induced neovascular sprouts. The authors further evaluated the kinetics of sprouts using time-lapse imaging and found that SDF-1/CXCR4 contributes to the elongation of neovascular sprouts and to the extension and retraction of leading edges. The movements of resident microglia after VEGF treatment were also reduced by SDF-1/CXCR4 inhibition. Interestingly, microglial depletion decreased VEGF-induced neovascular sprouts with the partial effects of SDF-1/CXCR4 blockade. CONCLUSIONS. SDF-1/CXCR4 promotes retinal angiogenesis by both tip cell activation and the indirect angiogenic effects of microglia.