Soluble Vascular Endothelial Growth Factor Receptor Research Articles

Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model

We investigated human cytomegalovirus pathogenesis by comparing infection with the low-passage, endotheliotropic strain VR1814 and the attenuated laboratory strain AD169 in human placental villi as explants in vitro and xenografts transplanted into kidney capsules of SCID mice (ie, mice with severe combined immunodeficiency). In this in vivo human placentation model, human cytotrophoblasts invade the renal parenchyma, remodel resident arteries, and induce a robust lymphangiogenic response. VR1814 replicated in villous and cell column cytotrophoblasts and reduced formation of anchoring villi in vitro. In xenografts, infected cytotrophoblasts had a severely diminished capacity to invade and remodel resident arteries. Infiltrating lymphatic endothelial cells proliferated, aggregated, and failed to form lymphatic vessels. In contrast, AD169 grew poorly in cytotrophoblasts in explants, and anchoring villi formed normally in vitro. Likewise, viral replication was impaired in xenografts, and cytotrophoblasts retained invasive capacity, but some partially remodeled blood vessels incorporated lymphatic endothelial cells and were permeable to blood. The expression of both vascular endothelial growth factor (VEGF)-C and basic fibroblast growth factor increased in VR1814-infected explants, whereas VEGF-A and soluble VEGF receptor-3 increased in those infected with AD169. Our results suggest that viral replication and paracrine factors could undermine vascular remodeling and cytotrophoblast-induced lymphangiogenesis, contributing to bleeding, hypoxia, and edema in pregnancies complicated by congenital human cytomegalovirus infection.

The Dynamics of Angiogenic Factors and Their Soluble Receptors in Relation to Organ Dysfunction in Disseminated Intravascular Coagulation Associated with Sepsis

We prospectively studied (1) the relationships between angiogenic factors, their soluble receptors and organ dysfunction and (2) the effects of disseminated intravascular coagulation (DIC)-induced platelet consumption, thrombin generation, and tissue hypoxia on the expression of the factors and receptors. Fifty patients with sepsis were classified into two subgroups: 37 patients with DIC and 13 patients without DIC. DIC patients showed higher Sequential Organ Failure Assessment (SOFA) scores, the prevalence of multiple organ dysfunction syndrome (MODS) and more increased soluble fibrin and lactate levels. We observed lower levels of vascular endothelial growth factor (VEGF), soluble VEGF receptor 2 (sVEGFR2), angiopoietin 1 (Ang1) and Ang1/Ang2, and higher sVEGFR1 and Ang2 levels in DIC patients, but not significant differences in soluble Tie2 expression during the study period. The levels of VEGF, sVEGFR1, and Ang2 in DIC patients correlated with the SOFA scores. Clear differences were observed in the levels of Ang2 in the DIC patients between survivors and nonsurvivors and between those with and without MODS. The area under receiver operating characteristic curves for predicting death and MODS by Ang2 were 0.710 and 0.784, respectively. The VEGF levels showed a marked correlation with the platelet counts. Soluble fibrin and lactate levels independently predicted increases in the levels of VEGF, sVEGFR1, and Ang2 in DIC patients. In conclusion, VEGF, sVEGFR1, Ang2, and Ang1/Ang2, especially Ang2, may have roles in the development of MODS in sepsis associated with DIC, and VEGF, sVEGFR1, and Ang2 serum levels correlated with the extent of DIC-induced platelet consumption, thrombin generation, and blood lactate levels.

Vitreous Levels of Proteins Implicated in Angiogenesis Are Modulated in Patients with Retinal or Choroidal Neovascularization

Aim: The aim of this study was to investigate the levels of pigment epithelium-derived factor (PEDF), angiopoietin 2, vascular endothelial growth factor (VEGF), and soluble VEGF receptor 1 (sVEGFR-1) in vitreous samples of patients suffering from age-related macular degeneration with choroidal neovascularization or from proliferative diabetic retinopathy (PDR). Methods: Proteins in vitreous samples of 29 patients were quantified via enzyme-linked immunosorbent assays. Results: Vitreous levels of sVEGFR-1 were significantly higher in age-related macular degeneration with choroidal neovascularization (p = 0.005) and in PDR (p = 0.003) versus controls. In analogue comparisons, PEDF was significantly decreased (p < 0.01). PDR was associated with significantly increased angiopoietin 2 and VEGF levels (p = 0.001 for both). Conclusion: The vitreous in retinal or choroidal neovascularization revealed a pro-angiogenic potential indicated by decreased PEDF or increased angiopoietin 2 levels compared to controls. However, higher amounts of sVEGFR-1 were concomitant, pointing to activation of an endogenous anti-angiogenic system in the protein network.

Vascular Endothelial Growth Factor (VEGF) and Soluble VEGF Receptor 1 (sFlt-1) Levels in Early and Mature Human Milk from Mothers of Preterm versus Term Infants

Vascular endothelial growth factor (VEGF) and its receptors regulate angiogenesis (formation of blood vessels). The soluble VEGF receptor 1 (sFlt-1) binds VEGF as a potent antagonist. The objective of this study was to compare VEGF and sFlt-1 levels in milk from mothers of preterm (n = 50) versus term (n = 49) infants in a longitudinal study. Milk samples were collected on days 3 and 28 of lactation. Vascular endothelial growth factor and sFlt-1 were quantified by sandwich-type enzyme-linked immunosorbent assay. Vascular endothelial growth factor and sFlt-1 were found in high concentrations in early milk (lactation day 3) from mothers of preterm and term infants and were lower in mature milk (lactation day 28). On day 3, median VEGF concentration was lower in preterm than in term milk (37.1 vs 53.9 ng/mL, P < .01). Otherwise, VEGF (day 28) and sFlt-1 (days 3 and 28) did not differ in preterm versus term milk. It was shown for the first time that sFlt-1 is present in human milk. Early human milk contains high concentrations of VEGF and sFlt-1, which decrease over the course of lactation.

Efficacy, Safety, and Biomarkers of Single-Agent Bevacizumab Therapy in Patients with Advanced Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly vascularized tumor in which neoangiogenesis contributes to growth and metastasis. We assessed the safety, efficacy, and potential biomarkers of activity of bevacizumab in patients with advanced HCC. In this phase II trial, eligible patients received bevacizumab, 5 mg/kg or 10 mg/kg every 2 weeks. The disease-control rate at 16 weeks (16W-DCR) was the primary endpoint. Circulating endothelial cells (CECs) and plasma cytokines and angiogenic factors (CAFs) were measured at baseline and throughout treatment. The 16W-DCR was 42% (95% confidence interval, 27%-57%). Six of the 43 patients who received bevacizumab achieved a partial response (objective response rate [ORR], 14%). Grade 3-4 asthenia, hemorrhage, and aminotransferase elevation occurred in five (12%), three (7%), and three (7%) patients, respectively. During treatment, placental growth factor markedly increased, whereas vascular endothelial growth factor (VEGF)-A dramatically decreased (p < .0001); soluble VEGF receptor-2 (p < .0001) and CECs (p = .03) transiently increased on day 3. High and increased CEC counts at day 15 were associated with the ORR (p = .04) and the 16W-DCR (p = .02), respectively. Lower interleukin (IL)-8 levels at baseline (p = .01) and throughout treatment (p ≤ .04) were associated with the 16W-DCR. High baseline IL-8 and IL-6 levels predicted shorter progression-free and overall survival times (p ≤ .04). Bevacizumab is active and well tolerated in patients with advanced HCC. The clinical value of CECs, IL-6, and IL-8 warrants further investigation.

Rosiglitazone enhances neovascularization in diabetic rat ischemic hindlimb model

There is increasing evidence that peroxisome proliferator-activated receptors (PPARs) may be involved in the regulation of angiogenesis. In this study, we examined whether rosiglitazone, a PPARγ agonist, can restore angiogenesis in a rat hindlimb ischemia model of diabetes. Male wistar rats were divided into four groups (n=6 each): control, diabetic and control and diabetic rats who received rosiglitazone (8 mg/kg/day). Diabetes was induced by streptozotocin (55 mg/kg; ip). After 21 days, serum concentrations of nitric oxide (NO), vascular endothelial growth factor (VEGF) and soluble VEGF receptor-2 (VEGFR-2) were measured and neovascularization in ischemic legs was evaluated by immunohistochemistry. Capillary density and capillary/fiber ratio in hindlimb ischemia of diabetic animals were significantly lower than the control group (P<0.05). Rosiglitazone significantly restored neovascularization in diabetic animals (P<0.05). rosiglitazone enhances neovascularization in diabetic ischemic skeletal muscle and could be considered for treatment of peripheral artery disease in diabetic subjects.

OS067. VEGF, a novel stimulator of aldosterone production

Adrenal aldosterone production depends upon capillary integrity. Inadequately explained by increased renin secretion, aldosterone is high in pregnancy, a proangiogenic state. In preeclampsia, low aldosterone levels coincide with disturbed endothelial integrity due to disrupted VEGF signaling. We hypothesized that the stimulation of adrenal aldosterone production is VEGF-sensitive. We cultured endothelial cells (EC) in the presence and absence of VEGF. The supernatent was transferred to cultured adrenal cells, either the cell line H295R or isolated primary human adrenal cells from zona glomerulosa. aldosterone synthase mRNA and protein expression, aldosterone synthesis was assessed by adding radioactive labeled precursors or measuring aldosterone in the supernatent by Elisa. Cells were cultured either with angiotensin II (Ang II), VEGF or a combination hereof. Adenovirus-based overexpression of the soluble VEGF receptor type 1 (sFlt-1) was used to simulated conditions of preeclampsia in rats and its effect on the adrenocortical vasculature and circulating aldosterone levels. EC conditioning in the presence of VEGF enhanced aldosterone synthase activity in human adrenocortical cells. VEGF either alone or combined with Ang II increased aldosterone synthase transcription, enzyme availability and aldosterone production in adrenal cells. Neuropilin-1 and VEGF receptor expression differed only for Flt-1 which was present in ECs but not in adrenocortical cells. In contrast to Ang II, VEGF did not upregulate the steroidogenic acute regulatory protein. In line with this observation, Ang II stimulated both aldosterone and cortisol synthesis from progesterone whereas VEGF preferably the former. In rats, overexpression of sFlt-1 which traps VEGF led to adrenocortical capillary rarefaction. Serum aldosterone concentrations inversely correlated with sFlt-1 levels. In conclusion, VEGF stimulates aldosterone production indirectly via ECs and directlyin adrenocortical cells a finding explaining the increased aldosterone/renin ratio in normal pregnancy. It is reasonable to assume that the inappropriately low aldosterone availability in preeclampsia is a consequence of the known disturbed VEGF signaling.

Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60–90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNFα, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNFα levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNFα mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients.These data indicate the involvement of inflammatory (TNFα and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD.

Role of miR-132 in Angiogenesis after Ocular Infection with Herpes Simplex Virus

MicroRNAs (miRNAs) are small regulatory molecules that control diverse biological processes that include angiogenesis. Herpes simplex virus (HSV) causes a chronic immuno-inflammatory response in the eye that may result in corneal neovascularization during blinding immunopathological lesion stromal keratitis (SK). miR-132 is a highly conserved miRNA that is induced in endothelial cells in response to growth factors, such as vascular endothelial growth factor (VEGF). In this study, we show that miR-132 expression was up-regulated (10- to 20-fold) after ocular infection with HSV, an event that involved the production of both VEGF-A and IL-17. Consequently, blockade of VEGF-A activity using soluble VEGF receptor 1 resulted in significantly lower levels of corneal miR-132 after HSV infection. In addition, low levels of corneal miR-132 were detected in IL-17 receptor knockout mice after HSV infection. In vivo silencing of miR-132 by the provision of anti-miR-132 (antagomir-132) nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished SK lesions. The anti-angiogenic effect of antagomir-132 was reflected by a reduction in angiogenic Ras activity in corneal CD31-enriched cells (presumably blood vessel endothelial cells) during SK. To our knowledge, this is one of the first reports of miRNA involvement in an infectious ocular disease. Manipulating miRNA expression holds promise as a therapeutic approach to control an ocular lesion that is an important cause of human blindness.

Exogenous vascular endothelial growth factor supplementation can restore the podocyte barrier‐forming capacity disrupted by sera of preeclamptic women

To determine whether exogenous vascular endothelial growth factor (VEGF) supplementation to sera from women with preeclampsia can restore the podocyte barrier-forming capacity disrupted after stimulation with sera from these women. Enzyme-linked immunosorbent assays (ELISAs) for sFlt-1 (soluble VEGF receptor 1) and VEGF were performed in the sera of preeclamptic and healthy pregnant women. VEGF-enriched preeclamptic serum was used to evaluate the capacity of such sera to restore the diminished podocyte barrier capacity secondary to stimulation with untreated preeclamptic sera. Two groups, preeclampsia (n=15) and healthy (n=15) pregnant women, were established. Median serum levels (pg/mL) of sFlt-1 and VEGF were significantly different between groups: 3987 versus 1432 and 6 versus 15.6 (P<0.001). Altered resistance values of podocytes stimulated with sera from women with preeclampsia were recovered after exogenous VEGF supplementation. The mean resistance value of cultured podocytes, an indicator of the integrity of the cytoskeleton and the capacity of these cells to form layers, is altered in women with preeclampsia. High levels of sFlt-1, an inhibitor of VEGF observed in high concentrations in the serum of these patients, could be the underlying cause of this alteration. We provide evidence that exogenous VEGF supplementation of the sera of these women could recover this indicator.

Effect of everolimus on angiogenic biomarkers in patients with tuberous sclerosis complex (TSC): Results from EXIST-1 and EXIST-2.

10619 Background: The efficacy and safety of everolimus, an oral mTOR inhibitor, was assessed in two randomized, double-blind, placebo-controlled, phase 3 trials: EXIST-1 (NCT00789828) and EXIST-2 (NCT00790400). EXIST-1 examined everolimus for the treatment of subependymal giant cell astrocytoma (SEGA) associated with TSC and EXIST-2 for the treatment of renal angiomyolipoma (AML) associated with either TSC or sporadic lymphangioleiomyomatosis. In each instance, everolimus was superior to placebo for the primary endpoints, SEGA and renal AML response rates. Inhibitors of mTOR have antiangiogenic effects on tumor growth in vitro and in vivo. Methods: Patients were randomized to receive everolimus (n=78) starting at 4.5 mg/m2/day (target trough, 5-15 ng/mL) or placebo (n=39) in EXIST-1 and 10 mg/day everolimus (n=79) or placebo (n=39) in EXIST-2. Plasma samples were taken at baseline and pre-dosing on day 1 of weeks 4, 12, 24, 36, and 48 of treatment. Angiogenic markers of interest were vascular endothelial growth factor (VEGF)-A and -D, placental growth factor (PlGF), soluble VEGF receptor-1 (sVEGFR1), soluble VEGF receptor 2 (sVEGFR2), c-Kit, and collagen type IV. Results: Compared with placebo, a sustained ~30% and ~60% increase in VEGF-A was observed in the everolimus arm of EXIST-1 and EXIST-2, respectively. A concomitant decrease in collagen type IV (~25% EXIST-1; ~45% EXIST-2) and sVEGFR2 (~25% both trials) was also observed in the everolimus arm. A sustained decrease (~60%) in VEGF-D was observed in the everolimus arm of EXIST-2, but not EXIST-1. In both studies, no change was observed in PlGF, sVEGFR1, or c-Kit plasma concentrations in the everolimus arm or any biomarkers evaluated in the placebo arm. Baseline sVEGFR2 and VEGF-D were ~40% and ~4-fold higher, respectively, while VEGF-A was ~50% lower in EXIST-2 compared with EXIST-1. A similar baseline plasma concentration for the other biomarkers was noted in both studies. Conclusions: Patients presenting with SEGA or renal AML associated with TSC had a reduction in plasma concentrations of sVEGFR2, collagen type IV, and VEGF-D (AML only) and an increase in VEGF-A. Everolimus may have antiangiogenic properties in TSC patients.

Randomized phase II study of docetaxel with or without ramucirumab (IMC-1121B) or icrucumab (IMC-18F1) in patients with urothelial transitional cell carcinoma (TCC) following progression on first-line platinum-based therapy.

TPS4675^ Background: The vascular endothelial growth factor (VEGF) pathway may play an important role in the pathogenesis of bladder cancer. Two antibodies (ramucirumab or icrucumab) in combination with docetaxel are being tested in this clinical study. Ramucirumab is a fully human IgG1 monoclonal antibody (MAb) that specifically binds with high affinity to VEGF receptor-2 (VEGFR-2), thereby blocking the interaction of VEGF ligands. Ramucirumab inhibits VEGF-mediated proliferation of human endothelial cells and migration of human leukemia cells. Icrucumab is a fully human IgG1 MAb that specifically binds with high affinity to VEGFR-1 and blocks the binding of VEGF-A, VEGF-B, and placental growth factor (PlGF) to the receptor, thereby inhibiting subsequent signaling. Blockage of VEGFR-1 and VEGFR-2 by antibodies demonstrates antiangiogenic and antitumor activity and prevents dissemination and growth of lung metastases in several tumor xenograft models. Methods: This study includes patients (pts) with TCC with progressive disease after prior platinum-based therapy. Pts are randomized equally to 1 of 3 open-label treatments given on a 21-day cycle: docetaxel (75 mg/m2) on Day 1 (Arm A); docetaxel on Day 1 and ramucirumab (10 mg/kg) on Day 1 (Arm B); or docetaxel on Day 1 and icrucumab (12 mg/kg) on Days 1 and 8 (Arm C). Randomization is stratified by the absence/presence of visceral metastases and receipt of prior antiangiogenic therapy. The primary endpoint is progression-free survival (PFS). Secondary outcome measures include response rate and duration of response, overall survival, and pharmacodynamic markers, including circulating levels of PlGF, VEGF-A, VEGF-B, soluble VEGFR-1, and soluble VEGFR-2. Exploratory analyses of VEGF, VEGFR-1, and VEGFR-2 genetic polymorphisms will be performed. As of 12 January 2012, approximately 20% of 138 planned pts were randomized in the US and Canada. The sample size will allow differentiation of an expected increase in median PFS from 3.0 months to 4.5 months with a one-sided alpha of 0.1 and a power of 71%. ClinicalTrials.gov identifier: NCT01282463.

Abstract 1938: Angiotensin-(1-7), a peptide hormone with therapeutic potential for the treatment of glioblastomas

Abstract Glioblastoma multiforme (GBM), the most common primary malignant brain tumor in adults, is characterized by aggressive proliferation throughout the brain parenchyma and enhanced angiogenesis. Despite multimodality treatment, GBM therapy is only palliative with a median patient survival of less than 10 to 12 months after diagnosis, indicating a need for novel agents with therapeutic potential. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, seven amino acid peptide hormone with anti-proliferative and anti-angiogenic properties which provides targeted therapy by activating mas, a unique G protein-coupled receptor. In this study, Ang-(1-7) effectively reduced the growth of human GBM in an orthotopic mouse model. Athymic mice with brain tumors resulting from injection of human G48a GBM cells, isolated from a grade 4 astrocytoma, were treated for 28 days with 24 μg/kg/h Ang-(1-7), delivered by osmotic mini-pump. The G48 GBM cells formed an aggressive and invasive phenotype, with outgrowths extending into the brain parenchyma. Ang-(1-7) administration markedly reduced the average tumor burden (a 75% reduction in tumor mass) as compared to untreated control mice (9.4 ± 1.6 x1010 vs 2.4 ± 1.3 x1010 p/sec/cm2/sr, p &amp;lt; 0.022). Histological analysis of brain tissue sections from untreated mice showed a significantly higher percent of Ki67 positive immunoreactivity in tumors as compared to tumor tissue from Ang-(1-7)-treated animals, suggesting that the heptapeptide hormone reduces cell proliferation. In support, proliferation of G48a cultured cells which express the mas receptor was reduced approximately 50% following incubation with 100 nM Ang-(1-7) for five days. Ang-(1-7) treatment also resulted in a marked decrease in MAP kinase ERK1/ERK2 phosphorylation with a concomitant 2-fold increase in the MAP kinase phosphatase DUSP1, suggesting that the heptapeptide hormone negatively regulates GBM cell growth in part by attenuating ERK1/ERK2 activation. Intratumoral vessel density in GBMs from mice administered Ang-(1-7) was attenuated more than 50% as compared to tumors from control animals. VEGF mRNA and protein were decreased over time with a maximal reduction at 8 h in G48a cells following treatment with 100 nM Ang-(1-7). Conversely, the soluble VEGF receptor sFlt-1 was significantly increased in cells incubated with the heptapeptide hormone. sFlt-1, produced by alternative mRNA splicing of Flt-1, is secreted into the circulation and serves as an endogenous trap that sequesters VEGF family ligands, making the angiogenic factors unavailable to the cell-bound VEGF receptors and preventing the downstream activation of pro-angiogenic signaling pathways. Taken together, these studies suggest that Ang-(1-7) inhibits the growth of aggressive, invasive GMBs by pleiotrophic mechanisms and may serve as a first-in-class, targeted, anti-proliferative, anti-angiogenic chemotherapeutic agent for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1938. doi:1538-7445.AM2012-1938

Effects of prior endurance exercise on postprandial lipemia‐induced changes in circulating angiogenic cytokines in young men

We examined the effect of prior endurance exercise on plasma angiogenic cytokine responses to a high fat meal as these may contribute to pathologic angiogenesis in atherosclerosis. Blood samples were obtained from 10 healthy men at baseline and 4 h postprandial with or without ~50 min of endurance exercise at 70% of VO2max the day before. Plasma levels of VEGF, basic fibroblast growth factor (bFGF), placental growth factor (PlGF), and soluble VEGF receptor (sFlt‐1) were measured by multiplex ELISA. Postprandial lipemia (PPL) significantly increased VEGF (~23%, P&lt;0.05) in each group (with and without prior exercise) as well as bFGF with (45%, P&lt;0.05) and without prior exercise (56%, P&lt;0.05). PlGF level decreased by 11% (P&lt;0.05) in the control trial but this effect of PPL was absent with prior exercise. sFlt‐1 level decreased (12%, P&lt;0.05) in the control trial but this difference was reduced to only a 4% decrease with prior exercise (P&gt;0.05). These findings indicate that PPL increases plasma VEGF and bFGF regardless of prior endurance exercise; however, PPL‐induced decreases in PlGF and sFlt‐1 were present only without prior endurance exercise. We speculate that prior exercise may reduce the pro‐atherogenic effects of PPL by attenuating the PPL‐induced changes in PlGF and sFLT‐1 to potentially inhibit pathological angiogenesis.Supported by NIH AGT3200068 and the American College of Sports Medicine Foundation.

The Role of Vascular Endothelial Growth Factor in Mechanically Stimulated Muscle Derived Stem Cell Repair

We have previously shown that muscle‐derived stem cells (MDSCs) are significantly better than myoblasts at maintaining cardiac function after infarction, which is likely due to higher survival and vascular endothelial growth factor (VEGF) secretion. Mechanical stimulation increases VEGF secretion, and after mechanically preconditioning MDSCs and injecting them into an acute model of cardiac infarction function is improved. Therefore, we hypothesized that the benefits seen from mechanical stimulation is due to an increase in VEGF, and if we block VEGF we will not see an improvement in repair. To test this we have mechanically stimulated MDSCs transduced with the soluble VEGF receptor antagonist sFlt1 and tested their regenerative potential in the skeletal muscle of a mouse model of Duchene Muscular Dystrophy. We have found that mechanically stimulating MDSCs resulted in an increase in angiogenesis in the engraftment area. The MDSCs transduced with sFlt1 resulted in less angiogenesis, and the mechanically stimulated MDSCs transduced with sFlt1 showed no difference in angiogenesis from the un‐stimulated sFlt1 group. There also appears to be little difference between fibrosis in the sFlt1 stimulated and un‐stimulated groups. Therefore, VEGF appears to be a critical factor in angiogenesis following cell transplantation in mechanically stimulated MDSCs.Grant Funding Source : DOD

Modeling the inhibition of breast cancer growth by GM-CSF

M-CSF is overexpressed in breast cancer and is known to stimulate macrophages to produce VEGF resulting in angiogenesis. It has recently been shown that the growth factor GM-CSF injected into murine breast tumors slowed tumor growth by secreting soluble VEGF receptor-1 (sVEGFR-1) that binds and inactivates VEGF. This study presents a mathematical model that includes all the components above, as well as MCP-1, tumor cells, and oxygen. The model simulations are representative of the in vivo data through predictions of tumor growth using different protocol strategies for GM-CSF for the purpose of predicting higher degrees of treatment success. For example, our model predicts that once a week dosing of GM-CSF would be less effective than daily, twice a week, or three times a week treatment because of the presence of essential factors required for the anti-tumor effect of GM-CSF.

Association of mast cell-derived VEGF and proteases in Dengue shock syndrome.

BackgroundRecent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls.Methodology/Principal FindingsThe study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9.ConclusionsAs mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-011-9249-6) contains supplementary material, which is available to authorized users.

VEGF-D Promotes Tumor Metastasis by Regulating Prostaglandins Produced by the Collecting Lymphatic Endothelium

Lymphatic metastasis is facilitated by lymphangiogenic growth factors VEGF-C and VEGF-D that are secreted by some primary tumors. We identified regulation of PGDH, the key enzyme in prostaglandin catabolism, in endothelial cells of collecting lymphatics, as a key molecular change during VEGF-D-driven tumor spread. The VEGF-D-dependent regulation of the prostaglandin pathway was supported by the finding that collecting lymphatic vessel dilation and subsequent metastasis were affected by nonsteroidal anti-inflammatory drugs (NSAIDs), known inhibitors of prostaglandin synthesis. Our data suggest a control point for cancer metastasis within the collecting lymphatic endothelium, which links VEGF-D/VEGFR-2/VEGFR-3 and the prostaglandin pathways. Collecting lymphatics therefore play an active and important role in metastasis and may provide a therapeutic target to restrict tumor spread.

Studies on Axenfeld–Rieger syndrome patients and mice reveal Foxc1's role in corneal neovascularization

Normal vision critically depends on the transparency of the cornea overlying the lens. This tissue is made up of precisely oriented layers of fibroblasts perfectly interwoven with the collagen and other ECM components that they produce. Importantly, in the healthy eye, there are no blood vessels or lymphatic vessels in the stroma, making it what is known as an “angiogenically privileged” site. However, in a number of human ocular disorders, and in response to injuries like chemical burns, blood vessels do grow into the cornea from around the junction between the iris and the sclera (the limbus). This pathological angiogenesis can severely disrupt clarity of vision and necessitate corneal transplants. Consequently, there is intense interest in understanding the processes that normally restrict blood vessel growth into the cornea. In PNAS, Seo et al. (1) provide compelling evidence from human and mouse studies that implicate one member of the large Forkhead box (Fox) transcription factor family [Forkhead box transcription factor c1 (Foxc1)] in the regulation of corneal avascularity. Specifically, they suggest that Foxc1 regulates the normal delicate balance between factors in the cornea that can promote angiogenesis (e.g., VEGF and VEGF receptor) and those that inhibit growth of blood vessels and lymphatics (e.g., soluble VEGF receptor).