Inhibition Of Vascular Endothelial Growth Factor Signaling Research Articles

VEGF affects mitochondrial ROS generation in glioma cells and acts as a radioresistance factor.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis. Anticancer therapy by inhibiting VEGF signaling is well established. However, the role of VEGF in cell-cell communication during the response to ionizing radiation is not well understood. Here, we examined the role of VEGF on radiosensitivity of cells. The addition of recombinant VEGF (rVEGF) on cultured rat C6 glioma cells showed a radioprotective effects on X-ray irradiation and reduced oxidative stress. These effects were also observed by endogenous VEGF in supernatant of C6 glioma cells. Reduction of oxidative stress by VEGF is suggested to underlie the radioprotective effects. The mechanism of VEGF-induced reduction of oxidative stress was indicated by a decreased oxygen consumption rate (OCR) in mitochondria. However, the number of DNA double-strand breaks (DSB) immediately after irradiation was not reduced by the treatment with VEGF. These results suggest that VEGF plays a role in cell survival after irradiation by controlling the oxidative condition through mitochondrial function that is independent of the efficiency of DSB induction.

COVID-19 instigates adipose browning and atrophy through VEGF in small mammals

Patients with COVID-19 frequently manifest adipose atrophy, weight loss and cachexia, which significantly contribute to poor quality of life and mortality1,2. Browning of white adipose tissue and activation of brown adipose tissue are effective processes for energy expenditure3–7; however, mechanistic and functional links between SARS-CoV-2 infection and adipose thermogenesis have not been studied. In this study, we provide experimental evidence that SARS-CoV-2 infection augments adipose browning and non-shivering thermogenesis (NST), which contributes to adipose atrophy and body weight loss. In mouse and hamster models, SARS-CoV-2 infection activates brown adipose tissue and instigates a browning or beige phenotype of white adipose tissues, including augmented NST. This browning phenotype was also observed in post-mortem adipose tissue of four patients who died of COVID-19. Mechanistically, high levels of vascular endothelial growth factor (VEGF) in the adipose tissue induces adipose browning through vasculature–adipocyte interaction. Inhibition of VEGF blocks COVID-19-induced adipose tissue browning and NST and partially prevents infection-induced body weight loss. Our data suggest that the browning of adipose tissues induced by COVID-19 can contribute to adipose tissue atrophy and weight loss observed during infection. Inhibition of VEGF signaling may represent an effective approach for preventing and treating COVID-19-associated weight loss.

Inhibitor of differentiation 1 in U87MG glioblastoma cells promotes HUVEC sprouting through endothelin-1.

Anti-angiogenesis therapy, a promising remedy against tumor progression, is now widely used to treat numerous types of cancer. Since vascular endothelial growth factor (VEGF) is the most vital factor in angiogenesis, most anti-angiogenesis drugs target the VEGF-related pathway. However, in glioblastoma (GBM), the therapeutic strategy involving the inhibition of VEGF signaling is ineffective. The present study demonstrated that the potential angiogenic function of endothelin-1 (EDN1) was upregulated by inhibitor of differentiation 1 (ID1) independent of VEGF during tumor angiogenesis. Anatomic structure transcriptomes of patients with GBM revealed that the expression levels of ID1 and EDN1 were specifically upregulated in the vascular-related region. The aortic ring assay and endothelial sprouting assay demonstrated that EDN1 more potently promoted endothelial sprouting ability than VEGF. The activity of EDN1 was induced by endothelin receptor, which seemed to mediate regulation via positive feedback. Finally, in patients with GBM who did not respond to bevacizumab, a VEGF antagonist, EDN1 expression was higher than that in bevacizumab responders. Collectively, the present study demonstrated that EDN1 is a potent angiogenic factor inducing endothelial sprouting and may be a novel target for inhibiting glioma angiogenesis.

Loss of apelin blocks the emergence of sprouting angiogenesis in experimental tumors.

Angiogenesis inhibitor drugs targeting vascular endothelial growth factor (VEGF) signaling to the endothelial cell (EC) are used to treat various cancer types. However, primary or secondary resistance to therapy is common. Clinical and pre-clinical studies suggest that alternative pro-angiogenic factors are upregulated after VEGF pathway inhibition. Therefore, identification of alternative pro-angiogenic pathway(s) is critical for the development of more effective anti-angiogenic therapy. Here we study the role of apelin as a pro-angiogenic G-protein-coupled receptor ligand in tumor growth and angiogenesis. We found that loss of apelin in mice delayed the primary tumor growth of Lewis lung carcinoma 1 and B16F10 melanoma when combined with the VEGF receptor tyrosine kinase inhibitor, sunitinib. Targeting apelin in combination with sunitinib markedly reduced the tumor vessel density, and decreased microvessel remodeling. Apelin loss reduced angiogenic sprouting and tip cell marker gene expression in comparison to the sunitinib-alone-treated mice. Single-cell RNA sequencing of tumor EC demonstrated that the loss of apelin prevented EC tip cell differentiation. Thus, apelin is a potent pro-angiogenic cue that supports initiation of tumor neovascularization. Together, our data suggest that targeting apelin may be useful as adjuvant therapy in combination with VEGF signaling inhibition to inhibit the growth of advanced tumors.

Anti-VEGF Effect of Bioactive Indolic Compounds and Hydroxytyrosol Metabolites.

Angiogenesis is a key process involved in both cancer and cardiovascular diseases, the vascular endothelial growth factor (VEGF) and its VEGF receptor-2 (VEGFR-2) being the main triggers. The aim of this study was to determine the molecular mechanism underlying the potent inhibition of VEGF signaling by hydroxytyrosol (HT) metabolites and indolic compounds and establish a relation between their structure and bioactivity. Experiments involved the evaluation of their potential to inhibit VEGF on human umbilical vein endothelial cells (HUVECs) by ELISA assay and their subsequent effect on the downstream signaling pathway (PLCγ1, Akt, and endothelial nitric oxide synthetase (eNOS)) by Western blot. Respectively, 3,4-dihydroxyphenylacetaldehyde (DOPAL) (100 µM) and indole pyruvic acid (IPy) (1 mM) were capable of inhibiting VEGFR-2 activation with an IC50 value of 119 µM and 1.037 mM. The anti-angiogenic effect of DOPAL and IPy is mediated via PLCγ1. Additionally, DOPAL significantly increases eNOS phosphorylation, while IPy maintained it. These data provide for the first time evidence of the anti-angiogenic effect of DOPAL and IPy for future use as potential bioactive food ingredients.

VEGF Maintains Maternal Vascular Space Homeostasis in the Mouse Placenta through Modulation of Trophoblast Giant Cell Functions.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that acts primarily on endothelial cells, but numerous studies suggest that VEGF also acts on non-endothelial cells, including trophoblast cells. Inhibition of VEGF signaling by excess production of the endogenous soluble VEGF receptor sFlt1 in trophoblast cells has been implicated in several pregnancy complications. Our previous studies and other reports have shown that VEGF directly regulates placental vascular development and functions and that excess VEGF production adversely affects placental vascular development. Trophoblast giant cells (TGCs) line the maternal side of the placental vasculature in mice and function like endothelial cells. In this study, we specifically examined the effect of excess VEGF signaling on TGC development associated with defective placental vascular development using two mouse models an endometrial VEGF overexpression model and a placenta-specific sFlt1 knockdown model. Placentas of endometrial VEGF-overexpressing dams at embryonic days (E) 11.5 and 14.5 showed dramatic enlargement of the venous maternal spaces in junctional zones. The size and number of the parietal TGCs that line these venous spaces in the placenta were also significantly increased. Although junctional zone venous blood spaces from control and VEGF-overexpressing dams were not markedly different in size at E17.5, the number and size of P-TGCs were both significantly increased in the placentas from VEGF-overexpressing dams. In sFlt1 knockdown placentas, however, there was a significant increase in the size of the sinusoidal TGC-lined, alkaline phosphatase-positive maternal blood spaces in the labyrinth. These results suggest that VEGF signaling plays an important role in maintaining the homeostasis of the maternal vascular space in the mouse placenta through modulation of TGC development and differentiation, similar to the effect of VEGF on endothelial cells in other vascular beds.

Vascular Sema3E-Plexin-D1 Signaling Reactivation Promotes Post-stroke Recovery through VEGF Downregulation in Mice

Post-stroke vascular remodeling, including angiogenesis, facilitates functional recovery. Proper vascular repair is important for efficient post-stroke recovery; however, the underlying mechanisms coordinating the diverse signaling pathways involved in vascular remodeling remain largely unknown. Recently, axon guidance molecules were revealed as key players in injured vessel remodeling. One such molecule, Semaphorin 3E (Sema3E), and its receptor, Plexin-D1, control vascular development by regulating vascular endothelial growth factor (VEGF) signaling. In this study, using a mouse model of transient brain infarction, we aimed to investigate whether Sema3E-Plexin-D1 signaling was involved in cerebrovascular remodeling after ischemic injury. We found that ischemic damage rapidly induced Sema3e expression in the neurons of peri-infarct regions, followed by Plexin-D1 upregulation in remodeling vessels. Interestingly, Plexin-D1 reemergence was concurrent with brain vessels entering an active angiogenic process. In line with this, Plxnd1 ablation worsened neurological deficits, infarct volume, neuronal survival rate, and blood flow recovery. Furthermore, reduced and abnormal vascular morphogenesis was caused by aberrantly increased VEGF signaling. In Plxnd1 knockout mice, we observed significant extravasation of intravenously administered tracers in the brain parenchyma, junctional protein downregulation, and mislocalization in regenerating vessels. This suggested that the absence of Sema3E-Plexin-D1 signaling is associated with blood–brain barrier (BBB) impairment. Finally, the abnormal behavioral performance, aberrant vascular phenotype, and BBB breakdown defects in Plxnd1 knockout mice were restored following the inhibition of VEGF signaling during vascular remodeling. These findings demonstrate that Sema3E-Plexin-D1 signaling can promote functional recovery by downregulating VEGF signaling in the injured adult brain.

Blocking VEGF signaling augments interleukin-8 secretion via MEK/ERK/1/2 axis in human retinal pigment epithelial cells.

To identify proangiogenic factors engaged in neovascular age-related macular degeneration (AMD) except vascular endothelial growth factor (VEGF) from human retinal pigment epithelial (hRPE) cells and investigate the underlying mechanisms. VEGF receptor 2 (VEGFR2) in ARPE-19 cells was depleted by siRNA transfection or overexpressed through adenovirus infection. The mRNA and the protein levels of interleukin-8 (IL-8) in ARPE-19 cells were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. The protein levels of AKT, p-AKT, MEK, p-MEK, ERK1/2, p-ERK1/2, JNK, p-JNK, p38 and p-p38 were detected by Western blotting. A selective chemical inhibitor, LY3214996, was employed to inhibit phosphorylation of ERK1/2. Cell viability was determined by MTT assay. Knockdown of VEGFR2 in ARPE-19 cells robustly augmented IL-8 production at both the mRNA and the protein levels. Silencing VEGFR2 substantially enhanced phosphorylation of MEK and ERK1/2 while exerted no effects on phosphorylation of AKT, JNK and p38. Inhibiting ERK1/2 phosphorylation by LY3214996 reversed changes in VEGFR2 knockdown-induced IL-8 upregulation at the mRNA and the protein levels with no effects on cell viability. VEGFR2 overexpression significantly reduced IL-8 generation at the mRNA and the protein levels. Blockade of VEGF signaling augments IL-8 secretion via MEK/ERK1/2 axis and overactivation of VEGF pathway decreases IL-8 production in hRPE cells. Upregulated IL-8 expression after VEGF signaling inhibition in hRPE cells may be responsible for being incompletely responsive to anti-VEGF remedy in neovascular AMD, and IL-8 may serve as an alternative therapeutic target for neovascular AMD.

AB0089 THE ANALYSIS FOR THE INHIBITION OF ANGIOGENESIS BY JAK INHIBITOR

Background:Many blood vessels are generated in the hyperplastic synovial tissue of patients with rheumatoid arthritis (RA), and lead to chronic tissue inflammation and joint destruction [1]. Janus kinase (JAK) family consisting of JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) are chain receptors which phosphorylate signal transducers and activators of transcription (STAT) and mediate inflammatory diseases including RA [2]. Nowadays, several JAK inhibitors such as Tofacitinib (TOF), Baricitinib (BAR) and Peficitinib (PEF) have been developed and demonstrated to have the inhibitory effects on inflammatory arthritis [3-5]. However, there were few reports concerning their effects on angiogenesis in vitro.Objectives:The purpose of the present study is to investigate the influence of JAK inhibitors on angiogenesis of human umbilical vein endothelial cell (HUVEC) activated by vascular endothelial growth factor (VEGF).Methods:The cell line of HUVECs were used for this study. The activity of proliferation and tube formation were analyzed by counting assay and tube formation assay, respectively.In counting assay, HUVECs (5 × 104cells/ml) were seeded onto 96-well cell culture plate with 20 ng/ml VEGF including various doses (0.1µM, 1µM, 5µM) of TOF, BAR or PEF. After 48 hours incubation at 37°C in a humidified atmosphere containing 5% CO2, cell proliferation of each groups was assessed using cell counting kit. In tube formation assay, HUVECs (5 × 104cells/ml) were treated with 20ng/ml VEGF including various dose (0.1µM, 1µM, 5µM) of TOF, BAR or PEF for 00 hours, then seeded onto 48-well plate applied with Matrigel. After 24 hours incubation on Matrigel, the capillary-like tube formation of each well was photographed using phase contrast microscopy. Tube formation were quantitated by measurement of the length of branch.Results:HUVECs were activated in proliferation and tube formation by VEGF treatment. And, the proliferation and tube formation of HUVECs activated by VEGF were suppressed by All of TOF, BAR and PEF. In particular, TOF and PEF could suppress them highly.Conclusion:This study showed the inhibitory effect of JAK inhibitors on proliferation and tube formation of HUVECs activated by VEGF. In particular, the angiogenesis of HUVECs activated by VEGF was highly suppressed by TOF and PEF. VEGF is reported to regulate the angiogenesis through multi JAK-STAT signaling pathways [6]. The inhibitory effects on angiogenesis of TOF, BAR and PEF might depend on the differences in their affinity for JAKs. VEGF has been shown to a have a central involvement in the angiogenic process in RA [7]. JAK inhibitors might suppress the angiogenesis in RA synovial tissues by inhibiting VEGF signaling.

PKA Inhibition Mimics Dopamine 2 receptor‐driven Inhibition of VEGF‐induced Proliferation of Human Retinal Microvascular Endothelial Cells

Diabetic retinopathy (DR), a leading cause of blindness, results in part from chronically increased expression of vascular endothelial growth factor (VEGF) in the eye in the diabetic state. VEGF increases proliferation of endothelial cells, a critical step in abnormal angiogenesis that contributes to the pathophysiology of DR. We have previously found that activation of dopamine receptors, specifically using a D2‐type receptor (D2R) agonist, inhibits VEGF signaling on human retinal microvascular endothelial cells (HRMECs) and that HRMECs express the dopamine receptor genes DRD1, 2, 3, and 5, but not DRD4. D2R activation normally inhibits the activity of PKA via activation of Gi‐alpha subunits. Here, we determined whether inhibition of PKA, the canonical downstream target of D2R‐agonist activation, suppresses VEGF‐induced proliferation of HRMECs.HRMECs, obtained from CSC, Inc. (Kirkland, WA), were plated in CSC 10% serum complete media in 96‐well plates. After overnight starvation in 0.5% serum media, wells were pretreated with either the D2R agonist quinpirole (Q: 50 nM), the PKA inhibitor 14–22 Amide (PKI: 1 μM), or only 0.5% serum media (C). This and all consecutive steps were performed in 0.5% serum media. After 15 min, VEGF (V: 25 ng/mL) was added to some wells pre‐treated with Q or PKI, resulting in the following groups: C, V, V+PKI, V+Q, Q, and PKI. Proliferation as a percent of control was determined using a WST‐1 assay after 48 hours. As anticipated, VEGF induced proliferation of HRMECs compared to control (1.21 ± 0.13 vs 1, normalized values). This proliferation was blocked by either Q or PKI treatment (0.96 ± 0.12 and 0.94 ± 0.12 vs 1.21 ± 0.13 respectively, p<0.05). Treatment with Q or PKI without VEGF had no effect on proliferation compared to control (1.10 ± 0.13 and 0.97 ± 0.18 respectively).In conclusion, application of the D2R agonist quinpirole or of the PKA inhibitor 14–22 Amide, suppressed VEGF‐induced HRMEC proliferation to control levels. These results suggest that targeting not only specific dopamine receptor activation but also cAMP and PKA signaling systems in HRMECs may be a useful adjunct treatment for blocking VEGF‐induced HRMEC activation in DR.Support or Funding InformationNIH R01 EY023290 and UIWRSO Faculty Development Grant

Vascular Endothelial Growth Factor-121 Administration Mitigates Halogen Inhalation-Induced Pulmonary Injury and Fetal Growth Restriction in Pregnant Mice.

BackgroundCirculating levels of sFLT‐1 (soluble fms‐like tyrosine kinase 1), the extracellular domain of vascular endothelial growth factor (VEGF) receptor 1, and its ratio to levels of placental growth factor are markers of the occurrence and severity of preeclampsia.Methods and ResultsC57BL/6 pregnant mice on embryonic day 14.5 (E14.5), male, and non‐pregnant female mice were exposed to air or to Br2 at 600 ppm for 30 minutes and were treated with vehicle or with VEGF‐121 (100 μg/kg, subcutaneously) daily, starting 48 hours post‐exposure. Plasma, bronchoalveolar lavage fluid, lungs, fetuses, and placentas were collected 120 hours post‐exposure. In Br2‐exposed pregnant mice, there was a time‐dependent and significant increase in plasma levels of sFLT‐1 which correlated with increases in mouse lung wet/dry weights and bronchoalveolar lavage fluid protein content. Supplementation of exogenous VEGF‐121 improved survival and weight gain, reduced lung wet/dry weights, decreased bronchoalveolar lavage fluid protein levels, enhanced placental development, and improved fetal growth in pregnant mice exposed to Br2. Exogenous VEGF‐121 administration had no effect in non‐pregnant mice.ConclusionsThese results implicate inhibition of VEGF signaling driven by sFLT‐1 overexpression as a mechanism of pregnancy‐specific injury leading to lung edema, maternal mortality, and fetal growth restriction after bromine gas exposure.

Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice.

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.

High-Altitude Cognitive Impairment Is Prevented by Enriched Environment Including Exercise via VEGF Signaling.

Exposure to hypobaric hypoxia at high altitude (above 2500 m asl) causes cognitive impairment, mostly attributed to changes in brain perfusion and consequently neuronal death. Enriched environment and voluntary exercise has been shown to improve cognitive function, to enhance brain microvasculature and neurogenesis, and to be neuroprotective. Here we show that high-altitude exposure (3540 m asl) of Long Evans rats during early adulthood (P48–P59) increases brain microvasculature and neurogenesis but impairs spatial and visual memory along with an increase in neuronal apoptosis. We tested whether enriched environment including a running wheel for voluntary exercise (EE) can prevent cognitive impairment at high-altitude and whether apoptosis is prevented. We found that EE retained spatial and visual memory at high altitude, and prevented neuronal apoptosis. Further, we tested whether vascular endothelial growth factor (VEGF) signaling is required for the EE-mediated recovery of spatial and visual memory and the reduction in apoptosis. Pharmacological inhibition of VEGF signaling by oral application of a tyrosine kinase inhibitor (Vandetanib) prevented the recovery of spatial and visual memory in animals housed in EE, along with an increase in apoptosis and a reduction in neurogenesis. Surprisingly, inhibition of VEGF signaling also caused impairment in spatial memory in EE-housed animals reared at low altitude, affecting mainly dentate gyrus microvasculature but not neurogenesis. We conclude that EE-mediated VEGF signaling is neuroprotective and essential for the maintenance of cognition and neurogenesis during high-altitude exposure, and for the maintenance of spatial memory at low altitude. Finally, our data also underlines the potential risk of cognitive impairment and disturbed high altitude adaption from the use of VEGF-signaling inhibitors for therapeutic purposes.

Role of Neuronal VEGF Signaling in the Prefrontal Cortex in the Rapid Antidepressant Effects of Ketamine.

The N-methyl-d-aspartate receptor antagonist ketamine produces rapid and sustained antidepressant actions even in patients with treatment-resistant depression. Vascular endothelial growth factor (VEGF) has been implicated in the effects of conventional monoamine-based antidepressants, but the role of VEGF in the rapid antidepressant actions of ketamine remains unclear. The authors examined whether neuronal VEGF signaling in the medial prefrontal cortex (mPFC) mediates the rapid antidepressant actions of ketamine. The authors used a combination of approaches, including conditional, neuron-specific knockout of VEGF or its receptor, Flk-1; antibody neutralization; viral-mediated knockdown of Flk-1; and pharmacological inhibitors. Further in vitro and in vivo experiments were performed to examine whether neuronal VEGF signaling was required for the neurotrophic and synaptogenic actions of ketamine that underlie its behavioral actions. The behavioral actions of systemic ketamine are blocked by forebrain excitatory neuron-specific deletion of either VEGF or Flk-1 or by intra-mPFC infusion of a VEGF neutralizing antibody. Moreover, intra-mPFC infusions of VEGF are sufficient to produce rapid ketamine-like behavioral actions, and these effects are blocked by neuron-specific Flk-1 deletion. The results also show that local knockdown of Flk-1 in mPFC excitatory neurons in adulthood blocks the behavioral effects of systemic ketamine. Moreover, inhibition of neuronal VEGF signaling blocks the neurotrophic and synaptogenic effects of ketamine. Together, these findings indicate that neuronal VEGF-Flk-1 signaling in the mPFC plays an essential role in the antidepressant actions of ketamine.

Triple-tyrosine kinase inhibition attenuates pulmonary arterial hypertension and neointimal formation

The present study examined the effects of simultaneous inhibition of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) receptor signaling with BIBF1000, a novel triple tyrosine kinase inhibitor on preventing and reversing the progression of severe pulmonary arterial hypertension (PAH) in an experimental model in rats. Left pneumonectomized male Wistar rats were injected with monocrotaline to induce PAH. Treatment with BIBF1000 from day 1 to day 21 after monocrotaline injection attenuated PAH development, as evidenced by lower values for pulmonary artery pressure (mPAP), right ventricular pressure (RVSP), pulmonary arterial neointimal formation, and the ratio of right ventricular weight to left ventricular and septum weight [RV/(LV+S)] on day 21 compared to control rats. Treatment with BIBF1000 from day 21 to day 42 after monocrotaline injection reversed established PAH as shown by normalized values for mPAP and RVSP, RV/(LV+S) ratio, pulmonary arterial occlusion scores, levels of heart and lung fibrosis, as well as improved survival. Treatment with BIBF1000 reduced inflammatory cell recruitment in bronchoalveolar lavage and lung tissues, reduced CD-68 positive macrophages and expression of proliferating cell nuclear antigen in the perivascular areas, and reduced TNF-α and growth factor productions, and inhibited the phosphorylation of AKT and GSK3β in lungs. In addition, BIBF1000 inhibited pulmonary artery smooth muscle cells migration and proliferation from rat pulmonary artery explant cultures. Simultaneous inhibition of VEGF, PDGF, and FGF receptor signaling by BIBF1000 prevents and reverses the progression of severe pulmonary arterial hypertension and vascular remodeling in this experimental model.

Gelatinolytic activity of autocrine matrix metalloproteinase-9 leads to endothelial de-arrangement in Moyamoya disease

Moyamoya disease (MMD) is a rare steno-occlusive cerebrovascular disorder. Mechanisms driving the formation of aberrant MMD vessels remain elusive. We collected serum and vessel specimens from MMD and atherosclerotic cerebrovascular disease (ACVD) patients serving as controls due to the same hypoxic stimulus but substantial differences in terms of vascular features. Based on patient material and an in vitro model mimicking ACVD and MMD conditions, matrix metalloproteinase-9 (MMP-9) and vascular-endothelial growth factor (VEGF) were tested for their potential involvement in cerebrovascular disintegration. While serum concentration of both molecules did not significantly differ in both patient groups, excessive collagenase activity and lowered collagen IV protein amount in MMD vessels pointed to a focal MMP-9 activity at the affected vessel sites. We observed overexpressed and autocrinely secreted MMP-9 and VEGF along with disturbances of EC–matrix interactions in MMD but not ACVD serum-treated cEND cells. These seemingly brain-specific effects were partially attenuated by VEGF signaling inhibition suggesting its role in the MMD etiology. In conclusion, our findings support the understanding of the high incidence of hemorrhagic and ischemic events in MMD and provide the basis for novel therapeutic strategies stopping or slowing the development of fragile cerebrovasculature or micro-bleeds characterizing the disease.

VEGFR (Vascular Endothelial Growth Factor Receptor) Inhibition Induces Cardiovascular Damage via Redox-Sensitive Processes.

Although VEGF (vascular endothelial growth factor) inhibitors (VEGFIs), are effective anticancer therapies, they cause hypertension through unknown mechanisms. We questioned whether changes in vascular redox state may be important, because VEGF signaling involves nitric oxide (NO) and reactive oxygen species. Molecular mechanisms, including NOS, NADPH oxidase (Nox)-derived reactive oxygen species, antioxidant systems, and vasoconstrictor signaling pathways, were probed in human endothelial cells and vascular smooth muscle exposed to vatalanib, a VEGFI. Vascular functional effects of VEGFI were assessed ex vivo in mouse arteries. Cardiovascular and renal in vivo effects were studied in vatalanib- or gefitinib (EGFI [epidermal growth factor inhibitor])-treated mice. In endothelial cells, vatalanib decreased eNOS (Ser1177) phosphorylation and reduced NO and H2O2 production, responses associated with increased Nox-derived O2- and ONOO- formation. Inhibition of Nox1/4 (GKT137831) or Nox1 (NoxA1ds), prevented vatalanib-induced effects. Nrf-2 (nuclear factor erythroid 2-related factor 2) nuclear translocation and expression of Nrf-2-regulated antioxidant enzymes were variably downregulated by vatalanib. In human vascular smooth muscles, VEGFI increased Nox activity and stimulated Ca2+ influx and MLC20 phosphorylation. Acetylcholine-induced vasodilatation was impaired and U46619-induced vasoconstriction was enhanced by vatalanib, effects normalized by N-acetyl-cysteine and worsened by L-NAME. In vatalanib-, but not gefitinib-treated mice vasorelaxation was reduced and media:lumen ratio of mesenteric arteries was increased with associated increased cardiovascular and renal oxidative stress, decreased Nrf-2 activity and downregulation of antioxidant genes. We demonstrate that inhibition of VEGF signaling induces vascular dysfunction through redox-sensitive processes. Our findings identify Noxs and antioxidant enzymes as novel targets underling VEGFI-induced vascular dysfunction. These molecular processes may contribute to vascular toxicity and hypertension in VEGFI-treated patients.

VEGF may contribute to macrophage recruitment and M2 polarization in the decidua.

It is increasingly evident that cytokines and growth factors produced in the decidua play a pivotal role in the regulation of the local immune microenvironment and the establishment of pregnancy. One of the major growth factors produced in the decidua is vascular endothelial growth factor (VEGF), which acts not only on endothelial cells, but also on multiple other cell types, including macrophages. We sought to determine whether decidua-derived VEGF affects macrophage recruitment and polarization using human endometrial/decidual tissue samples, primary human endometrial stromal cells (ESCs), and the human monocyte cell line THP1. In situ hybridization was used for assessment of local VEGF expression and immunohistochemistry was used for identification and localization of CD68-positive endometrial macrophages. Macrophage migration in culture was assessed using a transwell migration assay, and the various M1/M2 phenotypic markers and VEGF expression were assessed using quantitative real-time PCR (qRT-PCR). We found dramatic increases in both VEGF levels and macrophage numbers in the decidua during early pregnancy compared to the secretory phase endometrium (non-pregnant), with a significant increase in M2 macrophage markers, suggesting that M2 is the predominant macrophage phenotype in the decidua. However, decidual samples from preeclamptic pregnancies showed a significant shift in macrophage phenotype markers, with upregulation of M1 and downregulation of M2 markers. In THP1 cultures, VEGF treatment significantly enhanced macrophage migration and induced M1 macrophages to shift to an M2 phenotype. Moreover, treatment with conditioned media from decidualized ESCs induced changes in macrophage migration and polarization similar to that of VEGF treatment. These effects were abrogated by the addition of a potent VEGF inhibitor. Together these results suggest that decidual VEGF plays a significant role in macrophage recruitment and M2 polarization, and that inhibition of VEGF signaling may contribute to the shift in macrophage polarity observed in different pregnancy disorders, including preeclampsia.

Inhibition of VEGF Signaling Reduces Diabetes-Exacerbated Brain Swelling, but Not Infarct Size, in Large Cerebral Infarction in Mice.

In light of repeated translational failures with preclinical neuroprotection-based strategies, this preclinical study reevaluates brain swelling as an important pathological event in diabetic stroke and investigates underlying mechanism of the comorbidity-enhanced brain edema formation. Type 2 (mild), type 1 (moderate), and mixed type 1/2 (severe) diabetic mice were subjected to transient focal ischemia. Infarct volume, brain swelling, and IgG extravasation were assessed at 3days post-stroke. Expression of vascular endothelial growth factor (VEGF)-A, endothelial-specific molecule-1 (Esm1), and the VEGF receptor 2 (VEGFR2) was determined in the ischemic brain. Additionally, SU5416, a VEGFR2 inhibitor, was treated in the type 1/2 diabetic mice, and stroke outcomes were determined. All diabetic groups displayed bigger infarct volume and brain swelling compared to nondiabetic mice, and the increased swelling was disproportionately larger relative to infarct enlargement. Diabetic conditions significantly increased VEGF-A, Esm1, and VEGFR2 expressions in the ischemic brain compared to nondiabetic mice. Notably, in diabetic mice, VEGFR2 mRNA levels were positively correlated with brain swelling, but not with infarct volume. Treatment with SU5416 in diabetic mice significantly reduced brain swelling. The study shows that brain swelling is a predominant pathological event in diabetic stroke and that an underlying event for diabetes-enhanced brain swelling includes the activation of VEGF signaling. This study suggests consideration of stroke therapies aiming at primarily reducing brain swelling for subjects with diabetes.

Mechanisms of VEGF (Vascular Endothelial Growth Factor) Inhibitor-Associated Hypertension and Vascular Disease.

The VEGF (vascular endothelial growth factor) pathway has emerged as an important target for cancer therapy, with VEGF signaling pathway (VSP) inhibitors having been approved for several different malignancies. Less anticipated has been the multitude of cardiovascular and renal effects of these therapies. Hypertension occurs in at least a quarter of patients starting VSP inhibitors, with virtually every patient having an absolute increase in blood pressure. Patients can also develop proteinuria, renal dysfunction, vascular events, or cardiomyopathy. Although these toxicities have been generally manageable, they have introduced cardiovascular and renal considerations for patient care. On the other hand, these sequelae have provided a growing appreciation for the critical role of VEGF in vascular and renal homeostasis in human biology. Although several recent articles have addressed practical considerations with respect to patient care with this class of drugs,1–4 this review will explore mechanisms associated with the vascular toxicities arising from VEGF signaling inhibition in cancer treatment. The human VEGF family consists of 5 related glycoproteins: VEGFA, VEGFB, VEGFC, VEGFD, and PIGF (placental growth factor). These are secreted to form homodimers, which interact with a family of 3 receptor tyrosine kinases: VEGFR1 (VEGF receptor 1), VEGFR2, and VEGFR3 (Figure 1). VEGFA and VEGFB bind to VEGFR1, VEGFA binds to VEGFR2, and VEGFC and VEGFD bind to both VEGFR2 and VEGFR3.1,5 PIGF primarily interacts with VEGFR1. The VEGFRs are found on a wide variety of cell types. VEGFR1, also called Flt-1 (fms-like tyrosine kinase 1), is found on vascular endothelial cells, hematopoietic stem cells, monocytes, and macrophages. VEGFR2, also called KDR (kinase insert domain) or Flk-1 (fetal liver kinase 1), is expressed on vascular and lymphatic endothelial cells; VEGFR3 (also called Flt-4) is restricted to lymphatic endothelial cells.5 On ligand binding, VEGFRs transduce intracellular signals through …