VEGF-B is a potent antioxidant.

VEGF-B was discovered a long time ago. However, unlike VEGF-A, whose function has been extensively studied, the function of VEGF-B and the mechanisms involved still remain poorly understood. Notwithstanding, drugs that inhibit VEGF-B and other VEGF family members have been used to treat patients with neovascular diseases. It is therefore critical to have a better understanding of VEGF-B function and the underlying mechanisms. Here, using comprehensive methods and models, we have identified VEGF-B as a potent antioxidant. Loss of Vegf-b by gene deletion leads to retinal degeneration in mice, and treatment with VEGF-B rescues retinal cells from death in a retinitis pigmentosa model. Mechanistically, we demonstrate that VEGF-B up-regulates numerous key antioxidative genes, particularly, Gpx1 Loss of Gpx1 activity largely diminished the antioxidative effect of VEGF-B, demonstrating that Gpx1 is at least one of the critical downstream effectors of VEGF-B. In addition, we found that the antioxidant function of VEGF-B is mediated mainly by VEGFR1. Given that oxidative stress is a crucial factor in numerous human diseases, VEGF-B may have therapeutic value for the treatment of such diseases.

VEGFB Promotes Myoblasts Proliferation and Differentiation through VEGFR1-PI3K/Akt Signaling Pathway.

It has been demonstrated that vascular endothelial growth factor B (VEGFB) plays a vital role in regulating vascular biological function. However, the role of VEGFB in regulating skeletal muscle cell proliferation and differentiation remains unclear. Thus, this study aimed to investigate the effects of VEGFB on C2C12 myoblast proliferation and differentiation and to explore the underlying mechanism. For proliferation, VEGFB significantly promoted the proliferation of C2C12 myoblasts with the upregulating expression of cyclin D1 and PCNA. Meanwhile, VEGFB enhanced vascular endothelial growth factor receptor 1 (VEGFR1) expression and activated the PI3K/Akt signaling pathway in a VEGFR1-dependent manner. In addition, the knockdown of VEGFR1 and inhibition of PI3K/Akt totally abolished the promotion of C2C12 proliferation induced by VEGFB, suggesting that VEGFB promoted C2C12 myoblast proliferation through the VEGFR1-PI3K/Akt signaling pathway. Regarding differentiation, VEGFB significantly stimulated the differentiation of C2C12 myoblasts via VEGFR, with elevated expressions of MyoG and MyHC. Furthermore, the knockdown of VEGFR1 rather than NRP1 eliminated the VEGFB-stimulated C2C12 differentiation. Moreover, VEGFB activated the PI3K/Akt/mTOR signaling pathway in a VEGFR1-dependent manner. However, the inhibition of PI3K/Akt/mTOR blocked the promotion of C2C12 myoblasts differentiation induced by VEGFB, indicating the involvement of the PI3K/Akt pathway. To conclude, these findings showed that VEGFB promoted C2C12 myoblast proliferation and differentiation via the VEGFR1-PI3K/Akt signaling pathway, providing new insights into the regulation of skeletal muscle development.

A cyclic peptide reproducing the α1 helix of VEGF-B binds to VEGFR-1 and VEGFR-2 and inhibits angiogenesis and tumor growth.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are pivotal regulators of angiogenesis. The VEGF-VEGFR system is therefore an important target of anti-angiogenesis therapy. Based on the X-ray structure of VEGF-B/VEGFR-1 D2, we designed a cyclic peptide (known as VGB1) reproducing the α1 helix and its adjacent region to interfere with signaling through VEGFR-1. Unexpectedly, VGB1 bound VEGFR-2 in addition to VEGFR-1, leading to inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells and 4T1 murine mammary carcinoma cells, which express VGEFR-1 and VEGFR-2, and U87 glioblastoma cells that mostly express VEGFR-2. VGB1 inhibited different aspects of angiogenesis, including proliferation, migration and tube formation of endothelial cells stimulated by VEGF-A through suppression of extracellular signal-regulated kinase 1/2 and AKT (Protein Kinase B) phosphorylation. In a murine 4T1 mammary carcinoma model, VGB1 caused regression of tumors without causing weight loss in association with impaired cell proliferation (decreased Ki67 expression) and angiogenesis (decreased CD31 and CD34 expression), and apoptosis induction (increased TUNEL staining and p53 expression, and decreased Bcl-2 expression). According to far-UV circular dichroism (CD) and molecular dynamic simulation data, VGB1 can adopt a helical structure. These results, for the first time, demonstrate that α1 helix region of VEGF-B recognizes both VEGFR-1 and VEGFR-2.

Vascular endothelial growth factor B inhibits lipid accumulation in C2C12 myotubes incubated with fatty acids.

To investigate (1) the effect of vascular endothelial growth factor B (VEGFB) on lipid accumulation and the alteration of fatty acids and fatty acid-related enzymes in C2C12 myotubes incubated with fatty acids and (2) the regulatory effect of VEGFB on skeletal muscle lipid metabolism. Mouse C2C12 myotubes were incubated with oleic acid (OA) and palmitic acid (PA), and differentiated mature C2C12 myotubes were treated with VEGFB. Oil-red O staining, BODIPY staining and cell triglycerides (TG) content were examined. Total RNA was isolated, and real-time PCR analysis was performed. Treatment with 100 µM OA and 50 µM PA induced lipid droplet accumulation and increased TG content (p < .01), and 100 ng/mL VEGFB reduced lipid droplet accumulation and decreased TG content (p < .01). Treatment with 100 ng/mL VEGFB significantly induced the mRNA expression of fatty acid transport protein 1 (FATP1) (p < .01) and FATP4 (p < .01). Treatment with 100 ng/mL VEGFB significantly induced the mRNA expression of adipose TG lipase and hormone-sensitive lipase (p < .01) as well as carnitine palmitoyltransferase I (p < .01), peroxisome proliferator-activated receptor-γ coactivator-1α (p < .01), acyl-coa dehydrogenase very long chain (p < .05), acyl-coa synthetase long-chain family member 1 (p < .01), peroxisomal acyl-coenzyme A oxidase 1 (p < .05), and mitochondrial uncoupling protein 3 (p < .01). VEGFB enhanced FATP1and FATP4 expression, promoted C2C12 myotube fatty acid oxidation and TG decomposition, and inhibited C2C12 myotube fatty acid re-esterification, thus inhibiting lipid accumulation in C2C12 myotubes incubated with fatty acids.

Novel function of VEGF-B as an antioxidant and therapeutic implications.

Oxidative stress, due to insufficiency of antioxidants or over-production of oxidants, can lead to severe cell and tissue damage. Oxidative stress occurs constantly and has been shown to be involved in innumerable diseases, such as degenerative, cardiovascular, neurological, and metabolic disorders, cancer, and aging, thus highlighting the vital need of antioxidant defense mechanisms. Vascular endothelial growth factor B (VEGF-B) was discovered a long time ago, and is abundantly expressed in most types of cells and tissues. VEGF-B remained functionally mysterious for many years and later on has been shown to be minimally angiogenic. Recently, VEGF-B is reported to be a potent antioxidant by boosting the expression of key antioxidant enzymes. Thus, one major role of VEGF-B lies in safeguarding tissues and cells from oxidative stress-induced damage. VEGF-B may therefore have promising therapeutic utilities in treating oxidative stress-related diseases. In this review, we discuss the current knowledge on the newly discovered antioxidant function of VEGF-B and the related molecular mechanisms, particularly, in relationship to some oxidative stress-related diseases, such as retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, glaucoma, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease.

VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism and serves as a marker of poor prognosis for cancer patients.

The biological functions of VEGF-B in cancer progression remain poorly understood. Here, we report that VEGF-B promotes cancer metastasis through the remodeling of tumor microvasculature. Knockdown of VEGF-B in tumors resulted in increased perivascular cell coverage and impaired pulmonary metastasis of human melanomas. In contrast, the gain of VEGF-B function in tumors led to pseudonormalized tumor vasculatures that were highly leaky and poorly perfused. Tumors expressing high levels of VEGF-B were more metastatic, although primary tumor growth was largely impaired. Similarly, VEGF-B in a VEGF-A-null tumor resulted in attenuated primary tumor growth but substantial pulmonary metastases. VEGF-B also led to highly metastatic phenotypes in Vegfr1 tk(-/-) mice and mice treated with anti-VEGF-A. These data indicate that VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism. High expression levels of VEGF-B in two large-cohort studies of human patients with lung squamous cell carcinoma and melanoma correlated with poor survival. Taken together, our findings demonstrate that VEGF-B is a vascular remodeling factor promoting cancer metastasis and that targeting VEGF-B may be an important therapeutic approach for cancer metastasis.

VEGF-B selectively regenerates injured peripheral neurons and restores sensory and trophic functions.

VEGF-B primarily provides neuroprotection and improves survival in CNS-derived neurons. However, its actions on the peripheral nervous system have been less characterized. We examined whether VEGF-B mediates peripheral nerve repair. We found that VEGF-B induced extensive neurite growth and branching in trigeminal ganglia neurons in a manner that required selective activation of transmembrane receptors and was distinct from VEGF-A-induced neuronal growth. VEGF-B-induced neurite elongation required PI3K and Notch signaling. In vivo, VEGF-B is required for normal nerve regeneration: mice lacking VEGF-B showed impaired nerve repair with concomitant impaired trophic function. VEGF-B treatment increased nerve regeneration, sensation recovery, and trophic functions of injured corneal peripheral nerves in VEGF-B-deficient and wild-type animals, without affecting uninjured nerves. These selective effects of VEGF-B on injured nerves and its lack of angiogenic activity makes VEGF-B a suitable therapeutic target to treat nerve injury.

Dual effects of VEGF-B on activating cardiomyocytes and cardiac stem cells to protect the heart against short- and long-term ischemia-reperfusion injury.

AIMS: To investigate whether vascular endothelial growth factor B (VEGF-B) improves myocardial survival and cardiac stem cell (CSC) function in the ischemia-reperfusion (I/R) heart and promotes CSC mobilization and angiogenesis. METHODS AND RESULTS: One hour after myocardial ischemia and infarction, rats were treated with recombinant human VEGF-B protein following 24 h or 7 days of myocardial reperfusion. Twenty-four hours after myocardial I/R, VEGF-B increased pAkt and Bcl-2 levels, reduced p-p38MAPK, LC3-II/I, beclin-1, CK, CK-MB and cTnt levels, triggered cardiomyocyte protection against I/R-induced autophagy and apoptosis, and contributed to the decrease of infarction size and the improvement of heart function during I/R. Simultaneously, an in vitro hypoxia-reoxygenation (H/R)-induced H9c2 cardiomyocyte injury model was used to mimic I/R injury model in vivo; in this model, VEGF-B decreased LDH release, blocked H/R-induced apoptosis by inhibiting cell autophagy, and these special effects could be abolished by the autophagy inducer, rapamycin. Mechanistically, VEGF-B markedly activated the Akt signaling pathway while slightly inhibiting p38MAPK, leading to the blockade of cell autophagy and thus protecting cardiomyocyte from H/R-induced activation of the intrinsic apoptotic pathway. Seven days after I/R, VEGF-B induced the expression of SDF-1α and HGF, resulting in the massive mobilization and homing of c-Kit positive cells, triggering further angiogenesis and vasculogenesis in the infracted heart and contributing to the improvement of I/R heart function. CONCLUSION: VEGF-B could contribute to a favorable short- and long-term prognosis for I/R via the dual manipulation of cardiomyocytes and CSCs.

Effects of vascular endothelial growth factor-b on the bioelectric activity of rat atrial myocardium under normal conditions and during gradual stretching.

Using a microelectrode technique we studied the effects of vascular endothelial growth factor-B on the activity of rat atrial myocardium under normal conditions and after gradual stretching of the tissue. It was shown that vascular endothelial growth factor-B increased duration of the action potential only at the level of 90% re-polarization. Effects on the frequency and force of contraction were absent. The repetition frequency of the action potentials did not change. Close observation of the vascular endothelial growth factor-B-induced mechanisms and stretch-induced alteration in action potential durations to 90% of repolarization, confirmed the existence of a link between the examining growth factor-B and stretch induced mechanisms.

Binding of VEGF-A is sufficient to abrogate the disturbing effects of VEGF-B together with VEGF-A on retinal endothelial cells.

PURPOSE: Inhibition of vascular endothelial growth factor (VEGF) is a promising strategy to treat retinal complications of diabetes. In contrast to VEGF-A binding ranibizumab, aflibercept also binds to other members of the VEGF family including VEGF-B, but potential effects of this factor on permeability and angiogenic processes are unclear. Therefore, we studied how VEGF-B variants as single agents or together with VEGF-A165 might affect proliferation, migration, or barrier function of retinal endothelial cells (REC). Also investigated was the normalization of REC properties with both VEGF-inhibitors to explore if additional targeting of VEGF-B is relevant. METHODS: Stimulation of proliferation or migration of immortalized bovine REC (iBREC) and disturbance of their barrier by exposure to VEGF-B variants (as single factors or together with VEGF-A165) was determined with or without VEGF-binding proteins being added. Permeability of iBREC was assessed by measuring their transendothelial resistance (TER) and expression of the tight junction protein claudin-1. RESULTS: VEGF-B167 and VEGF-B186 enhanced proliferation of iBREC but these isoforms did not affect cell migration. Interestingly, ranibizumab completely blocked both migration and proliferation induced by VEGF-A plus VEGF-B. Both VEGF-B variants did also not affect barrier function or claudin-1 expression in a normal or high-glucose environment. Accordingly, binding VEGF-A was enough to normalize a reduced TER and reinstate claudin-1 lost during treatment with this factor in combination with VEGF-B. CONCLUSIONS: Important properties and functions of REC seem not to be affected by any VEGF-B variant and targeting the key factor VEGF-A is sufficient to normalize growth factor-disturbed cells of this type.

Vascular endothelial growth factor B prevents the shift in the ocular dominance distribution of visual cortical neurons in monocularly deprived rats.

A key model for examining the activity-dependent development of primary visual cortex (V1) involves the imbalance in activity between the two eyes induced by monocular deprivation (MD). MD early in life causes dramatic changes in the functional and structural organization of mammalian visual cortex. The molecular signals that mediate the effects of MD on the development of visual cortex are not well defined. Neurotrophic factors are important in regulating the plasticity of visual cortex, but the choice of an appropriate growth factor as well as its delivery has proven difficult. Although vascular endothelial growth factor-B (VEGF-B) is a homolog of the angiogenic factor VEGF-A, it has only minimal angiogenic activity, raising the question of whether this factor has other (more relevant) biological properties. Intrigued by the possibility that VEGF family members affect neuronal cells, we explored whether VEGF-B has a role in the nervous system. In rats, VEGF-B infusion during monocular deprivation (MD) counteracted the normally occurring ocular dominance (OD) shift toward the non-deprived eye so that the deprived eye dominated the VEGF-B-treated cortex after MD. In particular, VEGF-B counteracted the effects of MD without causing detectable alterations in spontaneous discharge or behavior. In conclusion, the simultaneous analysis of visual cortical cell discharge and ocular dominance plasticity suggests that VEGF-B has important effects on the functional architecture of the visual cortex. Therefore, VEGF-B is a new candidate trophic challenge molecule for the visual cortex.

Overexpression of VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, leads to insufficient angiogenesis in patients with systemic sclerosis.

RATIONALE: Systemic sclerosis (SSc) is characterized by widespread microangiopathy, fibrosis, and autoimmunity. Despite the lack of angiogenesis, the expression of vascular endothelial growth factor A (VEGF) was shown to be upregulated in SSc skin and circulation; however, previous studies did not distinguish between proangiogenic VEGF(165) and antiangiogenic VEGF(165)b isoforms, which are generated by alternative splicing in the terminal exon of VEGF pre-RNA. OBJECTIVE: We investigated whether VEGF isoform expression could be altered in skin and circulation of patients with SSc. METHODS AND RESULTS: Here, we show that the endogenous antiangiogenic VEGF(165)b splice variant is selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF(165)b expression correlated with increased expression of profibrotic transforming growth factor-ß1 and serine/arginine protein 55 splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. Circulating levels of VEGF(165)b were significantly higher in patients with SSc than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF(165)b than healthy MVECs. Transforming growth factor-ß1 upregulated the expression of VEGF(165)b and serine/arginine protein 55 in both SSc and healthy MVECs. In SSc MVECs, VEGF receptor-2 was overexpressed, but its phosphorylation was impaired. Recombinant VEGF(165)b and SSc-MVEC-conditioned medium inhibited VEGF(165)-mediated VEGF receptor-2 phosphorylation and capillary morphogenesis in healthy MVECs. The addition of anti-VEGF(165)b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC-conditioned medium. Capillary morphogenesis was severely impaired in SSc MVECs and could be ameliorated by treatment with recombinant VEGF(165) and anti-VEGF(165)b blocking antibodies. CONCLUSIONS: In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia.

Role of VEGFB in electrical pulse stimulation inhibits apoptosis in C2C12 myotubes.

Skeletal muscle is the major effector organ for exercise. It has been proposed that VEGFB is significantly related to apoptosis in various cell types but not yet in skeletal muscle. We hypothesize that the decrease of VEGFB in skeletal muscle participates in the occurrence of skeletal muscle apoptosis and that exercise inhibits apoptosis by elevating the expression of VEGFB in skeletal muscle cells. Based on this hypothesis, we developed in vitro experiments to mimic the effect of exercise through electrical pulse stimulation (EPS) to observe the effect of EPS on apoptosis and the change in VEGFB expression in differentiated myotubes. In addition, we employed RNA interference to explore whether VEGFB is directly involved in the regulation of myotube apoptosis during EPS. Our results showed that exogenous VEGFB167 significantly inhibited C2C12 myotube apoptosis induced by TNF-α treatment and that endogenous VEGFB in differentiated C2C12 myotubes was significantly upregulated by EPS. In addition, EPS significantly changed the expression of the apoptotic indicators Bax and Bcl-2 at the mRNA level and downregulated the protein expression of cleaved caspase-3. The antiapoptotic effect of EPS weakened substantially as VEGFB in C2C12 myotubes was inhibited. Taken together, these results indicate that exercise-like EPS inhibits apoptosis by increasing the expression of C2C12 myotube-derived VEGFB.

VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.

Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.

Improving high-fat diet-induced obesity and fatty liver by adipose tissue targeted delivery of vascular endothelial growth factor-B.

Impaired vascularization of adipose tissue leads to local hypoxia and results in chronic inflammation and obesity-related metabolic disorders. We have recently constructed an engineered protein named tPep-VEGF-B by bridging vascular endothelial growth factor (VEGF-B) with an adipose-targeted peptide. Here, we reported tPep-VEGF-B diminishes obesity and alleviates metabolic syndrome. High fat diet (HFD) treated mice had reduced adipose vascular density and showed adipose hypoxia and metabolic complications. In contrast, the treatment of tPep-VEGF-B repressed HFD-induced body weight gain, which led to increased adipose vasculature and reduced hypoxia. This treatment also alleviated obesity associated hyperlipidemia and fatty liver disease. This study provided a leading molecule for the treatment of type 2 diabetes and other metabolic diseases. It also provided experimental support for the theory that modulation of angiogenesis plays a key role in the treatment of metabolic diseases.

The balance of autocrine VEGF-A and VEGF-C determines podocyte survival.

Podocytes are an important component of the glomerular filtration barrier and are the major source of vascular endothelial growth factor (VEGF) in the glomerulus. The role of VEGF for the phenotype of the glomerular endothelium has been intensely studied; however, the direct effects of autocrine VEGF on the podocyte are largely unknown. In this study we characterized the expression of VEGF isoforms and VEGF receptors in cultured human podocytes and examined direct effects on cell signaling and apoptosis after stimulation with exogenous VEGF or ablation of autocrine VEGF. We identified VEGF-A and VEGF-C as the dominant isoforms in human podocytes and showed that autocrine levels of both are important for the intracellular activation of antiapoptotic phosphoinositol 3-kinase/AKT and suppression of the proapoptotic p38MAPK via VEGFR-2. We demonstrated that ablation of VEGF-A or VEGF-C as well as treatment with bevacizumab or a VEGFR-2/-3 tyrosine kinase inhibitor led to reduced podocyte survival. In contrast, ablation of VEGF-B had no effect on podocyte survival. Treatment with exogenous VEGF-C reversed the effect of VEGF-A neutralization, and exogenous VEGF-A abrogated the effect of VEGF-C ablation in human podocytes. Our results underline the importance of autocrine VEGF for podocyte survival and indicate the delicate balance of VEGF-A and VEGF-C to influence progression of glomerular diseases.

[Effect of VEGF-B on neuroprotection in mouse retinal ganglion cells].

OBJECTIVE: To investigate the expression of vascular endothelial growth factor-B (VEGF-B) in retina and its effect on neuroprotection in mouse retinal ganglion cells (RGCs). METHODS: It was a experimental study. 35 of C57BL/6 mice (adult male) were used. Optic nerve crush injury was made in 28 mice, 7 as control. Expression of VEGF-B in retina was detected by hybridization in situ in each group of 2 at 6 hours, 1 day, 1 and 2 weeks after optic nerve crush. A quantitative analysis of VEGF-B mRNA was determined by real time reverse transcription polymerase chain reaction (RT-PCR) in each group of 5 at 6 hours, 1 day, 1 and 2 weeks after optic nerve crush. Additional 36 mice were divided into 4 groups: control (8), optic nerve crush (9), optic nerve crash plus PBS intravitreal injection (7), and optic nerve crash plus VEGF-B (450 mg/L) intravitreal injection (12). RGCs were counted by retrograde tracing of Fluorogold to evaluate the effect of VEGF-B on neuroprotection by microscope. RESULTS: Compared with control group, the expression of VEGF-B was significantly increased in the retina of mice and peaked at one week after the optic nerve crush. The number of RGCs was 1.7 fold higher in optic nerve crush alone and 1.9 fold higher in optic nerve crush combined with intravitreal injection of VEGF-B, which reached statistically significance (t = 0.001, P < 0.01 and t = 0.001, P < 0.01, respectively). CONCLUSIONS: Our results indicate that VEGF-B is involved in the retinal recovery processes and plays a potent role of neuroprotection in RGCs after the optic nerve crush.

Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells.

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

Vascular Endothelial Growth Factor Isoform-B Stimulates Neurovascular Repair After Ischemic Stroke by Promoting the Function of Pericytes via Vascular Endothelial Growth Factor Receptor-1.

Ischemic stroke triggers endogenous angiogenic mechanisms, which correlates with longer survival in patients. As such, promoting angiogenesis appears to be a promising approach. Experimental studies investigated mostly the potent angiogenic factor vascular endothelial growth factor isoform-A (VEGF-A). However, VEGF-A increases the risk of destabilizing the brain microvasculature, thus hindering the translation of its usage in clinics. An attractive alternative VEGF isoform-B (VEGF-B) was recently reported to act as a survival factor rather than a potent angiogenic factor. In this study, we investigated the therapeutic potential of VEGF-B in ischemic stroke using different in vivo and in vitro approaches. We showed that the delayed intranasal administration of VEGF-B reduced neuronal damage and inflammation. Unexpectedly, VEGF-B stimulated the formation of stable brain microvasculature within the injured region by promoting the interaction between endothelial cells and pericytes. Our data indicate that the effects of VEGF-B were mediated via its specific receptor VEGF receptor-1 (VEGFR-1) that is predominately expressed in brain pericytes. Importantly, VEGF-B promoted the survival of pericytes, and not brain endothelial cells, by inducing expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and the main protein involved in energy homeostasis AMP-activated protein kinase α (AMPKα). Moreover, we showed that VEGF-B stimulated the pericytic release of factors stimulating a "reparative angiogenesis" that does not compromise microvasculature stability. Our study unraveled hitherto unknown role of VEGF-B/VEGFR-1 signaling in regulating the function of pericytes. Furthermore, our findings suggest that brain microvasculature stabilization via VEGF-B constitutes a safe therapeutic approach for ischemic stroke.

Coordinated activation of VEGFR-1 and VEGFR-2 is a potent arteriogenic stimulus leading to enhancement of regional perfusion.

OBJECTIVE: The process of arteriogenesis is driven by various growth factors including vascular endothelial growth factor (VEGF)-A, which mediates its activity through VEGFR-2 (Flk-1/KDR) on endothelial cells and through VEGFR-1 (Flt-1) on endothelial cells and monocytes. The purpose of this study was to identify which of the VEGF receptors are involved in arteriogenesis in vivo. METHODS: Collateral vessel growth was induced by femoral artery ligation in a mouse model of hindlimb ischemia. Following ligation, Balb/c mice were treated with different growth factors (VEGF-A, VEGF-E, PlGF-2, VEGF-E plus PlGF-2 or VEGF-A plus PlGF-2, activating either VEGFR-1, VEGFR-2, or both). After 1 week of treatment, hindlimb perfusion was assessed by perfusion scintigraphy using Tc-99m-MIBI. RESULTS: The strongest improvement of regional perfusion was achieved by simultaneous activation of VEGFR-1 and VEGFR-2, using either VEGF-A or VEGF-A plus PlGF-2, with elevation of relative perfusion in the ischemic limbs from 0.61 to 0.83. The partial restoration in perfusion was associated with morphological changes typical for arteriogenesis. Moreover, specific inhibition of both VEGF-receptors using ZK 202650 resulted in a significant inhibition of arteriogenesis, indicating an active role of the VEGF system in compensatory arteriogenesis. CONCLUSION: The coordinated activation of both VEGFR-1 and VEGFR-2 represents a more potent arteriogenic stimulus compared to the isolated activation of either one of these two receptors. These data imply that the activation of both monocytes and endothelial cells is necessary to obtain a maximal VEGF-induced activation of arteriogenesis.