Neuroprotective role of vascular endothelial growth factor: signalling mechanisms, biological function, and therapeutic potential

Vascular endothelial growth factor (VEGF or VEGF-A) and its receptors play essential roles in the formation of blood vessels during embryogenesis and in disease. Most biological effects of VEGF are mediated via two receptor tyrosine kinases, VEGFR1 and VEGFR2, but specific VEGF isoforms also bind neuropilins (NP) 1 and 2, non-tyrosine kinase receptors originally identified as receptors for semaphorins, polypeptides with essential roles in neuronal patterning. There is abundant evidence that VEGF-A has neurotrophic and neuroprotective effects on neuronal and glial cells in culture and in vivo, and can stimulate the proliferation and survival of neural stem cells. VEGFR2 and NP1 are the major VEGF receptors expressed on neuronal cells and, while the mechanisms mediating neuroprotective effects of VEGF are not fully understood, VEGF stimulates several signalling events in neuronal cell types, including activation of phospholipase C-gamma, Akt and ERK. Findings in diverse models of nerve damage and disease suggest that VEGF has therapeutic potential as a neuroprotective factor. VEGF is a key mediator of the angiogenic response to cerebral and peripheral ischaemia, and promotes nerve repair following traumatic spinal injury. Recent work has revealed a role for reduced VEGF expression in the pathogenesis of amyotrophic lateral sclerosis, a rare neurodegenerative disease caused by selective loss of motor neurons. In many instances, the neuroprotective effects of VEGF appear to result from a combination of the indirect consequences of increased angiogenesis, and the direct stimulation of neuronal function. However, more work is required to determine the specific functional role of direct neuronal effects of VEGF.     

Vascular endothelial growth factor-A: A multifunctional molecular player in diabetic retinopathy

Vascular endothelial growth factor-A (VEGF-A), first described as “vascular permeability factor”, is a critical molecule in the pathogenesis of diabetic retinopathy at several levels. Previous studies have outlined the importance of VEGF-A in mediating vascular pathology in both experimental models and clinical diabetic retinopathy, which are characterized by retinal vascular leakage, preretinal neovascularisation and neuronal degeneration. Paradoxically, recent reports have emphasized the potential neurotrophic effects of VEGF-A on the quiescent vasculature, as well as its direct and indirect protective effects on retinal neurons. VEGF-A has also been identified as an important signalling regulator in the normal central nervous system. Consequently, anti-VEGF therapy for diabetic retinopathy has become a controversal issue. This review outlines recently developed concepts relating to the role of VEGF-A in the pathogenesis of diabetic retinopathy, with particular emphasis on its implications for clinical practice.     

VEGF in the nervous system

Vascular endothelial growth factor (VEGF, VEGFA) is critical for blood vessel growth in the developing and adult nervous system of vertebrates. Several recent studies demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection and glial growth. For example, VEGF treatment of cultured neurons enhances survival and neurite growth independently of blood vessels. Moreover, evidence is emerging that VEGF guides neuronal migration in the embryonic brain and supports axonal and arterial co-patterning in the developing skin. Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, VEGF can be considered a promising tool to promote neuronal health and nerve repair.Key words: VEGF, neuron, neurogenesis, glia, endothelial cell, blood vessel, angiogenic niche     

VEGF in the nervous system

Vascular endothelial growth factor (VEGF, VEGFA) is critical for blood vessel growth in the developing and adult nervous system of vertebrates. Several recent studies demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection and glial growth. For example, VEGF treatment of cultured neurons enhances survival and neurite growth independently of blood vessels. Moreover, evidence is emerging that VEGF guides neuronal migration in the embryonic brain and supports axonal and arterial co-patterning in the developing skin. Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, VEGF can be considered a promising tool to promote neuronal health and nerve repair.

Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 91-103.     

Inhibition of VEGFR-2 by SU5416 increases neonatally glutamate-induced neuronal damage in the cerebral motor cortex and hippocampus

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A–E), receptor types (VEGFR1–3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity.     

Vascular endothelial growth factor (VEGF) signaling regulates hippocampal neurons by elevation of intracellular calcium and activation of calcium/calmodulin protein kinase II and mammalian target of rapamycin

The present study was undertaken to characterize neuronal activity-dependent expression and release of vascular endothelial growth factor (VEGF) from rat hippocampal neurons and its contribution to neuronal functions. Increased levels of VEGF164 mRNA were evident both in cultured neurons and slices, but not astrocytes, following membrane depolarization with KCl. Activity-dependent expression of VEGF, as well as its release, was dependent on the activation of the N-methyl-d-aspartate receptors or L-type voltage-activated calcium channels. A brief (10 min) application of recombinant VEGF165 to neurons elicited a slow rise in cytosolic Ca2+ in a VEGFR2 dependent manner. The VEGF-induced Ca2+ responses required Ca2+ influx, phospholipase Cgamma and Ca2+ stores. An inhibitor of transient receptor potential canonical channels reduced the VEGF-induced Ca2+ responses by 50%, suggesting the involvement of transient receptor potential canonical channels in the VEGF-mediated responses. The same brief stimulus with VEGF led to long-term synaptic enhancement dependent on protein synthesis. VEGF had prominent effects on the activation calcium/calmodulin protein kinase II and cAMP responsive element binding protein as well as extracellular signal-regulated protein kinase and mammalian target of rapamycin-all in a VEGFR2 dependent manner. Our findings suggest that VEGF released from neuronal cells plays a local role in Ca2+ influx and synaptic transmission that may influence the generation of long-term changes in synaptic efficacy.     

Signaling through the vascular endothelial growth factor receptor VEGFR-2 protects hippocampal neurons from mitochondrial dysfunction and oxidative stress

Vascular endothelial growth factor VEGF (VEGF-A or VEGF₁₆₅) is a potent angiogenic factor that also signals neuroprotection through activation of its cognate receptor VEGFR-2. In this capacity, VEGF signaling can rescue neurons from the damage induced by stressful stimuli many of which elicit oxidative stress. However, the regulatory role that VEGFR-2 plays in providing neuroprotection remains elusive. Therefore, we investigated the effects of VEGFR-2 inhibition on primary cultures of mature hippocampal neurons undergoing nutritional stress. We found that neurons cultured under nutritional stress had increased expression of VEGF and its receptors, VEGFR-1, VEGFR-2, and NP-1, as well as enhanced levels of VEGFR-2 phosphorylation. These neurons also showed increased activation of the prosurvival pathways for MEK/ERK1/2 and PI3K/Akt, enhanced phosphorylation (inactivation) of the proapoptotic BAD, and higher levels of the antiapoptotic protein Bcl-xL, all of which were augmented by treatments with exogenous VEGF and blocked by VEGFR-2 inhibition. The blockade of VEGFR-2 function also elicited a cytotoxicity that was accompanied by caspase-3 activation, induction of hemeoxygenase-1 (HO-1), oxidative stress, and a collapse in the mitochondrial membrane potential (ΔΨ_m). Knockdown of VEGFR-2 by siRNA generated a similar pattern of redox change and mitochondrial impairment. Pretreatments with VEGF, VEGF-B, or the antioxidant N-acetylcysteine (NAC) rescued SU1498 or siRNA-treated neurons from the mitochondrial dysfunction and oxidative stress induced by VEGFR-2 inhibition in a timely fashion. These findings suggested that VEGF or VEGF-B can provide neuroprotection by signaling through an alternate VEGF receptor. Together, our findings suggest that VEGF signaling through VEGFR-2 plays a critical regulatory role in protecting stressed hippocampal neurons from the damaging effects of an oxidative insult. These findings also implicate VEGFR-1 or NP-1 as compensatory receptors that mediate neuroprotection when VEGFR-2 function is blocked.     

Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system.

Recent data have demonstrated that vascular endothelial growth factor (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within the developing cerebral cortex. Here we investigate the characteristics of VEGF expression by neurons and test the hypothesis that VEGF may serve both paracrine and autocrine functions in the developing central nervous system. To begin to address these questions, we assayed expression of VEGF and one of its potential receptors, Flk-1 (VEGFR-2), in the embryonic mouse forebrain and embryonic cortical neurons grown in vitro. Both VEGF and Flk-1 are present in subsets of post-mitotic neurons in vivo and in vitro. Moreover, VEGF levels are up-regulated in neuronal cultures subjected to hypoxia, consistent with our previous results in vivo. While the abundance of Flk-1 is unaffected by hypoxia, the receptor exhibits a higher level of tyrosine phosphorylation, as do downstream signaling kinases, including extracellular signal-regulated protein kinase, p90RSK and STAT3a, demonstrating activation of the VEGF pathway. These same signaling components also exhibited higher tyrosine phosphorylation levels in response to exogenous addition of rVEGFA(165). This activation was diminished in the presence of specific inhibitors of Flk-1 function and agents that sequester VEGF, resulting in a dose-dependent increase in apoptosis in these neuronal cultures. Further, inhibition of MEK resulted in increased apoptosis, while inhibition of phosphatidylinositol 3-kinase had no appreciable affect. In addition to the novel function for VEGF that we describe in neuronal survival, neuronal VEGF also affected the organization and differentiation of brain endothelial cells in a three-dimensional culture paradigm, consistent with its more traditional role as a vascular agent. Thus, our in vitro data support a role for neuronal VEGF in both paracrine and autocrine signaling in the maintenance of neurons and endothelia in the central nervous system.     

Fast rearrangement of the neuronal growth cone’s actin cytoskeleton following VEGF stimulation

The neuronal growth cone plays a crucial role in the development of the nervous system. This highly motile structure leads the axon to its final destination by translating guidance cues into cytoskeletal rearrangements. Recently, vascular endothelial growth factor (VEGF), which is essential for angiogenesis and vascular sprouting, has been found to exert a trophic activity also on neurons, leading to an increased axonal outgrowth, similar to the well-known nerve growth factor (NGF). The neurotrophic properties of VEGF are likely to be promoted via the VEGF receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1). In the long term, VEGF attracts and influences the growth cone velocity and leads to growth cone enlargement. The present study focuses on immediate VEGF effects using RFP-actin and GFP-NF-M microinjected chicken dorsal root ganglia for live cell imaging of the neuronal growth cone. We analyzed actin and neurofilament dynamics following VEGF and NGF treatment and compared the effects. Furthermore, key signaling pathways of VEGF were investigated by specific blocking of VEGFR-2 or NRP-1. With the aid of confocal laser scanning microscopy and stimulated emission depletion microscopy, we show for the first time that VEGF has a quick effect on the actin-cytoskeleton, since actin rearrangements were identifiable within a few minutes, leading to a dramatically increased motion. Moreover, these effects were strongly enhanced by adding both VEGF and NGF. Most notably, the effects were inhibited by blocking VEGFR-2, therefore we propose that the immediate effects of VEGF on the actin-cytoskeleton are mediated through VEGFR-2.     

VEGF111b,a new member of VEGFxxxb isoforms and induced by mitomycin C,inhibits angiogenesis

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA_3.1 empty vector, pcDNA_3.1-VEGF111b or pcDNA_3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.     

Prolonged blockade of VEGF family members does not cause identifiable damage to retinal neurons or vessels

Several ocular diseases complicated by neovascularization are being treated by repeated intraocular injections of vascular endothelial growth factor (VEGF) antagonists. While substantial benefits have been documented, there is concern that unrecognized damage may be occurring, because blockade of VEGF may damage the fenestrated vessels of the choroicapillaris and deprive retinal neurons of input from a survival factor. One report has suggested that even temporary blockade of all isoforms of VEGF-A results in significant loss of retinal ganglion cells. In this study, we utilized double transgenic mice with doxycycline-inducible expression of soluble VEGF receptor 1 coupled to an Fc fragment (sVEGFR1Fc), a potent antagonist of several VEGF family members, including VEGF-A, to test the effects of VEGF blockade in the retina. Expression of sVEGFR1Fc completely blocked VEGF-induced retinal vascular permeability and significantly suppressed the development of choroidal neovascularization at rupture sites in Bruch's membrane, but did not cause regression of established choroidal neovascularization. Mice with constant expression of sVEGFR1Fc in the retina for 7 months had normal electroretinograms and normal retinal and choroidal ultrastructure including normal fenestrations in the choroicapillaris. They also showed no significant difference from control mice in the number of ganglion cell axons in optic nerve cross sections and the retinal level of mRNA for 3 ganglion cell-specific genes. These data indicate that constant blockade of VEGF for up to 7 months has no identifiable deleterious effects on the retina or choroid and support the use of VEGF antagonists in the treatment of retinal diseases.     

Pleiotropic role of VEGF-A in regulating fetal pulmonary mesenchymal cell turnover

Tight regulation of VEGF-A production and signaling is important for the maintenance of lung development and homeostasis. VEGF null mice have provided little insight into the role of VEGF during the later stages of lung morphogenesis. Therefore, we examined the in vitro effects of autocrine and paracrine VEGF-A production and the inhibition of VEGF-A signaling on a Flk-1-negative subset of fetal pulmonary mesenchymal cells (pMC). We hypothesized that VEGF-A receptor signaling regulates turnover of fetal lung mesenchyme in a cell cycle-dependent manner. VEGF receptor blockade with SU-5416 caused cell spreading and decreased proliferation and bcl-2 localization. Nuclear expression of the cell cycle inhibitory protein, p21, was increased with SU-5416 treatment, and p27 was absent. Autocrine VEGF production by pMC resulted in proliferation and p21/p27-dependent contact inhibition. In contrast, exogenous VEGF-A increased cell progression through the cell cycle. Selective activation of Flt by placental growth factor demonstrated the importance of this receptor/kinase in the VEGF-A responsiveness of pMC. The expression and localization of the survival factor bcl-2 was dependent on VEGF. These results provide evidence that VEGF-A plays a critical role in the regulation of fetal pulmonary mesenchymal proliferation, survival, and the subsequent development of normal lung architecture through bcl-2 and p21/p27-dependent cell cycle control.     

Crosstalk between VEGF-A/VEGFR2 and GDNF/RET signaling pathways

Vascular endothelial growth factor (VEGF-A) plays multiple roles in kidney development: stimulates cell proliferation, survival, tubulogenesis, and branching morphogenesis. However, the mechanism that mediates VEGF-A induced ureteric bud branching is unclear. Glial-derived neurotrophic factor (GDNF) signaling through tyrosine kinase c-RET is the major regulator of ureteric bud branching. Here we examined whether VEGF-A regulates RET signaling. We determined that ureteric bud-derived cells express the main VEGF-A signaling receptor, VEGFR2 and RET, by RT-PCR, immunoblotting, and immunocytochemistry. We show that the VEGF-A isoform VEGF(165) induces RET-tyr(1062) phosphorylation in addition to VEGFR2 autophosphorylation, that VEGF(165) and GDNF have additive effects on RET-tyr(1062) phosphorylation, and that VEGFR2 and RET co-immunoprecipitate. Functionally, VEGF(165) induces ureteric bud cell proliferation and branching morphogenesis. Similarly, in embryonic kidney explants VEGF(165) induces RET-tyr(1062) phosphorylation and upregulates GDNF. These findings provide evidence for a novel cooperative interaction between VEGFR2 and RET that mediates VEGF-A functions in ureteric bud cells.     

Autocrine functions of VEGF in breast tumor cells: Adhesion,survival, migration and invasion

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.     

PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis

Vascular endothelial growth factor (VEGF), a specific pro-angiogenic peptide, has shown neuroprotective effects in the Parkinson’s disease (PD) models, but the underlying mechanisms remain elusive. In this study, the neuroprotective properties of VEGF on 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced neurotoxicity in primary cerebellar granule neurons were investigated. Pretreatment of VEGF prevented MPP⁺-induced neuronal apoptosis in a concentration- and time-dependent manner. And this prevention was blocked by PTK787/ZK222584, a VEGF receptor-2 specific inhibitor. Both inhibition of the Akt pathway and activation of the extracellular signal-regulated kinase (ERK) pathway contribute to MPP⁺-induced neuronal apoptosis. VEGF reversed the inhibition of phosphoinositide 3-kinase (PI3-K)/Akt pathway caused by MPP⁺, but further enhanced the activation of ERK induced by MPP⁺. Interestingly, VEGF and PD98059 (an ERK kinase inhibitor) play a synergistic role in protecting neurons from MPP⁺-induced toxicity. Collectively, these findings suggest that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP⁺-induced neuronal apoptosis. This finding offers not only a new and clinically significant modality as to how VEGF exerts its neuroprotective effects but also a novel therapeutic strategy for PD by differentially regulating PD-associated signaling pathways.     

Autocrine functions of VEGF in breast tumor cells

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.     

Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and -2 heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A(165) inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A(165) promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI(2)). However, it is unknown whether VEGF-A(165) is mediating PGI(2) synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A(165) activity. In this study, PGI(2) synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F(1alpha), 6-keto PGF(1alpha)) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A(165) (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A(121) (VEGFR-1 and VEGFR-2 agonist) (up to 10(-9) m), increased PGI(2) synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI(2) synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI(2) release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI(2) release mediated by VEGF-A(165) and VEGF-A(121), and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121) up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121), which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A(165) to NRP-1 potentiates its capacity to promote PGI(2) synthesis.     

Vascular endothelial growth factor (VEGF)-D and VEGF-A differentially regulate KDR-mediated signaling and biological function in vascular endothelial cells

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.