The role of VEGF receptors in angiogenesis; complex partnerships

Abstract.: Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis but also have profound effects on neural cells. VEGFs are predominantly produced by endothelial, hematopoietic and stromal cells in response to hypoxia and upon stimulation with growth factors such as transforming growth factors, interleukins or platelet-derived growth factor. VEGFs bind to three variants of type III receptor tyrosine kinases, VEGF receptor 1, 2 and 3. Each VEGF isoform binds to a particular subset of these receptors giving rise to the formation of receptor homo- and heterodimers that activate discrete signaling pathways. Signal specificity of VEGF receptors is further modulated upon recruitment of coreceptors, such as neuropilins, heparan sulfate, integrins or cadherins. Here we summarize the knowledge accumulated since the discovery of these proteins more than 20years ago with the emphasis on the signaling pathways activated by VEGF receptors in endothelial cells during cell migration, growth and differentiatio

A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2

Abstract.: The development of functional blood and lymphatic vessels requires spatio-temporal coordination of the production and release of growth factors such as vascular endothelial growth factors (VEGFs). VEGF family proteins are produced in multiple isoforms with distinct biological properties and bind to three types of VEGF receptors. A VEGF-A splice variant, VEGF-A165b, has recently been isolated from kidney epithelial cells. This variant is identical to VEGF-A165 except for the last six amino acids encoded by an alternative exon. VEGF-A165b and VEGF-A165 bind VEGF receptors 1 and 2 with similar affinity. VEGF-A165b elicits drastically reduced activity in angiogenesis assays and even counteracts signaling by VEGF-A165. VEGF-A165b weakly binds to heparan sulfate and does not interact with neuropilin-1, a coreceptor for VEGF receptor 2. To determine the molecular basis for altered signaling by VEGF-A165b we measured VEGF receptor 2 and ERK kinase activity in endothelial cells in culture. VEGF-A165 induced strong and sustained activation of VEGF receptor 2 and ERK-1 and −2, while activation by VEGF-A165b was only weak and transient. Taken together these data show that VEGF-A165b has attenuated signaling potential through VEGF receptor 2 defining this new member of the VEGF family as a partial receptor agonis

The control of alternative splicing by SRSF1 in myelinated afferents contributes to the development of neuropathic pain

Neuropathic pain results from neuroplasticity in nociceptive neuronal networks. Here we demonstrate that control of alternative pre-mRNA splicing, through the splice factor serine-arginine splice factor 1 (SRSF1), is integral to the processing of nociceptive information in the spinal cord. Neuropathic pain develops following a partial saphenous nerve ligation injury, at which time SRSF1 is activated in damaged myelinated primary afferent neurons, with minimal found in small diameter (IB4 positive) dorsal root ganglia neurons. Serine arginine protein kinase 1 (SRPK1) is the principal route of SRSF1 activation. Spinal SRPK1 inhibition attenuated SRSF1 activity, abolished neuropathic pain behaviors and suppressed central sensitization. SRSF1 was principally expressed in large diameter myelinated (NF200-rich) dorsal root ganglia sensory neurons and their excitatory central terminals (vGLUT1 + ve) within the dorsal horn of the lumbar spinal cord. Expression of pro-nociceptive VEGF-Axxxa within the spinal cord was increased after nerve injury, and this was prevented by SRPK1 inhibition. Additionally, expression of anti-nociceptive VEGF-Axxxb isoforms was elevated, and this was associated with reduced neuropathic pain behaviors. Inhibition of VEGF receptor-2 signaling in the spinal cord attenuated behavioral nociceptive responses to mechanical, heat and formalin stimuli, indicating that spinal VEGF receptor-2 activation has potent pro-nociceptive actions. Furthermore, intrathecal VEGF-A165a resulted in mechanical and heat hyperalgesia, whereas the sister inhibitory isoform VEGF-A165b resulted in anti-nociception. These results support a role for myelinated fiber pathways, and alternative pre-mRNA splicing of factors such as VEGF-A in the spinal processing of neuropathic pain. They also indicate that targeting pre-mRNA splicing at the spinal level could lead to a novel target for analgesic development

Diabetes-induced microvascular complications at the level of the spinal cord: a contributing factor in diabetic neuropathic pain

Abnormalities of neurovascular interactions within the central nervous system of diabetic patients is associated with the onset of many neurological disease states. However, to date, the link between the neurovascular network within the spinal cord and regulation of nociception has not been investigated despite neuropathic pain being common in diabetes. We hypothesised that hyperglycaemia-induced endothelial degeneration in the spinal cord, due to suppression of VEGF-A/VEGFR2 signalling, induces diabetic neuropathic pain. Nociceptive pain behaviour was investigated in a chemically induced model of type 1 diabetes (streptozotocin induced, insulin supplemented; either vehicle or VEGF-A165b treated) and an inducible endothelial knockdown of VEGFR2 (tamoxifen induced). Diabetic animals developed mechanical allodynia and heat hyperalgesia. This was associated with a reduction in the number of blood vessels and reduction in Evans blue extravasation in the lumbar spinal cord of diabetic animals versus age-matched controls. Endothelial markers occludin, CD31 and VE-cadherin were downregulated in the spinal cord of the diabetic group versus controls, as well as a concurrent reduction of VEGF-A165b expression. In diabetic animals, VEGF-A165b treatment (biweekly intraperitoneal, 20ng/g) restored normal Evans blue extravasation and prevented vascular degeneration, diabetes-induced central neuron activation and neuropathic pain. Inducible knockdown of VEGFR2 (tamoxifen treated Tie2CreERT2-vegfr2flfl mice) led to a reduction in blood vessel network volume in the lumbar spinal cord and development of heat hyperalgesia. These findings indicate that hyperglycaemia leads to a reduction in the VEGF-A/VEGFR2 signalling cascade resulting in endothelial dysfunction in the spinal cord, which could be an undiscovered contributing factor to diabetic neuropathic pain

Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

Background and Purpose: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165a to NanoLuciferase-tagged VEGF Receptor 2 (VEGFR2) in living cells is that the bioluminescence resonance energy transfer (BRET) signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations.Experimental Approach: Ligand binding kinetics were monitored in both intact HEK293T cells and membranes (expressing NanoLuciferase tagged VEGFR2) using BRET between the tagged receptor and fluorescent analogues of VEGF165a, VEGF165b and VEGF121a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging.Key Results: Quantitiative analysis of the effect of fluorescent VEGF-A isoforms onVEGFR2 endocytosis in cells demonstrated that they produced a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand binding association curves were maintained for the duration of the 90 minute experiment. Measurement of koff at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.Conclusions and Implications: These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/koff) as VEGFR2 moves from the plasma membrane to intracellular endosomes