What Type of VEGF Do You Need?
Authors: Chris R.J. Kennedy
Journal: Journal of the American Society of Nephrology (2010)

Typically considered a mediator of angiogenesis and vascular permeability, vascular endothelial growth factor (VEGF) has emerged as an important modulator of glomerular health and disease. The pleiotropic nature VEGF was dramatically illustrated in cases in which patients who were undergoing chemotherapy with VEGF antibodies (e.g., bevacizumab) developed proteinuria1 and thrombotic microangiopathy,2 indicating damage to the glomerular filtration barrier. In addition, individuals with preeclampsia, a condition characterized by excess circulating levels of a soluble form of the VEGF receptor, can likewise develop proteinuria,3 and increased serum levels of sFlt may contribute to endothelial dysfunction in chronic kidney disease.4 These studies suggest that a threshold of VEGF must be met to ensure glomerular health. Accordingly, in this editorial space just 4 years ago, Mathieson asked, "How much VEGF do you need?"5 At the time, the question was prompted by several studies demonstrating that VEGF gene dose is critical to glomerular development, structure, and function. Quaggin's group6,7 was reporting the generation of a series of gene-targeted mice with podocyte-specific VEGF reduction, with expression ranging from 0 to 50% of normal. This exquisite control over VEGF expression yielded a striking variety of phenotypes: When VEGF expression is ablated entirely, glomerular development fails and perinatal lethality ensues; low expression levels (approximately 25% of normal) results in a cascade of glomerular cell attrition beginning with endothelial and proceeding to mesangial cells shortly after birth, and intermediate VEGF levels (approximately 50% of normal) recapitulates an endotheliosis and proteinuria reminiscent of preeclampsia. VEGF ablation in adult mice yields a pronounced thrombotic glomerular injury, similar to that encountered with patients who have cancer and are on anti-VEGF therapy. A postdevelopmental role for podocyte-derived VEGF was subsequently demonstrated using a strategy of inducible gene deletion.2 Essentially, the less podocyte-derived VEGF mice have, the more severe the pathology. If too little VEGF is detrimental to glomerular health, then more must be beneficial, right? A cursory survey of the literature quickly dismisses this notion. Overexpression of an isoform of VEGF (VEGF164) in murine podocytes during renal development promotes features seen in HIV-associated collapsing FSGS,6,8 possibly in a Stat3-dependent manner.9 Induced overexpression of VEGF164 in podocytes of adult mice results in rapid and widespread diabetic nephropathy–like glomerular injury with proteinuria, glomerulomegaly, glomerular basement membrane thickening, mesangial expansion, and podocyte foot process effacement with loss of slit diaphragms.10 From these studies, a picture of VEGF glomerular biology begins to emerge—one requiring tight control over the timing and degree of VEGF expression during development and throughout adulthood. Other studies detailed how podocyte-derived VEGF contributes to endothelial and mesangial cell migration, survival, and differentiation. Conditions promoting either loss or gain of VEGF may lead to a variety of podocyte, endothelial, or mesangial defects, thereby rendering the glomerular filtration barrier dysfunctional. Although the glomerular chapter of the VEGF story is unfolding ever more clearly, new findings suggest the plot will thicken. VEGF is not merely a family of proangiogenic factors but can be subdivided into both proangiogenic and antiangiogenic subtypes. The former are distinguished from the latter by alternative splicing of terminal exon 8 that codes for six amino acids.11 VEGF isoforms that contain exon 8a are proangiogenic, whereas those spliced with 8b are antiangiogenic. Differences in amino acid charge properties between 8a and 8b alter the mature protein's binding and activation of VEGF receptors and likely account for the opposing effects on angiogenesis and endothelial permeability.12 Despite being identified in 2002, little is known regarding the glomerular actions of VEGF-8b–containing isoforms. Mathieson5 asked us to watch this space for future developments in the VEGF story. The latest includes a new study in this issue of JASN by Harper's group13 in Bristol. This group asked what would happen if an antiangiogenic isoform of VEGF (VEGF165b) were overexpressed in the podocytes of mice. Unlike overexpression of proangiogenic VEGF, which yields features reminiscent of HIV-collapsing FSGS,6 transgenic expression of VEGF165b in podocytes results in a filtration barrier that is less permeable to water. This phenotype associates with remarkable changes to endothelial fenestral ultrastructure that effectively reduce both density and number of open fenestrations, essentially plugging a proportion of the holes in the first level of the glomerular filtration barrier. These data are consistent with findings obtained with conditionally immortalized human glomerular endothelial cells, in which VEGF165b reduces monolayer permeability.14 These new findings raise a number of intriguing questions: Are glomerular levels of antiangiogenic VEGF modulated in human renal disease? If so, then is there an impact on the endothelial fenestral ultrastructure? Similarly, because of its antiangiogenic properties, novel strategies that implement the use of recombinant VEGF165b as anticancer therapeutics are on the horizon. Will they have an impact on the structure of the glomerular endothelial cell fenestrae? Perhaps they will but in a beneficial way. Salmon and colleagues15 have already reported that overexpression of renal VEGF165b alleviates increased water permeability and proteinuria of streptozotocin-induced diabetic mice. Such findings are encouraging in light of recent data showing that soluble Flt-gene therapy, targeting both pro- and antiangiogenic isoforms of VEGF, reduces proteinuria but exacerbates tubulointerstitial injury in diabetic mice.16 Moreover, most current VEGF antiangiogenic antibodies used in clinical practice, including bevacizumab,1 do not discriminate between exon 8a– and 8b–containing VEGF family members; however, the push is on to develop antibodies or small molecules that will target exon 8a VEGF to improve efficacy of treatment. Will VEGF 8a–specific antibodies or inhibitory small molecules preserve glomerular integrity as they shift the balance of VEGF signaling to an exclusively VEGF 8b pattern? How cells regulate the relative abundance of these splice variants is a key question. In differentiated podocytes, levels of exon 8b–containing VEGF predominate over exon 8a–containing isoforms17; however, podocytes in patients with Denys-Drash syndrome (Wilms' tumor, genital anomalies, and nephropathy) exhibit a reversal of this ratio.18 Harper and colleagues13 suggest that the Wilms' tumor-1 transcription factor, which is mutated in Denys-Drash, may influence the splicing of VEGF in podocytes because it acts as a repressor for a splice factor kinase and thereby downregulates VEGF165b. Last, given the opposing actions of VEGF exon 8a– and exon 8b–containing isoforms, it would be informative to test whether the glomerular phenotype of VEGF164-overexpressing mice can be rescued by intercrossing them with VEGF165b mice. Moreover, all of the VEGF knockout studies to date are deletions of both pro- and antiangiogenic VEGF isoforms, thereby complicating interpretations of the phenotypic role played by each splice variant. Thus, future studies could settle this issue by deleting individual splice variants by knock-in of nonsplicing exon 818containing constructs into the VEGF gene locus. The biology of the VEGF family is indeed complex. The nature of the interplay between the pro- and antiangiogenic classes of VEGF in glomerular development, maintenance, and function awaits more work. The challenge may be to develop approaches that regulate the balance between VEGF types. Taken together, these recent findings compel us to ask not only how much but what type of VEGF do we need. The answers to each of these questions are likely to be interdependent and could dictate future modes of therapy in both renal and nonrenal disease. Disclosures None.