Rebecca R. Foster

VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant Mechanism of Action, In vivo Effect On Angiogenesis and Endogenous Protein Expression

Growth of new blood vessels (angiogenesis), required for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is up-regulated in all known solid tumors but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms have been universally described as proangiogenic cytokines. Here, we show that an endogenous splice variant, VEGF165b, is expressed as protein in normal cells and tissues and is circulating in human plasma. We also present evidence for a sister family of presumably inhibitory splice variants. Moreover, these isoforms are down-regulated in prostate cancer. We also show that VEGF165b binds VEGF receptor 2 with the same affinity as VEGF165 but does not activate it or stimulate downstream signaling pathways. Moreover, it prevents VEGF165-mediated VEGF receptor 2 phosphorylation and signaling in cultured cells. Furthermore, we show, with two different in vivo angiogenesis models, that VEGF165b is not angiogenic and that it inhibits VEGF165-mediated angiogenesis in rabbit cornea and rat mesentery. Finally, we show that VEGF165b expressing tumors grow significantly more slowly than VEGF165-expressing tumors, indicating that a switch in splicing from VEGF165 to VEGF165b can inhibit tumor growth. These results suggest that regulation of VEGF splicing may be a critical switch from an antiangiogenic to a proangiogenic phenotype.

Nephrotic Plasma Alters Slit Diaphragm–Dependent Signaling and Translocates Nephrin, Podocin, and CD2 Associated Protein in Cultured Human Podocytes

Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the slit diaphragm (SD) proteins nephrin, podocin, and CD2-associated protein (CD2AP) to function optimally. The effects of normal human plasma and nephrotic plasma on podocytes were tested, focusing particularly on the SD complex. With the use of a conditionally immortalized human podocyte cell line, it first was shown that exposure to normal and non-nephrotic human plasma leads to a concentration of nephrin, podocin, CD2AP, and actin at the cell surface. Next, the effects of plasma from patients with nephrotic conditions to non-nephrotic conditions were compared. When exposed to all nephrotic plasma samples (and a non-human serum control), nephrin podocin and CD2AP assumed a cytoplasmic distribution; nephrin and synaptopodin were selectively downregulated, and the relocation of nephrin induced by nephrotic plasma could be rescued back to the plasma membrane by co-incubation with non-nephrotic plasma. Furthermore, intracellular calcium signaling was altered by nephrotic plasma, which was mediated by tyrosine kinase phosphorylation. With the use of nephrin mutant human cell lines, it was shown that this signaling and translocation response to normal plasma is nephrin dependent. This work demonstrates that nephrotic plasma seems to be deficient in factors that act via the podocyte SD complex, which are essential in maintaining its physiologic function.

The Alternatively Spliced Anti-Angiogenic Family of VEGF Isoforms VEGFxxxb in Human Kidney Development

Background/Aim: Vascular endothelial growth factor (VEGF), required for renal development, is generated by alternative splicing of 8 exons to produce two families, pro-angiogenic VEGFxxx, formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGFxxxb, generated by distal splicing in exon 8 (exon 8b). VEGF165b, the first described exon 8b-containing isoform, antagonises VEGF165 and is anti-angiogenic in vivo. Methods: Using VEGFxxxb-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF165b on renal endothelial and epithelial cells. Results: VEGFxxxb formed 45% of total VEGF protein in adult renal cortex, and VEGF165b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGFxxxb was expressed in the condensed vesicles of the metanephros, epithelial cells of the comma-shaped bodies, invading endothelial cells and epithelial cells of the S-shaped body, and in the immature podocytes. Expression reduced as the glomerulus matured. Conclusion: These results show that the anti-angiogenic VEGFxxxb isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGFxxxb family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGFxxx isoforms.

Reactive oxygen species modulate the barrier function of the human glomerular endothelial glycocalyx.

Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S35 and 3H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.

High glucose causes dysfunction of the human glomerular endothelial glycocalyx

The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes.

Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.

Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca(2+)](i)) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod). Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca(2+)](i)) were calculated. 200microM FFA induced an increase in [Ca(2+)](i) in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca(2+)](i), which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca(2+)](i) activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca(2+)](i) in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6. In conclusion, FFA activates TRPC6 [Ca(2+)](i) signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.

Vascular Endothelial Growth Factor-C, a Potential Paracrine Regulator of Glomerular Permeability, Increases Glomerular Endothelial Cell Monolayer Integrity and Intracellular Calcium

We have previously reported expression of vascular endothelial growth factor (VEGF)-A and -C in glomerular podocytes and actions of VEGF-A on glomerular endothelial cells (GEnC) that express VEGF receptor-2 (VEGFR-2). Here we define VEGFR-3 expression in GEnC and investigate the effects of the ligand VEGF-C. Renal cortex and cultured GEnC were examined by microscopy, and both cell and glomerular lysates were assessed by Western blotting. VEGF-C effects on trans-endothelial electrical resistance and albumin flux across GEnC monolayers were measured. The effects of VEGF-C156S, a VEGFR-3-specific agonist, and VEGF-A were also studied. VEGF-C effects on intracellular calcium ([Ca2+]i) were measured using a fluorescence technique, receptor phosphorylation was examined by immunoprecipitation assays, and phosphorylation of myosin light chain-2 and VE-cadherin was assessed by blotting with phospho-specific antibodies. GEnC expressed VEGFR-3 in tissue sections and culture, and VEGF-C increased trans-endothelial electrical resistance in a dose-dependent manner with a maximal effect at 120 minutes of 6.8 Omega whereas VEGF-C156S had no effect. VEGF-C reduced labeled albumin flux by 32.8%. VEGF-C and VEGF-A increased [Ca2+]i by 15% and 39%, respectively. VEGF-C phosphorylated VEGFR-2 but not VEGFR-3, myosin light chain-2, or VE-cadherin. VEGF-C increased GEnC monolayer integrity and increased [Ca2+]i, which may be related to VEGF-C-S particular receptor binding and phosphorylation induction characteristics. These observations suggest that podocytes direct GEnC behavior through both VEGF-C and VEGF-A.

Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor α: a contributor to endothelial cell glycocalyx dysfunction

The endothelial surface glycocalyx is a hydrated mesh in which proteoglycans are prominent. It is damaged in diseases associated with elevated levels of tumor necrosis factor α (TNF‐α). We investigated the mechanism of TNF‐α‐induced disruption of the glomerular endothelial glycocalyx. We used conditionally immortalized human glomerular endothelial cells (GEnCs), quantitative PCR arrays, Western blotting, immunoprecipitation, immunofluorescence, and dot blots to examine the effects of TNF‐α. TNF‐α induced syndecan 4 (SDC4) mRNA up‐regulation by 2.5‐fold, whereas cell surface SDC4 and heparan sulfate (HS) were reduced by 36 and 30%, respectively, and SDC4 and sulfated glycosaminoglycan in the culture medium were increased by 52 and 65%, respectively, indicating TNF‐α‐induced shedding. Small interfering (siRNA) knockdown of SDC4 (by 52%) caused a corresponding loss of cell surface HS of similar magnitude (38%), and immunoprecipitation demonstrated that SDC4 and HS are shed as intact proteoglycan ectodomains. All of the effects of TNF‐α on SDC4 and HS were abrogated by the metalloproteinase (MMP) inhibitor batimastat. Also abrogated was the associated 37% increase in albumin passage across GEnC monolayers. Specific MMP9 knockdown by siRNA similarly blocked TNF‐α effects. SDC4 is the predominant HS proteoglycan in the GEnC glycocalyx. TNF‐α‐induced MMP9‐mediated shedding of SDC4 is likely to contribute to the endothelial glycocalyx disruption observed in diabetes and inflammatory states.—Ramnath, R., Foster, R. R., Qiu, Y., Cope, G., Butler, M. J., Salmon, A. H., Mathieson, P. W., Coward, R. J., Welsh, G. I., Satchell, S. C., Matrix metalloproteinase 9‐mediated shedding of syndecan 4 in response to tumor necrosis factor α: a contributor to endothelial cell glycocalyx dysfunction. FASEB J. 28, 4686–4699 (2014). www.fasebj.org

The importance of cellular VEGF bioactivity in the development of glomerular disease

The bioactivity of glomerular VEGF (or activity of available VEGF) is critical to the physiological maintenance of the glomerular filtration barrier. Disturbances in glomerular VEGF expression have been linked to numerous glomerulopathies, highlighting its importance in disease progression within the kidney. However, the changes in expression are not consistent between conditions; enhanced expression sometimes appears to have a renoprotective effect, yet at other times it appears destructive. Also, the level of expression can change with the progression of disease. This review focuses on how other cellular factors, such as TGF-β and nitric oxide, work in concert to affect the bioactivity, which is not necessarily the same as the expression of VEGF, in different glomerulopathies and attempts to explain some of the paradoxes between glomerulopathies. In conclusion, the bioactivity of glomerular VEGF is regulated by many factors that are themselves moderated by changes in the local glomerular environment, such as mechanical strain and hyperglycaemia. Thus, to understand VEGF signalling in glomerular disease progression, we must examine it in the context of other appropriate cellular factors.