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.

Vascular endothelial growth factor (VEGF) is expressed by renal glomerular epithelial cells (podocytes) and is thought to be protective against nephrotoxic agents. VEGF has been shown to be an autocrine survival factor in neuropilin-1-positive, VEGF receptor-negative breast carcinoma cells. Normal human podocytes are also known to express neuropilin-1, VEGF, and are VEGF-R2 negative. Here, we investigated whether a similar VEGF autocrine loop may exist in podocytes. Podocyte cytosolic calcium concentration ([Ca²⁺]_i) was analyzed in primary cultured and conditionally immortalized podocytes using ratiometric fluorescence measurement. Cytotoxicity was determined by lactate dehydrogenase assay, proliferation by [³H]-thymidine incorporation, and cell counts by hemocytometric assay. VEGF decreased [Ca²⁺]_iin primary podocytes (from 179 ± 36 to 121 ± 25 nM, P < 0.05) and conditionally immortalized podocytes (from 95 ± 10 to 66 ± 8 nM, P < 0.02) in the absence of extracellular calcium. The type III receptor tyrosine-kinase inhibitor PTK787/ZK222584 abolished this reduction. VEGF increased podocyte [³H]-thymidine incorporation (3,349 ± 283 cpm, control 2,364 ± 301 cpm, P < 0.05) and cell number (4.5 ± 0.7 × 10⁴/ml, control 2.6 ± 0.5 × 10⁴/ml, P < 0.05) and decreased cytotoxicity (5.9 ± 0.7%, control 12 ± 3%, P < 0.05), whereas a monoclonal antibody to VEGF increased cytotoxicity. Electron microscopy of normal human glomeruli demonstrated that the glomerular VEGF is mostly podocyte cell membrane associated. These results indicate that one of the functions of VEGF secreted from podocytes may be to act as an autocrine factor on calcium homeostasis and cell survival.

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

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.

Key points: We have developed novel techniques for paired, direct, real‐time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability. Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel. The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth. Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real‐time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17–3.02 μm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 μm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time‐dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin.

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.