Reduced Glomerular Filtration in Diabetes Is Attributable to Loss of Density and Increased Resistance of Glomerular Endothelial Cell Fenestrations.

BACKGROUND: Glomerular endothelial cell (GEnC) fenestrations are recognized as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease. METHODS: We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular Kf and GFR in diabetic mice (BTBR ob-/ob- ). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explored its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells. RESULTS: Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knockdown b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation. CONCLUSIONS: This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease.

Novel hemodynamic structures in the human glomerulus.

To investigate human glomerular structure under conditions of physiological perfusion, we have analyzed fresh and perfusion-fixed normal human glomeruli at physiological hydrostatic and oncotic pressures using serial resin section reconstruction, confocal, multiphoton, and electron microscope imaging. Afferent and efferent arterioles (21.5 ± 1.2 µm and 15.9 ± 1.2 µm diameter), recognized from vascular origins, lead into previously undescribed wider regions (43.2 ± 2.8 µm and 38.4 ± 4.9 µm diameter) we have termed vascular chambers (VCs) embedded in the mesangium of the vascular pole. Afferent VC (AVC) volume was 1.6-fold greater than efferent VC (EVC) volume. From the AVC, long nonbranching high-capacity conduit vessels ( n = 7) (Con; 15.9 ± 0.7 µm diameter) led to the glomerular edge, where branching was more frequent. Conduit vessels have fewer podocytes than filtration capillaries. VCs were confirmed in fixed and unfixed specimens with a layer of banded collagen identified in AVC walls by multiphoton and electron microscopy. Thirteen highly branched efferent first-order vessels (E1; 9.9 ± 0.4 µm diameter) converge on the EVC, draining into the efferent arteriole (15.9 ± 1.2 µm diameter). Banded collagen was scarce around EVCs. This previously undescribed branching topology does not conform to the branching of minimum energy expenditure (Murray's law), suggesting that even distribution of pressure/flow to the filtration capillaries is more important than maintaining the minimum work required for blood flow. We propose that AVCs act as plenum manifolds possibly aided by vortical flow in distributing and balancing blood flow/pressure to conduit vessels supplying glomerular lobules. These major adaptations to glomerular capillary structure could regulate hemodynamic pressure and flow in human glomerular capillaries.

VEGF-A165 b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney.

KEY POINTS: Progressive depletion of all vascular endothelial growth factor A (VEGF-A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over-expression of VEGF-A165 b only. VEGF-A165 b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub-podocyte space coverage, produced by VEGF-A depletion. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF-A165 b has opposite effects to VEGF-A165 on the expression of genes involved in endothelial cell migration and proliferation. ABSTRACT: Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF-A). However, little is known about the contribution of VEGF-A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF-A165 b (resulting from alternative usage of a 3' splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad-transgenic model, that over-expression of VEGF-A165 b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF-A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub-podocyte space coverage is reduced when VEGF-A is depleted, all of which are rescued in VEGF-A165 b over-expressors. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule-1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down-regulates genes involved in migration and proliferation of endothelial cells, otherwise up-regulated by the canonical isoform VEGF-A165 . The results of the present study indicate that manipulation of VEGF-A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function.

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. ABSTRACT: 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.

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.

The carboxyl terminus of VEGF-A is a potential target for anti-angiogenic therapy.

Anti-VEGF-A therapy has become a mainstay of treatment for ocular neovascularisation and in cancer; however, their effectiveness is not universal, in some cases only benefiting a minority of patients. Anti-VEGF-A therapies bind and block both pro-angiogenic VEGF-Axxx and the partial agonist VEGF-Axxxb isoforms, but their anti-angiogenic benefit only comes about from targeting the pro-angiogenic isoforms. Therefore, antibodies that exclusively target the pro-angiogenic isoforms may be more effective. To determine whether C-terminal-targeted antibodies could inhibit angiogenesis, we generated a polyclonal antibody to the last nine amino acids of VEGF-A165 and tested it in vitro and in vivo. The exon8a polyclonal antibody (Exon8apab) did not bind VEGF-A165b even at greater than 100-fold excess concentration, and dose dependently inhibited VEGF-A165 induced endothelial migration in vitro at concentrations similar to the VEGF-A antibody fragment ranibizumab. Exon8apab can inhibit tumour growth of LS174t cells implanted in vivo and blood vessel growth in the eye in models of age-related macular degeneration, with equal efficacy to non-selective anti-VEGF-A antibodies. It also showed that it was the VEGF-Axxx levels specifically that were upregulated in plasma from patients with proliferative diabetic retinopathy. These results suggest that VEGF-A165-specific antibodies can be therapeutically useful.

VEGF-A165b is an endogenous neuroprotective splice isoform of vascular endothelial growth factor A in vivo and in vitro.

Vascular endothelial growth factor (VEGF) A is generated as two isoform families by alternative RNA splicing, represented by VEGF-A165a and VEGF-A165b. These isoforms have opposing actions on vascular permeability, angiogenesis, and vasodilatation. The proangiogenic VEGF-A165a isoform is neuroprotective in hippocampal, dorsal root ganglia, and retinal neurons, but its propermeability, vasodilatatory, and angiogenic properties limit its therapeutic usefulness. In contrast, a neuroprotective effect of endogenous VEGF-A165b on neurons would be advantageous for neurodegenerative pathologies. Endogenous expression of human and rat VEGF-A165b was detected in hippocampal and cortical neurons. VEGF-A165b formed a significant proportion of total VEGF-A in rat brain. Recombinant human VEGF-A165b exerted neuroprotective effects in response to multiple insults, including glutamatergic excitotoxicity in hippocampal neurons, chemotherapy-induced cytotoxicity of dorsal root ganglion neurons, and retinal ganglion cells (RGCs) in rat retinal ischemia-reperfusion injury in vivo. Neuroprotection was dependent on VEGFR2 and MEK1/2 activation but not on p38 or phosphatidylinositol 3-kinase activation. Recombinant human VEGF-A165b is a neuroprotective agent that effectively protects both peripheral and central neurons in vivo and in vitro through VEGFR2, MEK1/2, and inhibition of caspase-3 induction. VEGF-A165b may be therapeutically useful for pathologies that involve neuronal damage, including hippocampal neurodegeneration, glaucoma diabetic retinopathy, and peripheral neuropathy. The endogenous nature of VEGF-A165b expression suggests that non-isoform-specific inhibition of VEGF-A (for antiangiogenic reasons) may be damaging to retinal and sensory neurons.

TNF-α-induced ICAM-1 expression and monocyte adhesion in human RPE cells is mediated in part through autocrine VEGF stimulation.

PURPOSE: Local inflammation at the RPE cell layer is associated with inflammatory cell migration and secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-α. TNF-α upregulates intercellular adhesion molecule (ICAM)-1 expression on the RPE, which allows lymphocyte function-associated antigen-1 (LFA-1) to bind on leukocytes that contribute to leukocyte adhesion at sites of inflammation. Vascular endothelial growth factor (VEGF)-A(165)b is generated by alternative splicing of VEGF-A in the terminal exon, exon 8. VEGF-A(165)b is cytoprotective and antiangiogenic, but its effects on inflammation have not yet been elucidated. Therefore, we tested the hypothesis that VEGF-A(165)b regulates TNF-α-induced ICAM-1 expression and monocyte adhesion in RPE cells. METHODS: Primary RPE cells were pretreated with TNF-α alone, VEGF-A(165)b alone, VEGF-A(165)b with anti-VEGF-A(165)b, or the VEGFR-2 inhibitor ZM323881 before exposure to TNF-α for 24 h. Western blotting and monocyte adhesion assays were performed. RESULTS: VEGF-A(165)b and ZM323881 inhibited TNF-α-induced upregulation of ICAM-1 in RPE cells. The effect of VEGF-A(165)b was neutralized by an antibody to VEGF-A(165)b. VEGF-A(165)b ameliorated TNF-α-induced monocyte-RPE adhesion. CONCLUSIONS: These findings indicate that VEGF-A(165)b inhibits TNF-α-mediated upregulation of ICAM-1 expression and increases monocyte-RPE cell adhesion, suggesting an anti-inflammatory property of VEGF-A(165)b in the eye.

The role of VEGF-A165b in trophoblast survival.

BACKGROUND: Pre-eclampsia remains a dominant cause of maternal and fetal mortality in developed countries. In a previous prospective study we identified a fall in the VEGF-A isoform VEGF-A165b in the plasma of patients in the first trimester to be a predictor of later pre-eclampsia. VEGF-A165b has been shown to have potent cytoprotective properties in many cell types. We therefore tested the hypothesis that VEGF-A165b may be cytoprotective for placental trophoblasts. METHODS: We used an immortalised first trimester trophoblast cell line exposed to chemical toxicity, and physiological (<2% O2) and atmospheric oxygen (21% O2) in the presence or absence of VEGF-A165b, angiogenic VEGF-A165a, a non-specific anti-VEGF-A blocking antibody (bevacizumab), or a specific anti-VEGF-A165b antibody. Cell viability and cytotoxicity were measured by trypan blue and LDH assay respectively. RESULTS: Under high (21%) levels of oxygen, trophoblast viability was increased, and cytotoxicity reduced by exogenous recombinant VEGF-A165b (p < 0.05, n = 10) or VEGF-A165a. The cytoprotective effect was not seen under lower (<2%) oxygen conditions, where VEGF-A165b was upregulated. However inhibition of VEGF-A with blocking antibodies (bevacizumab or anti-VEGF-A165b) had marked cytotoxic effects under low oxygen conditions presumably through the blockade of autocrine survival pathways. CONCLUSIONS: These results show that when trophoblasts are exposed to lower oxygen tensions (as they are early in the 1st trimester) endogenous VEGF-A165b contributes to their survival through an autocrine pathway. In contrast in high oxygen conditions exogenous VEGF-A isoforms have a greater effect on trophoblast survival.

Loss of the endothelial glycocalyx links albuminuria and vascular dysfunction.

Patients with albuminuria and CKD frequently have vascular dysfunction but the underlying mechanisms remain unclear. Because the endothelial surface layer, a meshwork of surface-bound and loosely adherent glycosaminoglycans and proteoglycans, modulates vascular function, its loss could contribute to both renal and systemic vascular dysfunction in proteinuric CKD. Using Munich-Wistar-Fromter (MWF) rats as a model of spontaneous albuminuric CKD, multiphoton fluorescence imaging and single-vessel physiology measurements revealed that old MWF rats exhibited widespread loss of the endothelial surface layer in parallel with defects in microvascular permeability to both water and albumin, in both continuous mesenteric microvessels and fenestrated glomerular microvessels. In contrast to young MWF rats, enzymatic disruption of the endothelial surface layer in old MWF rats resulted in neither additional loss of the layer nor additional changes in permeability. Intravenous injection of wheat germ agglutinin lectin and its adsorption onto the endothelial surface layer significantly improved glomerular albumin permeability. Taken together, these results suggest that widespread loss of the endothelial surface layer links albuminuric kidney disease with systemic vascular dysfunction, providing a potential therapeutic target for proteinuric kidney disease.

Mineralocorticoid receptor antagonism in diabetes reduces albuminuria by preserving the glomerular endothelial glycocalyx.

The glomerular endothelial glycocalyx (GEnGlx) forms the first part of the glomerular filtration barrier. Previously, we showed that mineralocorticoid receptor (MR) activation caused GEnGlx damage and albuminuria. In this study, we investigated whether MR antagonism could limit albuminuria in diabetes and studied the site of action. Streptozotocin-induced diabetic Wistar rats developed albuminuria, increased glomerular albumin permeability (Ps'alb), and increased glomerular matrix metalloproteinase (MMP) activity with corresponding GEnGlx loss. MR antagonism prevented albuminuria progression, restored Ps'alb, preserved GEnGlx, and reduced MMP activity. Enzymatic degradation of the GEnGlx negated the benefits of MR antagonism, confirming their dependence on GEnGlx integrity. Exposing human glomerular endothelial cells (GEnC) to diabetic conditions in vitro increased MMPs and caused glycocalyx damage. Amelioration of these effects confirmed a direct effect of MR antagonism on GEnC. To confirm relevance to human disease, we used a potentially novel confocal imaging method to show loss of GEnGlx in renal biopsy specimens from patients with diabetic nephropathy (DN). In addition, patients with DN randomized to receive an MR antagonist had reduced urinary MMP2 activity and albuminuria compared with placebo and baseline levels. Taken together, our work suggests that MR antagonists reduce MMP activity and thereby preserve GEnGlx, resulting in reduced glomerular permeability and albuminuria in diabetes.

VEGF165b overexpression restores normal glomerular water permeability in VEGF164-overexpressing adult mice.

Vascular endothelial growth factor (VEGF)-A, a family of differentially spliced proteins produced by glomerular podocytes, maintains glomerular filtration barrier function. The expression of VEGF molecules is altered in human nephropathy. We aimed to determine the roles of the angiogenic VEGF(164) isoform, and the antiangiogenic VEGF(165)b isoform in mature, adult glomeruli in vivo using conditional, inducible transgenic overexpression systems in mice. Podocyte-specific VEGF(164) overexpression (up to 100 days) was induced by oral administration of doxycycline to adult podocin-rtTA/TetO-VEGF(164) double transgenic mice. The consequences of simultaneous overexpression of VEGF(164) and VEGF(165)b were assessed in triple-transgenic podocin-rtTA/TetO-VEGF(164)/nephrin-VEGF(165)b mice. Persistent VEGF(164) overexpression did not cause proteinuria but did increase glomerular ultrafiltration coefficient between days 3 and 7. Despite persistently increased VEGF(164) levels, glomerular ultrafiltration coefficient normalized by day 14 and remained normal up to 100 days. Decreased subpodocyte space (SPS) coverage of the glomerular capillary wall accompanied increased glomerular hydraulic conductivity in VEGF(164)-overexpressing mice. The changes in glomerular ultrafiltration coefficient and SPS coverage induced by 7 days of overexpression of VEGF(164) were not present in triple transgenic VEGF(164) and VEGF(165)b overexpressing mice. These results indicate that 1) the adult mouse glomerulus is relatively resistant to induced VEGF(164) overexpression. VEGF(164) overexpression altered glomerular permeability but did not cause proteinuria in these mature, adult animals; 2) the SPS is a dynamic VEGF-responsive modulator of glomerular function; and 3) the balance of VEGF isoforms plays a critical role in the regulation of glomerular permeability. VEGF(165)b is capable of preventing VEGF(164)-induced changes in glomerular permeability and ultrastructure in vivo.

Regulation of vascular endothelial growth factor (VEGF) splicing from pro-angiogenic to anti-angiogenic isoforms: a novel therapeutic strategy for angiogenesis.

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF(165), and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF(165)b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF(165) and less VEGF(165)b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.

A human neutralizing antibody specific to Ang-2 inhibits ocular angiogenesis.

OBJECTIVE: Angiogenesis, a critical contributor to ocular as well as neoplastic diseases, is stimulated by endothelial production of angiopoietin-2 (Ang2). Our objective was to determine the requirement of ocular angiogenesis for Ang2 in animal models of disease. METHODS: We developed and compared the effect of a novel human Ang2 antibody with a pan-angiopoietin strategy on angiogenesis in ocular angiogenesis in animal models of oxygen-induced retinopathy, and laser photocoagulation and confirmed its efficacy in xenografted human colorectal tumors. RESULTS: Human anti-Ang2 and anti-angiopoietin1(Ang1)/Ang2 antibodies blocked colorectal carcinoma growth in immuno-compromised mice (p < 0.001, n = 6). Injection of 1 µg of Ang2 or Ang2/Ang1 antibody-inhibited angiogenesis in models of retinal (p < 0.001, n = 6), and choroidal neovascularization (p < 0.001, n = 11-13 per group) to levels similar to that with anti-VEGF antibodies. There was no difference between Ang2 specific and Ang1/Ang2 bi-specific antibodies. In vitro, Ang2 antibodies showed no cytotoxicity and did not inhibit endothelial cell migration or proliferation. CONCLUSION: Thus, human Ang2 antibodies are potentially therapeutic agents for ocular neovascularization in models of retinal and choroidal neovascularization, in the absence of VEGF inhibition.

Hypogammaglobulinemia and bronchiectasis in mycophenolate mofetil-treated renal transplant recipients: an emerging clinical phenomenon?

BACKGROUND: Mycophenolate mofetil (MMF) inhibits T- and B-cell proliferation and can cause acquired or secondary hypogammaglobulinemia. This finding and the subsequent development of opportunistic infection, including pneumonia, have been reported in patients receiving MMF. Chronic pulmonary infection and hypogammaglobulinemia predispose to bronchiectasis, and we aimed to establish the incidence and clinical pattern of this condition within our MMF-treated renal transplant population. METHODS: We performed a retrospective analysis of MMF-treated transplant recipients. Two hundred and eighty-nine patients were identified and for each, demographic, clinical, radiological and laboratory data from case notes and electronic records were collected. RESULTS: Twenty-three of 289 patients had recurrent severe chest infections (>2 episodes) between 12 and 95 months after the introduction of MMF. The mean age was 53 ± 17yr. Pulmonary lesions fulfilled clinical, radiographic and computerized tomography criteria for bronchiectasis in 7/289 (2.4%). All seven patients with bronchiectasis had low serum IgG levels. Three patients had sufficient samples available for B-cell phenotype analysis but no conclusive results emerged. No cases of post-transplant bronchiectasis were identified in our transplant population not receiving MMF. DISCUSSION: We report seven cases of bronchiectasis in renal transplant patients receiving MMF. We speculate that low immunoglobulin levels may contribute to the development of this significant pulmonary disease.

Balance of pro- versus anti-angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth.

Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro. VEGF(165) has been shown to be the predominant angiogenic isoform expressed in NB cell lines and tumours. In this study, we investigated the anti-angiogenic isoform of VEGF-A, generated from distal splice site selection in the terminal exon of VEGF (VEGF(165)b) and shown to be down-regulated in epithelial malignancies. The expression of both the pro- (VEGF(xxx)) and the anti-angiogenic (VEGF(xxx)b) isoforms was compared in a range of NB and ganglioneuroma (GN) tumours. Whereas VEGF(xxx)b and VEGF(xxx) were both expressed in GN, specific up-regulation of the VEGF(xxx) isoforms was seen in NB at RNA and protein levels. Highly tumourigenic NB cell lines also showed up-regulation of the angiogenic isoforms relative to VEGF(xxx)b compared to less tumourigenic cell lines, and the isoforms were differentially secreted. These results indicate that VEGF(165) is up-regulated in NB and that there is a difference in the balance of isoform expression from anti-angiogenic VEGF(165)b to angiogenic VEGF(165). Treatment with recombinant human VEGF(165)b significantly reduced the growth rate of established xenografts of SK-N-BE(2)-C cells (4.24 +/- 1.01 fold increase in volume) compared with those treated with saline (9.76 +/- 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF(165)b-treated tumours (19.4 +/- 1.9 vessels/mm(3)) in contrast to the saline-treated tumours (45.5 +/- 8.6 vessels/mm(3)). VEGF(165)b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK-N-BE(2)-C cells after 48 h. In conclusion, VEGF(165)b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of VEGF(165)b in NB and other paediatric malignancies.

VEGF in the lung: a role for novel isoforms.

Vascular endothelial cell growth factor (VEGF) is a potent mitogen and permogen that increases in the plasma and decreases in the alveolar space in respiratory diseases such as acute respiratory distress syndrome (ARDS). This observation has led to controversy over the role of this potent molecule in lung physiology and disease. We hypothesized that some of the VEGF previously detected in normal lung may be of the anti-angiogenic family (VEGF(xxx)b) with significant potential effects on VEGF bioactivity. VEGF(xxx)b protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGF(xxx)b was also detected by immunoblotting in normal lung tissue, primary human alveolar type II (ATII) cells, and bronchoalveolar lavage (BAL) fluid in normal subjects and by ELISA in normal, "at risk," and ARDS subjects. The effect of VEGF(165) and VEGF(165)b on both human primary endothelial cells and alveolar epithelial cell proliferation was assessed by [(3)H]thymidine uptake. We found that VEGF(165)b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF(121)b and VEGF(165)b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF(165) on both human primary endothelial cells and human alveolar epithelial cells was significantly inhibited by VEGF(165)b (P < 0.01). These data demonstrate that the novel VEGF(xxx)b family members are expressed in normal lung and are reduced in ARDS. A specific functional effect on primary human endothelial and alveolar epithelial cells has also been shown. These data suggest that the VEGF(xxx)b family may have a role in repair after lung injury.

Molecular diversity of VEGF-A as a regulator of its biological activity.

The vascular endothelial growth factor (VEGF) family of proteins regulates blood flow, growth, and function in both normal physiology and disease processes. VEGF-A is alternatively spliced to form multiple isoforms, in two subfamilies, that have specific, novel functions. Alternative splicing of exons 5-7 of the VEGF gene generates forms with differing bioavailability and activities, whereas alternative splice-site selection in exon 8 generates proangiogenic, termed VEGF(xxx), or antiangiogenic proteins, termed VEGF(xxx)b. Despite its name, emerging roles for VEGF isoforms on cell types other than endothelium have now been identified. Although VEGF-A has conventionally been considered to be a family of proangiogenic, propermeability vasodilators, the identification of effects on nonendothelial cells, and the discovery of the antiangiogenic subfamily of splice isoforms, has added further complexity to their regulation of microvascular function. The distally spliced antiangiogenic isoforms are expressed in normal human tissue, but downregulated in angiogenic diseases, such as cancer and proliferative retinopathy, and in developmental pathologies, such as Denys Drash syndrome and preeclampsia. Here, we examine the molecular diversity of VEGF-A as a regulator of its biological activity and compare the role of the pro- and antiangiogenic VEGF-A splice isoforms in both normal and pathophysiological processes.

The anti-angiogenic isoforms of VEGF in health and disease.

Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys-Drash syndrome and pre-eclampsia). Administration of recombinant VEGF(165)b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGF(xxx)b isoforms to the pro-angiogenic VEGF(xxx) isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGF(xxx)b isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.

New aspects of glomerular filtration barrier structure and function: five layers (at least) not three.

PURPOSE OF REVIEW: Three structures (glomerular endothelial fenestrae, glomerular basement membrane and podocyte interfoot process/slit diaphragms) have traditionally been considered as the major determinants of glomerular permeability. We review recent work demonstrating the functional importance of two additional layers: the endothelial surface layer (ESL) and the subpodocyte space (SPS). RECENT FINDINGS: Removing glomerular endothelial cell monolayer ESL in vitro significantly alters monolayer permeability, supporting previous in-vivo demonstrations of the importance of the ESL in determining glomerular permeability. Whether fenestral diaphragms are present to support the ESL in healthy adult glomeruli has been examined in a recent report. On the downstream side of the glomerular filtration barrier, the SPS is a recently described structure that covers approximately two-thirds of the barrier, has highly restrictive dimensions and contributes to the hydraulic resistance and ultrafiltration characteristics of the glomerulus. Different layers of the barrier have also been shown to influence the permeability characteristics of one another, either through biophysical interactions, or through the activities of ligand-receptor axes that cross the various layers of the barrier. SUMMARY: The structure and function of the glomerular filtration barrier remains an area of significant new discovery, and recent work continues to highlight the complexity of this dynamic multilayered watershed.