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.

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase.

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.

Overexpression of VEGF165b in podocytes reduces glomerular permeability.

The observation that therapeutic agents targeting vascular endothelial growth factor-A (VEGF-A) associate with renal toxicity suggests that VEGF plays a role in the maintenance of the glomerular filtration barrier. Alternative mRNA splicing produces the VEGF(xxx)b family, which consists of antiangiogenic peptides that reduce permeability and inhibit tumor growth; the contribution of these peptides to normal glomerular function is unknown. Here, we established and characterized heterozygous and homozygous transgenic mice that overexpress VEGF(165)b specifically in podocytes. We confirmed excess production of glomerular VEGF(165)b by reverse transcriptase-PCR, immunohistochemistry, and ELISA in both heterozygous and homozygous animals. Macroscopically, the mice seemed normal up to 18 months of age, unlike the phenotype of transgenic podocyte-specific VEGF(164)-overexpressing mice. Animals overexpressing VEGF(165)b, however, had a significantly reduced normalized glomerular ultrafiltration fraction with accompanying changes in ultrastructure of the glomerular filtration barrier on the vascular side of the glomerular basement membrane. These data highlight the contrasting properties of VEGF splice variants and their impact on glomerular function and phenotype.

Comparing Point of Care International Normalised Ratio testing with laboratory testing methods in a cardiac inpatient population.

AIMS AND OBJECTIVE: To compare agreement between International Normalised Ratio results from Point of Care testing with laboratory testing for cardiac inpatients receiving warfarin sodium. BACKGROUND: Availability of point of care technology for International Normalised Ratio testing offers considerable benefits to patients and health care staff across a range of context. DESIGN: Prospective comparison study. METHOD: Setting--Four cardiac wards in a regional referral hospital in New South Wales, Australia. Participants--50 cardiovascular inpatients receiving warfarin therapy, including those patients being converted from intravenous heparin sodium. Intervention-Point of Care International Normalised Ratio testing via finger prick using the CoaguChek®XS attended within one hour of laboratory International Normalised Ratio testing. Paired International Normalised Ratio results were compared using spearman rank and Mann-Whitney rank sum. Bland-Altman plots were used to demonstrate agreement. RESULTS: One hundred and seventeen blinded paired tests were carried out, 44 on patients receiving intravenous heparin. Laboratory and Point of Care International Normalised Ratio testing were highly significantly correlated (r = 0.953, p < 0.0001, n = 117). There was close agreement between Point of Care International Normalised Ratio and laboratory International Normalised Ratio results for patients receiving warfarin regardless of whether they were receiving heparin sodium. There was a mean bias of +0.2 units (95% CI 0.145-0.246). The presence of diabetes significantly reduced the difference between paired tests. Bias significantly increased above an International Normalised Ratio of 4.5 units. Ninety-seven per cent of all values fell between 20% limits of agreement after accounting for the mean bias of +0.2 units. CONCLUSION: Results indicated Point of Care International Normalised Ratio testing can be used for clinical decision making for cardiovascular inpatients receiving warfarin. Clinical guidelines need to be developed and tested in appropriate population groups and across different contexts, because of the potential for significant patient benefit. RELEVANCE TO CLINICAL PRACTICE: Point of Care International Normalised Ratio results in time and procedural efficiency, care responsiveness, cost saving, increased patient comfort and reduced handling errors (Pharmacotherapy 22; 2002: 677), as well as the potential for continuity of care.

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.

Mammary alveolar development during lactation is inhibited by the endogenous antiangiogenic growth factor isoform, VEGF165b.

Extensive tissue remodeling occurs in breast tissue during pregnancy, resulting in growth and development of the mammary gland associated with extensive vascular remodeling, which is thought to be dependent on vascular endothelial growth factor (VEGF). We show here that the endogenous antiangiogenic splice isoform of VEGF, VEGF(165)b, is normally expressed in nonlactating human and mouse breast, and is down-regulated in WT mice during lactation. To demonstrate the physiological role of VEGF(165)b in mammary tissue, we generated transgenic (TG) mice expressing VEGF(165)b, under the control of the mouse mammary tumor virus (MMTV) enhancer/promoter. These mice increase expression of VEGF(165)b in mammary tissue during mammary development. The offspring of TG mothers, but not TG fathers, die shortly after birth. The female TG mice have fewer blood vessels, less blood in the mammary tissue, and impaired alveolar coverage of the fat pad, and do not produce sufficient milk for nourishment of their pups. These findings demonstrate that endogenous overexpression of VEGF(165)b in the mammary gland inhibits physiological angiogenesis and that the regulation of the balance of VEGF isoforms is a requirement for mammary alveolar development and milk production. This study provides the first evidence for the role of endogenous antiangiogenic VEGF isoforms in normal physiology--their down-regulation is required for effective milk production.

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 VEGF(xxx), formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGF(xxx)b, generated by distal splicing in exon 8 (exon 8b). VEGF(165)b, the first described exon 8b-containing isoform, antagonises VEGF(165) and is anti-angiogenic in vivo. METHODS: Using VEGF(xxx)b-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF(165)b on renal endothelial and epithelial cells. RESULTS: VEGF(xxx)b formed 45% of total VEGF protein in adult renal cortex, and VEGF(165)b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGF(xxx)b 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 VEGF(xxx)b isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGF(xxx)b family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGF(xxx) isoforms.

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, VEGF(165)b, 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 VEGF(165)b binds VEGF receptor 2 with the same affinity as VEGF(165) but does not activate it or stimulate downstream signaling pathways. Moreover, it prevents VEGF(165)-mediated VEGF receptor 2 phosphorylation and signaling in cultured cells. Furthermore, we show, with two different in vivo angiogenesis models, that VEGF(165)b is not angiogenic and that it inhibits VEGF(165)-mediated angiogenesis in rabbit cornea and rat mesentery. Finally, we show that VEGF(165)b expressing tumors grow significantly more slowly than VEGF(165)-expressing tumors, indicating that a switch in splicing from VEGF(165) to VEGF(165)b can inhibit tumor growth. These results suggest that regulation of VEGF splicing may be a critical switch from an antiangiogenic to a proangiogenic phenotype.

VEGF-A165b is cytoprotective and antiangiogenic in the retina.

PURPOSE: A number of key ocular diseases, including diabetic retinopathy and age-related macular degeneration, are characterized by localized areas of epithelial or endothelial damage, which can ultimately result in the growth of fragile new blood vessels, vitreous hemorrhage, and retinal detachment. VEGF-A(165), the principal neovascular agent in ocular angiogenic conditions, is formed by proximal splice site selection in its terminal exon 8. Alternative splicing of this exon results in an antiangiogenic isoform, VEGF-A(165)b, which is downregulated in diabetic retinopathy. Here the authors investigate the antiangiogenic activity of VEGF(165)b and its effect on retinal epithelial and endothelial cell survival. METHODS: VEGF-A(165)b was injected intraocularly in a mouse model of retinal neovascularization (oxygen-induced retinopathy [OIR]). Cytotoxicity and cell migration assays were used to determine the effect of VEGF-A(165)b. RESULTS: VEGF-A(165)b dose dependently inhibited angiogenesis (IC(50), 12.6 pg/eye) and retinal endothelial migration induced by 1 nM VEGF-A(165) across monolayers in culture (IC(50), 1 nM). However, it also acts as a survival factor for endothelial cells and retinal epithelial cells through VEGFR2 and can stimulate downstream signaling. Furthermore, VEGF-A(165)b injection, while inhibiting neovascular proliferation in the eye, reduced the ischemic insult in OIR (IC(50), 2.6 pg/eye). Unlike bevacizumab, pegaptanib did not interact directly with VEGF-A(165)b. CONCLUSIONS: The survival effects of VEGF-A(165)b signaling can protect the retina from ischemic damage. These results suggest that VEGF-A(165)b may be a useful therapeutic agent in ischemia-induced angiogenesis and a cytoprotective agent for retinal pigment epithelial cells.

The endogenous anti-angiogenic family of splice variants of VEGF, VEGFxxxb, are down-regulated in pre-eclamptic placentae at term.

PET (pre-eclamptic toxaemia) has recently been linked with alterations in production of a VEGFR1 [VEGF (vascular endothelial growth factor) receptor 1] splice variant that acts as a circulating inhibitor. We have recently described a family of naturally occurring splice variants of VEGF, termed VEGFxxxb, that also appear to act as inhibitors of conventional VEGFxxx-mediated angiogenesis. To determine whether alteration in splicing of VEGF-VEGFR family members extended beyond VEGFR1, we investigated the effect of pre-eclampsia on placental VEGFxxxb mRNA and protein expression. VEGFxxx and VEGFxxxb mRNA and protein were both found in normal human term placentae. VEGFxxx protein formed the majority of the total VEGF protein (980+/-195 pg/mg), whereas VEGFxxxb (11.5 pg/mg) was found to form a small part of the total VEGF protein expression (1.5+/-0.24%). Evidence for VEGF165b, VEGF121b and VEGF145b expression was found. In pre-eclamptic placentae, there was a significant down-regulation of VEGFxxxb isoforms, but a small up-regulation of VEGFxxx isoforms. In normal placenta VEGFxxxb and VEGFxxx concentrations were positively correlated (r=0.69, P<0.02), whereas in pre-eclamptic placentae, there was a significant negative correlation between VEGFxxxb and VEGFxxx protein expression (r=-0.8, P<0.02), indicating that there was a significant uncoupling of the splicing regulation of the VEGF isoforms. Combined with previous studies showing increased soluble VEGFR1 isoforms in human pre-eclampsia, these data suggest that there may be a common mechanism in pre-eclampsia that involves dysregulation of mRNA splicing of members of the VEGF-VEGFR axis.