Steven J Harper

University of Bristol, Bristol, England, United Kingdom

Are you Steven J Harper?

Claim your profile

Publications (94)451.33 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Targeting activating mutations in the proto-oncogene B-Raf, in melanoma, has led to increases in progression free survival. Treatment with vemurafenib, which inhibits the most common activating-mutated form of B-Raf (B-Raf(V600E)), eventually results in resistance to therapy. VEGF-A is the principal driver of angiogenesis in primary and metastatic lesions. The bioactivity of VEGF-A is dependent upon alternative RNA splicing and pro-angiogenic isoforms of VEGF-A are upregulated in many disease states dependent upon angiogenesis, including cancers. Using techniques including RT-PCR, Western blotting, ELISA and luciferase reporter assays, the effect of vemurafenib on proliferation, ERK1/2 phosphorylation and the levels of pro- and anti-angiogenic VEGF-A isoforms was investigated in melanoma cell types expressing either wild-type B-Raf or B-Raf(V600E), including a primary melanoma culture derived from a highly vascularised and active nodule taken from a patient with a V600E mutant melanoma. The primary melanoma culture was characterised and found to have reverted to wild-type B-Raf. In B-Raf(V600E) A375 cells ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA, total VEGF-A protein expression and VEGF-A 3'UTR activity were all decreased in a concentration-dependent manner by vemurafenib. Conversely vemurafenib treatment of wild-type B-Raf cells significantly increased ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA and total VEGF-A expression in a concentration-dependent manner. A switch to pro-angiogenic VEGF-A isoforms, with a concomitant upregulation of expression by increasing VEGF-A mRNA stability, may be an additional oncogenic and pathological mechanism in B-Raf(V600E) melanomas, which promotes tumor-associated angiogenesis and melanoma-genesis. We have also identified the genetic reversal of B-Raf(V600E) to wild-type in an active melanoma nodule taken from a V600E-positive patient and continued vemurafenib treatment for this patient is likely to have had a detrimental effect by promoting B-Raf(WT) activity.
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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. Copyright © 2014 by the American Society of Nephrology.
    Journal of the American Society of Nephrology 12/2014; DOI:10.1681/ASN.2014040350 · 9.47 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Angiogenesis is required for tumour growth and is induced principally by vascular endothelial growth factor A (VEGF-A). VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer (PCa). In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by serine-arginine protein kinase 1 (SRPK1). Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared with benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in PCa may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards the anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knockdown (KD). PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1-KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Small-molecule inhibitors of SRPK1 switched splicing towards the anti-angiogenic isoform VEGF165b in PC-3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of PCa. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumour angiogenesis by regulation of VEGF splicing can alter prostate tumour growth and supports further studies for the use of SRPK1 inhibition as a potential anti-angiogenic therapy in PCa.Oncogene advance online publication, 10 November 2014; doi:10.1038/onc.2014.360.
    Oncogene 11/2014; DOI:10.1038/onc.2014.360 · 8.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Angiogenesis 10/2014; DOI:10.1007/s10456-014-9444-3 · 4.41 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that by blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a- prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.
    Neurobiology of Disease 08/2014; 71. DOI:10.1016/j.nbd.2014.08.012 · 5.20 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    BMC Pregnancy and Childbirth 08/2014; 14(1):278. DOI:10.1186/1471-2393-14-278 · 2.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy.
    Molecular Oncology 08/2014; 9(1). DOI:10.1016/j.molonc.2014.07.017 · 5.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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)-A165b is generated by alternative splicing of VEGF-A in the terminal exon, exon 8. VEGF-A165b is cytoprotective and antiangiogenic, but its effects on inflammation have not yet been elucidated. Therefore, we tested the hypothesis that VEGF-A165b regulates TNF-α-induced ICAM-1 expression and monocyte adhesion in RPE cells.
    Molecular vision 06/2014; 20:781-9. · 2.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.
    PLoS ONE 07/2013; 8(7):e68399. DOI:10.1371/journal.pone.0068399 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A<sub>165</sub>a, and anti-angiogenic isoforms typified by VEGF-A<sub>165</sub>b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A<sub>165</sub>b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the imp
    PLoS ONE 07/2013; 8(7):e68399. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: PURPOSE. Exudative age related macular degeneration (wet AMD) is treated by monthly injection into the eye of anti-vascular endothelial growth factor (VEGF) proteins. VEGF is alternatively spliced to produce numerous isoforms that differ in angiogenic activity. Serine-rich protein kinase-1 (SRPK1) has been identified as a regulator of pro-angiogenic VEGF splicing by phosphorylating serine-rich splicing factor-1 (SRSF1), which binds to VEGF pre-mRNA. We tested the hypothesis that topical (eye drop) SRPK1 selective inhibitors could be generated that reduce pro-angiogenic isoforms, and prevent choroidal neovascularization in vivo. METHODS. Novel inhibitors were tested for SRPK inhibition in vitro, pro-angiogenic VEGF production in RPE cells by PCR and ELISA, and for inhibition of choroidal neovascularisation in mice and rats. RESULTS. A novel disubstituted furan inhibitor was selective for the SRPK family of kinases and reduced expression of pro-angiogenic but not anti-angiogenic VEGF isoforms. This inhibitor and previously identified SRPK inhibitors significantly reduced choroidal neovascularisation in vivo. Topical adminstration of SRPK inhibitors dose dependently blocked CNV with an EC50 of 9µM. CONCLUSIONS. These results indicate that novel SRPK1 selective inhibitors could be potential novel topical (eye drop) therapeutics for wet AMD.
    Investigative ophthalmology & visual science 07/2013; 54(9). DOI:10.1167/iovs.13-12422 · 3.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    American Journal Of Pathology 07/2013; 27(3). DOI:10.1016/j.ajpath.2013.05.031 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: PURPOSE. We tested the hypothesis that recombinant human VEGF-A165b and the serine arginine protein kinase (SRPK) inhibitor, SRPIN340, which controls splicing of the VEGF-A pre-mRNA, prevent neovascularisation in a rodent model of retinopathy of prematurity (ROP). METHODS. In the 50/10 oxygen-induced retinopathy (50/10 OIR) model that exposes newborn rats to repeated cycles of 24 h of 50% oxygen alternating with 24 h of 10% oxygen, pups received intraocular injections of SRPIN340, vehicle, VEGF165b, anti-VEGF antibody or saline. Wholemounts of retinas were prepared for isolectin immunohistochemistry, and pre-retinal or intravitreal neovascularisation (PRNV) determined by clock hour analysis. RESULTS. The anti-VEGF antibody (p<0.04), rhVEGF165b (p<0.001) and SRPIN340 (p<0.05) significantly reduced PRNV compared with control eyes. SRPIN340 reduced the expression of pro-angiogenic VEGF165 without affecting VEGF165b expression. CONCLUSIONS These results suggest that splicing regulation through selective downregulation of pro-angiogenic VEGF isoforms (via SRPK1 inhibition) or competitive inhibition of VEGF signalling by rhVEGF165b has the potential to be an effective alternative to potential cyto- and neuro- toxic anti-VEGF agents in the treatment of pathological neovascularisation in the eye.
    Investigative ophthalmology & visual science 06/2013; 54(8). DOI:10.1167/iovs.13-11634 · 3.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: PURPOSE: Bevacizumab improves survival for patients with metastatic colorectal cancer with chemotherapy, but no proven predictive markers exist. The VEGF-A splice form, VEGF(165)b, anti-angiogenic in animal models, binds bevacizumab. We tested the hypothesis that prolonged progression-free survival (PFS) would occur only in patients with low relative VEGF(165)b levels treated with bevacizumab.EXPERIMENTAL DESIGN: Blinded tumor samples from the phase III trial of FOLFOX4 ± bevacizumab were assessed for VEGF(165)b and VEGF(total) by immunohistochemistry and scored relative to normal tissue. A predictive index (PI) was derived from the ratio of VEGF(165)b:VEGF(total) for 44 samples from patients treated with FOLFOX + bevacizumab (arm A) and 53 samples from patients treated with FOLFOX4 (arm B), and PFS, and overall survival (OS) analyzed on the basis of PI relative to median ratio.RESULTS: Unadjusted analysis of PFS showed significantly better outcome for individuals with VEGF(165)b:VEGF(total) ratio scores below median treated with FOLFOX4 + bevacizumab compared with FOLFOX4 alone (median, 8.0 vs. 5.2 months; P < 0.02), but no effect of bevacizumab on PFS in patients with VEGF(165)b:VEGF(total) ratio >median (5.9 vs. 6.3 months). These findings held after adjustment for other clinical and demographic features. OS was increased in arm A (median, 13.6 months) compared with arm B (10.6 months) in the low VEGF(165)b group, but this did not reach statistical significance. There was no difference in the high VEGF(165)b:VEGF(total) group between FOLFOX + bevacizumab (10.8 months) and FOLFOX alone (11.3months).CONCLUSION: Low VEGF(165)b:VEGF(total) ratio may be a predictive marker for bevacizumab in metastatic colorectal cancer, and individuals with high relative levels may not benefit. Clin Cancer Res; 1-8. ©2012 AACR.
    Clinical Cancer Research 10/2012; DOI:10.1158/1078-0432.CCR-12-2223 · 8.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    AJP Renal Physiology 07/2012; 303(7):F1026-36. DOI:10.1152/ajprenal.00410.2011 · 4.42 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of the American Society of Nephrology 07/2012; 23(8):1339-50. DOI:10.1681/ASN.2012010017 · 9.47 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Angiogenesis and vascular regression are critical for the female ovulatory cycle. They enable progression and regression of follicular development, and corpora lutea formation and regression. Angiogenesis in the ovary occurs under the control of the vascular endothelial growth factor-A (VEGFA) family of proteins, which are generated as both pro-(VEGF(165)) and anti(VEGF(165)b)-angiogenic isoforms by alternative splicing. To determine the role of the VEGF(165)b isoforms in the ovulatory cycle, we measured VEGF(165)b expression in marmoset ovaries by immunohistochemistry and ELISA, and used transgenic mice over-expressing VEGF(165)b in the ovary. VEGF(165)b was expressed in the marmoset ovaries in granulosa cells and theca, and the balance of VEGF(165)b:VEGF(165) was regulated during luteogenesis. Mice over-expressing VEGF(165)b in the ovary were less fertile than wild-type littermates, had reduced secondary and tertiary follicles after mating, increased atretic follicles, fewer corpora lutea and generated fewer embryos in the oviduct after mating, and these were more likely not to retain the corona radiata. These results indicate that the balance of VEGFA isoforms controls follicle progression and luteogenesis, and that control of isoform expression may regulate fertility in mammals, including in primates.
    Reproduction 01/2012; 143(4):501-11. DOI:10.1530/REP-11-0091
  • [Show abstract] [Hide abstract]
    ABSTRACT: Angiogenesis is regulated by the balance of proangiogenic VEGF(165) and antiangiogenic VEGF(165)b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF(165)b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF(165)b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth.
    Cancer cell 12/2011; 20(6):768-80. DOI:10.1016/j.ccr.2011.10.016 · 23.89 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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. 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. 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. Thus, human Ang2 antibodies are potentially therapeutic agents for ocular neovascularization in models of retinal and choroidal neovascularization, in the absence of VEGF inhibition.
    Microcirculation (New York, N.Y.: 1994) 08/2011; 18(7):598-607. DOI:10.1111/j.1549-8719.2011.00120.x · 2.26 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pre-eclampsia is diagnosed by hypertension and proteinuria, probably caused by endothelial dysfunction, resulting in symptoms including oedema, inflammation and altered metabolism. Vascular endothelial growth factor A (VEGF-A) is detected at higher concentrations in plasma from patients with pre-eclampsia than in plasma from normotensive pregnant patients when determined by radioimmunoassay. This study tested the hypothesis that circulating VEGF-A in pre-eclamptic plasma is biologically active in vivo, and aimed to identify specific isoforms responsible for this activity. Plasma from pre-eclamptic (n = 17) and normotensive (n = 10) pregnant women was perfused into Rana mesenteric microvessels, and the subsequent change in microvascular permeability was measured using a single-vessel perfusion micro-occlusion technique. Pre-eclamptic but not normotensive plasma resulted in a 5.25 ± 0.8-fold acute increase in vascular permeability (P = 0.0003). This increase could be blocked by the incubation of plasma with bevacizumab, an antibody to VEGF-A (n = 7; P = 0012), and by VEGF-A receptor inhibition by SU5416 at doses specific to VEGF-A receptor-1 (VEGFR1), but not by the VEGF-A receptor-2 inhibitor, ZM323881. Although VEGF(165) b levels were not significantly altered in the PET samples, the increase in permeability was also inhibited by incubation of pre-eclamptic plasma with an inhibitory monoclonal antibody specific for VEGF₁₆₅b (n=6; P<0.01), or by the addition of placental growth factor 1 (PlGF-1; n = 3; P < 0.001). PlGF-1 was detected at lower concentrations in pre-eclamptic plasma than in normotensive plasma. These findings suggest that circulating VEGF-A levels in pre-eclampsia are biologically active because of a loss of repression of VEGFR1 signalling by PlGF-1, and VEGF₁₆₅b may be involved in the increased vascular permeability of pre-eclampsia.
    BJOG An International Journal of Obstetrics & Gynaecology 06/2011; 118(10):1253-61. DOI:10.1111/j.1471-0528.2011.02925.x · 3.86 Impact Factor

Publication Stats

3k Citations
451.33 Total Impact Points

Institutions

  • 2001–2015
    • University of Bristol
      • • School of Veterinary Sciences
      • • School of Physiology and Pharmacology
      • • School of Clinical Sciences
      Bristol, England, United Kingdom
  • 2012
    • Bristol Urological Institute
      Bristol, England, United Kingdom
  • 2007
    • Bristol Hospital
      Bristol, Connecticut, United States
  • 2004
    • Università degli Studi di Siena
      Siena, Tuscany, Italy