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1.  Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms 
The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumor cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function.
PMCID: PMC3399765  PMID: 22822423
2.  Topical Antiangiogenic SRPK1 Inhibitors Reduce Choroidal Neovascularization in Rodent Models of Exudative AMD 
Exudative AMD (wet AMD) is treated by monthly injection into the eye of anti-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.
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.
A novel disubstituted furan inhibitor was selective for the SRPK family of kinases and reduced expression of pro-angiogenic but not antiangiogenic VEGF isoforms. This inhibitor and previously identified SRPK inhibitors significantly reduced choroidal neovascularisation in vivo. Topical administration of SRPK inhibitors dose-dependently blocked CNV with an EC50 of 9 μM.
These results indicate that novel SRPK1 selective inhibitors could be a potentially novel topical (eye drop) therapeutic for wet AMD.
Exudative AMD results from excess angiogenic VEGF production. Switching VEGF splicing to the antiangiogenic isoforms by inhibiting serine-rich protein kinase-1 (SRPK1) can be achieved by topical application of novel SRPK1 inhibitors. This could result in novel treatments that do not require injection in wet AMD.
PMCID: PMC3771558  PMID: 23887803
VEGF; splicing; AMD
3.  Detection of VEGF-Axxxb Isoforms in Human Tissues 
PLoS ONE  2013;8(7):e68399.
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.
PMCID: PMC3729684  PMID: 23935865
4.  Association between VEGF splice isoforms and progression free survival in metastatic colorectal cancer patients treated with bevacizumab 
Bevacizumab improves survival for metastatic colorectal cancer patients with chemotherapy, but no proven predictive markers exist. The VEGF-A splice form, VEGF165b, 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 VEGF165b levels treated with bevacizumab.
Blinded tumor samples from the phase-III trial of FOLFOX4±bevacizumab were assessed for VEGF165b and VEGFtotal by immunohistochemistry and scored relative to normal tissue. A predictive index (PI) was derived from the ratio of VEGF165b:VEGFtotal 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) analysed based on PI relative to median ratio.
Unadjusted analysis of PFS showed significantly better outcome for individuals with VEGF165b:VEGFtotal ratio scores below median treated with FOLFOX4+bevacizumab compared to FOLFOX4 alone (median 8.0 months vs 5.2 months, p<0.02), but no effect of bevacizumab on PFS in patients with VEGF165b:VEGFtotal ratio >median (5.9 months vs 6.3 months). These findings held after adjustment for other clinical and demographic features. Overall survival (OS) was increased in Arm A (median 13.6 months) compared with Arm B (10.6 months) in the low VEGF165b group, but this did not reach statistical significance. There was no difference in the high VEGF165b:VEGFtotal group between FOLFOX+bevacizumab (10.8 months) and FOLFOX alone (11.3months).
Low VEGF165b:VEGFtotal ratio may be a predictive marker for bevacizumab in metastatic colorectal cancer, and individuals with high relative levels may not benefit.
PMCID: PMC3602975  PMID: 23104894
5.  WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing 
Cancer cell  2011;20(6):768-780.
Angiogenesis is regulated by the balance of pro-angiogenic VEGF165 and anti-angiogenic VEGF165b splice isoforms. Mutations in WT1, the Wilms’ tumour suppressor gene, suppress VEGF165b and cause abnormal gonadogenesis, renal failure and Wilms’ tumours. In WT1 mutant cells, reduced VEGF165b 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 pro-angiogenic. Altered VEGF splicing was reversed by wildtype WT1, knockdown of SRSF1 or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumour growth.
PMCID: PMC3574979  PMID: 22172722
6.  VEGF-A splicing 
Nature reviews. Cancer  2008;8(11):880-887.
The physiology of microvessels limits the growth and development of tumours. Tumours gain nutrients and excrete waste through growth-associated microvessels. New anticancer therapies target this microvasculature by inhibiting vascular endothelial growth factor A (VEGF-A) splice isoforms that promote microvessel growth. However, certain VEGF-A splice isoforms in normal tissues inhibit growth of microvessels. Thus, it is the VEGF-A isoform balance, which is controlled by mRNA splicing, that orchestrates angiogenesis. Here, we highlight the functional differences between the pro-angiogenic and the anti-angiogenic VEGF-A isoform families and the potential to harness the synthetic capacity of cancer cells to produce factors that inhibit, rather than aid, cancer growth.
PMCID: PMC2613352  PMID: 18923433
7.  VEGF-A165b Is Cytoprotective and Antiangiogenic in the Retina 
The alternative splice variant of VEGF, VEGF165b, is cytoprotective for endothelial and epithelial cells and is antiangiogenic, making it a new candidate for the treatment of ischemic retinopathies.
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-A165, 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-A165b, which is downregulated in diabetic retinopathy. Here the authors investigate the antiangiogenic activity of VEGF165b and its effect on retinal epithelial and endothelial cell survival.
VEGF-A165b 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-A165b.
VEGF-A165b dose dependently inhibited angiogenesis (IC50, 12.6 pg/eye) and retinal endothelial migration induced by 1 nM VEGF-A165 across monolayers in culture (IC50, 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-A165b injection, while inhibiting neovascular proliferation in the eye, reduced the ischemic insult in OIR (IC50, 2.6 pg/eye). Unlike bevacizumab, pegaptanib did not interact directly with VEGF-A165b.
The survival effects of VEGF-A165b signaling can protect the retina from ischemic damage. These results suggest that VEGF-A165b may be a useful therapeutic agent in ischemia-induced angiogenesis and a cytoprotective agent for retinal pigment epithelial cells.
PMCID: PMC2910648  PMID: 20237249
8.  Recombinant Human VEGF165b Inhibits Experimental Choroidal Neovascularization 
The alternative splice form of VEGF, VEGF-A165b, inhibits choroidal neovascularization at very low doses in mice, indicating that it may be an effective therapy for age-related macular degeneration, comparable with or better than existing anti-VEGF therapy.
Vascular endothelial growth factor (VEGF-A) is the principal stimulator of angiogenesis in wet age-related macular degeneration (AMD). However, VEGF-A is generated by alternate splicing into two families, the proangiogenic VEGF-Axxx family and the antiangiogenic VEGF-Axxxb family. It is the proangiogenic family that is responsible for the blood vessel growth seen in AMD.
To determine the role of antiangiogenic isoforms of VEGF-A as inhibitors of choroidal neovascularization, the authors used a model of laser-induced choroidal neovascularization in the mouse eye and investigated VEGF-A165b effects on endothelial cells and VEGFRs in vitro.
VEGF-A165b inhibited VEGF-A165–mediated endothelial cell migration with a dose effect similar to that of ranibizumab and bevacizumab and 200-fold more potent than that of pegaptanib. VEGF-A165b bound both VEGFR1 and VEGFR2 with affinity similar to that of VEGF-A165. After laser injury, mice were injected either intraocularly or subcutaneously with recombinant human VEGF-A165b. Intraocular injection of rhVEGF-A165b gave a pronounced dose-dependent inhibition of fluorescein leakage, with an IC50 of 16 pg/eye, neovascularization (IC50, 0.8 pg/eye), and lesion as assessed by histologic staining (IC50, 8 pg/eye). Subcutaneous administration of 100 μg twice a week also inhibited fluorescein leakage and neovascularization and reduced lesion size.
These results show that VEGF-A165b is a potent antiangiogenic agent in a mouse model of age-related macular degeneration and suggest that increasing the ratio of antiangiogenic-to-proangiogenic isoforms may be therapeutically effective in this condition.
PMCID: PMC2910649  PMID: 20237252
9.  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 VEGFxxx, or antiangiogenic proteins, termed VEGFxxxb. 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.
PMCID: PMC2929464  PMID: 19521900
VEGF; splicing; angiogenesis
10.  The anti-angiogenic isoforms of VEGF in health and disease 
Biochemical Society transactions  2009;37(Pt 6):1207-1213.
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 VEGF165b 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 VEGFxxxb isoforms to the pro-angiogenic VEGFxxx 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 VEGFxxxb 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.
PMCID: PMC2882696  PMID: 19909248
angiogenesis; carcinoma sample; Denys-Drash syndrome; human vitreous fluid; rheumatoid arthritis; vasculogenesis
11.  Therapeutic potential of manipulating VEGF splice isoforms in oncology 
Anti-angiogenic therapies currently revolve around targeting vascular endothelial growth factor-A (VEGF-A) or its receptors. These therapies are effective to some degree, but have low response rates and poor side-effect profiles. Part of these problems is likely to be due to their lack of specificity between pro- and anti-angiogenic isoforms, and their nonspecific effects on proactive, pleiotropic survival and maintenance roles of VEGF-A in endothelial and other cell types. An alternative approach, and one which has recently been shown to be effective in animal models of neovascularization in the eye, is to target the mechanisms by which the cell generates pro-angiogenic splice forms of VEGF-A, its receptors and, co-incidentally, by targeting the upstream processes, other oncogenes that have antagonistic splice isoforms. The concept here is to target the splicing mechanisms that control splice site choice in the VEGF-A mRNA. Recent evidence on the pharmacological possibilities of such splice factors is described.
PMCID: PMC2879319  PMID: 19519209
angiogenesis; anti-angiogenic; cancer; eye disease; splicing; VEGF; VEGF165b
12.  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 (VEGFxxxb) with significant potential effects on VEGF bioactivity. VEGFxxxb protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGFxxxb 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 VEGF165 and VEGF165b on both human primary endothelial cells and alveolar epithelial cell proliferation was assessed by [3H]thymidine uptake. We found that VEGF165b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF121b and VEGF165b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF165 on both human primary endothelial cells and human alveolar epithelial cells was significantly inhibited by VEGF165b (P < 0.01). These data demonstrate that the novel VEGFxxxb 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 VEGFxxxb family may have a role in repair after lung injury.
PMCID: PMC2886605  PMID: 20228180
ARDS; vascular endothelial growth factor
13.  Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms 
The Journal of Biological Chemistry  2009;285(8):5532-5540.
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 VEGF165, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF165b. 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 VEGF165 and less VEGF165b. 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.
PMCID: PMC2820781  PMID: 19906640
Growth Factors; RNA/Splicing; ASF/SF2; Angiogenesis; SRPK1; VEGF
14.  Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx 
Cardiovascular Research  2009;83(1):24-33.
In this study, we wished to determine whether angiopoietin-1 (Ang1) modified the permeability coefficients of non-inflamed, intact continuous, and fenestrated microvessels in vivo and to elucidate the underlying cellular mechanisms.
Methods and results
Permeability coefficients were measured using the Landis–Michel technique (in frog and rat mesenteric microvessels) and an oncopressive permeability technique (in glomeruli). Ang1 decreased water permeability (LP: hydraulic conductivity) in continuous and fenestrated microvessels and increased the retention of albumin (σ: reflection coefficient) in continuous microvessels. Endothelial glycocalyx is common to these anatomically distinct microvascular beds, and contributes to the magnitude of both LP and σ. Ang1 treatment increased the depth of endothelial glycocalyx in intact microvessels and increased the content of glycosaminoglycan of cultured microvascular endothelial cell supernatant. Ang1 also prevented the pronase-induced increase in LP (attributable to selective removal of endothelial glycocalyx by pronase) by restoration of glycocalyx at the endothelial cell surface. The reduction in permeability was inhibited by a cell transport inhibitor, Brefeldin.
Ang1 modifies basal microvessel permeability coefficients, in keeping with previous reports demonstrating reduced solute flux in inflamed vessels. Anatomical, biochemical, and physiological evidence indicates that modification of endothelial glycocalyx is a novel mechanism of action of Ang1 that contributes to these effects.
PMCID: PMC2695703  PMID: 19297368
Permeability; Angiopoietin-1; Glycocalyx; Microvessel; Glomerulus
15.  The Alternatively Spliced Anti-Angiogenic Family of VEGF Isoforms VEGFxxxbin Human Kidney Development 
Nephron. Physiology  2008;110(4):p57-p67.
Vascular endothelial growth factor (VEGF), required for renal development, is generated by alternative splicing of 8 exons to produce two families, pro-angiogenic VEGFxxx, formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGFxxxb, generated by distal splicing in exon 8 (exon 8b). VEGF165b, the first described exon 8b-containing isoform, antagonises VEGF165 and is anti-angiogenic in vivo.
Using VEGFxxxb-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF165b on renal endothelial and epithelial cells.
VEGFxxxb formed 45% of total VEGF protein in adult renal cortex, and VEGF165b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGFxxxb was expressed in the condensed vesicles of the metanephros, epithelial cells of the comma-shaped bodies, invading endothelial cells and epithelial cells of the S-shaped body, and in the immature podocytes. Expression reduced as the glomerulus matured.
These results show that the anti-angiogenic VEGFxxxb isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGFxxxb family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGFxxx isoforms.
PMCID: PMC2635558  PMID: 19039247
Kidney development; Vascular endothelial growth factor 165b; Angiogenesis
16.  Failure to upregulate VEGF165b in maternal plasma is a first trimester predictive marker for pre-eclampsia 
Pre-eclampsia is a pregnancy related condition characterised by hypertension, proteinuria and endothelial dysfunction. VEGF165b, formed by alternative splicing of vascular endothelial growth factor (VEGF) pre-mRNA inhibits VEGF165 mediated vasodilatation and angiogenesis, but has not been quantified in pregnancy. Enzyme-linked immunoassays (ELISA) were used to measure mean±SEM plasma VEGF165b, soluble endoglin (sEng) and soluble Flt1 (sFlt-1). At 12 weeks gestation the plasma VEGF165b concentration was significantly upregulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group 4.90 ±1.6 ng/ml, p<0.01 Mann-Whitney U test) compared with non-pregnant women (0.40±0.22ng/ml). In contrast, in patients who later developed pre-eclampsia VEGF165b levels were lower than in the normotensive group (0.467 ±0.21 ng/ml) but no greater than non-pregnant women. At term plasma VEGF165b concentrations was greater than normal in both pre-eclamptic (3.75 ±2.24 ng/ml) and normotensive pregnancies (10.6ng/ml±3.84 ng/ml p>0.1 compared with pre-eclampsia). Patients with a lower than median plasma VEGF165b at 12 weeks, had elevated soluble fms-like tyrosine kinase receptor 1 (sFlt-1) and soluble endoglin (sEng) pre-delivery. Concentrations of sFlt-1 (1.20±0.07 ng/ml and 1.27±0.18ng/ml) and sEng (4.4±0.18 vs 4.1±0.5) were similar at 12 weeks gestation in the normotensive and pre-eclamptic groups, respectively. Plasma VEGF165b levels were elevated in pregnancy, but this elevation is delayed in women that subsequently develop pre-eclampsia. Low VEGF165b may therefore be a clinically useful first trimester plasma marker for increased pre-eclampsia risk.
PMCID: PMC2635543  PMID: 18826376
vascular endothelial growth factor165b; pre-eclampsia; splice variant; plasma marker; vascular permeability; angiogenesis
17.  Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors 
Journal of cell science  2008;121(Pt 20):3487-3495.
Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in pro-angiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFα treatment favoured PSS (increasing VEGFxxx) whereas TGFβ1 favoured DSS, increasing VEGFxxxb levels. TGFβ1 induced DSS selection was prevented by inhibition of p38 MAPK and the Clk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3′UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro- versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFβ1-induced DSS selection, and identify SRp55 as a key regulatory splice factor.
PMCID: PMC2613349  PMID: 18843117
VEGF; VEGF165b; splicing; VEGFxxxb; SRp55; TGFβ1; IGF1; Clk1/sty (CLK1); CLK4
18.  A Role for the Endothelial Glycocalyx in Regulating Microvascular Permeability in Diabetes Mellitus 
Diabetic angiopathy is a major cause of morbidity and mortality in diabetes mellitus. Endothelial dysfunction and associated alterations in blood flow, pressure and permeability are widely accepted phenomena in the diabetic milieu and are understood to lead to microangiopathy. Despite the clinical importance of diabetic microangiopathy, the mechanisms of pathogenesis remain elusive. In particular, much is yet to be understood about the nature of the putative increased permeability with respect to diabetes. Microvessel permeability is intrinsically difficult to measure and a surrogate (solute or solvent flux) is usually reported, the measurement of which is hampered by haemodynamic factors, such as flow rate, hydrostatic pressure gradient, solute concentration and surface area available for exchange. Very few studies describing the measurement of permeability with respect to diabetes have controlled for all these factors. As a result, the nature of the increased microvessel permeability in diabetes mellitus and indeed its causes are poorly understood. Recent studies have shown that hyperglycaemia can alter the glycocalyx structure, and parallel findings have shown that the apparent increase in permeability demonstrated in hyperglycaemia may be due to an increase in the permeability of the vessels to water, and not an increase in protein permeability, an effect attributable to altered glycocalyx. This review focuses on the current understanding of microvascular permeability in terms of the endothelial glycocalyx- fibre-matrix theory, those methods used to determine permeability in the context of diabetes, and the more recent developments in our understanding of elevated microvascular permeability in the diabetic circulation.
PMCID: PMC2613348  PMID: 17906361
19.  VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis 
Cardiovascular research  2007;78(2):315-323.
Vascular endothelial growth factor-C (VEGF-C) has been shown to stimulate both angiogenesis and lymphangiogenesis in some but not all models where VEGF-C is over-expressed. Our aim was to investigate the interaction between lymphangiogenesis and angiogenesis in adult tissues regulated by VEGF-C and identify evidence of polarized growth of lymphatics driven by specialized cells at the tip of the growing sprout.
Methods and results
We used an adult model of lymphangiogenesis in the rat mesentery. The angiogenic effect of VEGF-C was markedly attenuated in the presence of a growing lymphatic network. Furthermore, we show that this growth of lymphatic vessels can occur both by recruitment of isolated lymphatic islands to a connected network and by filopodial sprouting. The latter is independent of polarized tip cell differentiation that can be generated all along lymphatic capillaries, independently of the proliferation status of the lymphatic endothelial cells.
These results both demonstrate a dependence of VEGF-C-mediated angiogenesis on lymphatic vascular networks and indicate that the mechanism of VEGF-C-mediated lymphangiogenesis is different from that of classical angiogenic mechanisms.
PMCID: PMC2613351  PMID: 18065770
Angiogenesis; Lymphangiogenesis; VEGF-C; Sprouting
20.  Arteriolar Genesis and Angiogenesis Induced by Endothelial Nitric Oxide Synthase Overexpression Results in a Mature Vasculature 
Generation of physiologically active vascular beds by delivery of combinations of growth factors offers promise for vascular gene therapy.
Methods and Results
In a mesenteric model of physiological angiogenesis, combining endothelial nitric oxide synthase (eNOS) (and hence NO production) with VEGF and angiopoietin-1 overexpression resulted in a more functional vascular phenotype than growth factor administration alone. eNOS gene delivery upregulated eNOS, VEGF, and Ang-1 to similar levels as gene transfer with VEGF or Ang-1. eNOS overexpression resulted in neovascularization to a similar extent as VEGF and Ang-1 combined, but not by sprouting angiogenesis. Whereas combining Ang-1 and VEGF increased both exchange vessels and conduit vessels, neither growth factor nor eNOS alone resulted in vessels with smooth muscle cell (SMC) coverage. In contrast, combining all three generated microvessels with SMCs (arteriolar genesis) and further increased functional vessels. Use of a vasodilator, prazosin, in combination with Ang1 and VEGF, but not alone, also generated SMC-positive vessels.
Coexpression of eNOS, VEGF, and Ang-1 results in a more mature vascularization of connective tissue, and generates new arterioles as well as new capillaries, and provides a more physiological therapeutic approach than single growth factor administration, by combining hemodynamic forces with growth factors.
PMCID: PMC2566940  PMID: 18497305
angiogenesis; arteriogenesis; VEGF; Ang-1; eNOS; pericyte; vascular smooth muscle
21.  Recombinant human VEGF165b protein is an effective anti-cancer agent in mice 
Tumour growth is dependent on angiogenesis, the key mediator of which is vascular endothelial growth factor-A (VEGF-A). VEGF-A exists as two families of alternatively spliced isoforms - pro-angiogenic VEGFxxx generated by proximal, and anti-angiogenic VEGFxxxb by distal splicing of exon 8. VEGF165b inhibits angiogenesis and is downregulated in tumours. Here, we show for the first time that administration of recombinant human VEGF165b inhibits colon carcinoma tumour growth and tumour vessel density in nude mice, with a terminal plasma half-life of 6.2 h and directly inhibited angiogenic parameters (endothelial sprouting, orientation and structure formation) in vitro. Intravenous injection of 125I-VEGF165b demonstrated significant tumour uptake lasting at least 24 h. No adverse effects on liver function or haemodynamics were observed. These results indicate that injected VEGF165b was taken up into the tumour as an effective anti-angiogenic cancer therapy, and provide proof of principle for the development of this anti-angiogenic growth factor splice isoform as a novel cancer therapy.
PMCID: PMC2565644  PMID: 18657413
VEGF; VEGF165b; Anti-angiogenesis; Cancer inhibition; Pharmacokinetics; Liver toxicity
22.  Failure to up-regulate VEGF165b in maternal plasma is a first trimester predictive marker for pre-eclampsia 
Pre-eclampsia is a pregnancy-related condition characterized by hypertension, proteinuria and endothelial dysfunction. VEGF165b, formed by alternative splicing of VEGF (vascular endothelial growth factor) pre-mRNA, inhibits VEGF165-mediated vasodilation and angiogenesis, but has not been quantified in pregnancy. ELISAs were used to measure means±S.E.M. plasma VEGF165b, sEng (soluble endoglin) and sFlt-1 (soluble fms-like tyrosine kinase-1). At 12 weeks of gestation, the plasma VEGF165b concentration was significantly up-regulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group, 4.90±1.6 ng/ml; P<0.01, as determined using a Mann-Whitney U test) compared with non-pregnant women (0.40±0.22 ng/ml). In contrast, in patients who later developed pre-eclampsia, VEGF165b levels were lower than in the normotensive group (0.467±0.209 ng/ml), but were no greater than non-pregnant women. At term, plasma VEGF165b concentrations were greater than normal in both pre-eclamptic (3.75±2.24 ng/ml) and normotensive (10.58 ng/ml±3.74 ng/ml; P>0.1 compared with pre-eclampsia) pregnancies. Patients with a lower than median plasma VEGF165b at 12 weeks had elevated sFlt-1 and sEng pre-delivery. Concentrations of sFlt-1 (1.20±0.07 and 1.27±0.18 ng/ml) and sEng (4.4±0.18 and 4.1±0.5 ng/ml) were similar at 12 weeks of gestation in the normotensive and pre-eclamptic groups respectively. Plasma VEGF165b levels were elevated in pregnancy, but this increase is delayed in women that subsequently develop pre-eclampsia. In conclusion, low VEGF165b may therefore be a clinically useful first trimester plasma marker for increased risk of pre-eclampsia.
PMCID: PMC2635543  PMID: 18826376
angiogenesis; plasma marker; pre-eclampsia; splice variant; vascular endothelial growth factor165b (VEGF165b); vascular permeability; AUC, area under the curve; BP, blood pressure; CV, coefficient of variation; EIA, enzyme immunoassay; cEIA, competitive EIA; Flt-1, fms-like tyrosine kinase-1; ROC, receiver operating characteristic; sEng, soluble endoglin; sFlt-1, soluble Flt-1; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor

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