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1.  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.
doi:10.1371/journal.pone.0068399
PMCID: PMC3729684  PMID: 23935865
2.  Association between VEGF splice isoforms and progression free survival in metastatic colorectal cancer patients treated with bevacizumab 
PURPOSE
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.
METHODS
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.
RESULTS
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).
CONCLUSION
Low VEGF165b:VEGFtotal ratio may be a predictive marker for bevacizumab in metastatic colorectal cancer, and individuals with high relative levels may not benefit.
doi:10.1158/1078-0432.CCR-12-2223
PMCID: PMC3602975  PMID: 23104894
3.  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.
doi:10.1038/nrc2505
PMCID: PMC2613352  PMID: 18923433
4.  VEGF-Mediated Elevated Intracellular Calcium and Angiogenesis in Human Microvascular Endothelial Cells In Vitro Are Inhibited by Dominant Negative TRPC6 
Objective
Vascular endothelial growth factor (VEGF)-induced vascular permeability has been shown to be dependent on calcium influx, possibly through a transient receptor potential cation channel (TRPC)-mediated cation channel with properties of the TRPC3/6/7 subfamily. To investigate further the involvement of this subfamily, we determined the effects of dominant negative TRPC6 overexpression on VEGF-mediated changes of human microvascular endothelial cell (HMVEC) calcium, proliferation, migration, and sprouting.
Methods
Cytoplasmic calcium concentration was estimated by fura-2 fluorescence spectrophotometry, migration by Boyden chamber assay, sprouting by immunofluorescence imaging of stimulated endothelial cells, and proliferation by flow cytometry.
Results
Overexpression of a dominant negative TRPC6 construct in HMVECs inhibited the VEGF-mediated increases in cytosolic calcium, migration, sprouting, and proliferation. In contrast, overexpression of a wild-type TRPC6 construct increased the proliferation and migration of HMVECs.
Conclusions
TRPC6 is an obligatory component of cation channels required for the VEGF-mediated increase in cytosolic calcium and subsequent downstream signaling that leads to processes associated with angiogenesis.
doi:10.1080/10739680802220323
PMCID: PMC2635545  PMID: 18800249
VEGF; TRPC6; calcium; angiogenesis
5.  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.
doi:10.1039/C0SC00297F
PMCID: PMC3399765  PMID: 22822423
6.  An unexpected tail of VEGF and PlGF in pre-eclampsia 
Biochemical Society transactions  2011;39(6):1576-1582.
Pre-eclamptic toxaemia (PET), characterised by pregnancy related hypertension and proteinuria, due to widespread endothelial dysfunction, is a primary cause of maternal morbidity. Altered circulating factors, particularly the VEGF family of proteins and their receptors, are thought to be key contributors to this disease. Plasma from patients with PET induces numerous cellular and physiological changes in endothelial cells indicating the presence of a circulating imbalance of the normal plasma constituents. These have been narrowed down to macromolecules of the VEGF family of proteins and receptors. It has been shown that responses of endothelial cells in intact vessels to plasma from patients with pre-eclampsia is VEGF dependent. It has recently been shown that this may be specific to the VEGF165b isoform, and blocked by addition of recombinant human PlGF. Taken together with results that show that sVEGFR1 levels are insufficient to bind VEGF-A in human plasma from patients with pre-eclampsia, and that other circulating macromolecules bind but do not inactivate VEGF-A, suggest that novel hypotheses involving altered bioavailability of VEGF isoforms resulting from either reduced, or bound PlGF, or increased sVEGFR1 increasing biological activity of circulating plasma could be tested. This suggests that knowing how to alter the balance of VEGF family members could prevent endothelial activation, and potentially some symptoms, of pre-eclampsia.
doi:10.1042/BST20110671
PMCID: PMC3399770  PMID: 22103490
7.  CCR7 mediates directed growth of melanomas towards lymphatics 
Objective
To determine whether chemotactic-metastasis, the preferential growth of melanomas towards areas of high lymphatic density, is CCL21/CCR7 dependent in vivo. Lymphatic endothelial cells (LECs) produce the chemokine CCL21. Metastatic melanoma cells express CCR7, its receptor, and exhibit chemotactic-metastasis, whereby metastatic cells recognise and grow towards areas of higher lymphatic density.
Methods
We used two in vivo models of directional growth towards depots of LECs of melanoma cells over-expressing CCR7. Injected LEC were tracked by intravital fluorescence microscopy, and melanoma growth by bioluminescence.
Results
Over-expression of the chemokine receptor CCR7 enables non-metastatic tumour cells to recognise and grow towards LECs (3.9 fold compared with control), but not blood endothelial cells (0.9 fold) in vitro and in vivo, in the absence of increased lymphatic clearance. Chemotactic metastasis was inhibited by a CCL21 neutralising antibody (4-17% of control). Furthermore, CCR7 expression in mouse B16 melanomas resulted in in-transit metastasis (50-100% of mice) that was less often seen with control tumours (0-50%) in vivo.
Conclusion
These results suggest that recognition of LEC by tumours expressing receptors for lymphatic specific ligands contributes towards the identification and invasion of lymphatics by melanoma cells, and provides further evidence for a chemotactic metastasis model of tumour spread.
doi:10.1111/j.1549-8719.2010.00074.x
PMCID: PMC3287291  PMID: 21166932
lymphatic endothelial cells; melanoma; metastasis; CCR7; lymph flow
8.  Topical Antiangiogenic SRPK1 Inhibitors Reduce Choroidal Neovascularization in Rodent Models of Exudative AMD 
Purpose.
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.
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 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.
Conclusions.
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.
doi:10.1167/iovs.13-12422
PMCID: PMC3771558  PMID: 23887803
VEGF; splicing; AMD
9.  Chemotrap-1: an engineered soluble receptor that blocks chemokine-induced migration of metastatic cancer cells in vivo 
Cancer research  2010;70(20):8138-8148.
Cancer and dendritic cells recognise and migrate towards chemokines secreted from lymphatics, and use this mechanism to invade the lymphatic system, and cancer cells, metastasise through it. The lymphatic secreted chemokine ligand CCL21 has been identified as a key regulatory molecule in the switch to a metastatic phenotype in melanoma and breast cancer cells. However, it is not known whether CCL21 inhibition is a potential therapeutic strategy for inhibition of metastasis. Here, we describe an engineered CCL21 soluble inhibitor, Chemotrap-1, which inhibits migration of metastatic melanoma cells in vivo. Two-hybrid, pull down and co-immunoprecipitation assays allowed us to identify a naturally occurring human zinc finger protein with CCL21 chemokine-binding properties. Further analyses revealed a short peptide (~ 70 amino-acids), with a predicted coiled-coil structure, which is sufficient for association with CCL21. This CCL21 chemokine-binding peptide was then fused to the Fc region of human IgG1 to generate Chemotrap-1, a human chemokine-binding Fc-fusion protein. Surface plasmon resonance (SPR) and chemotaxis assays showed that Chemotrap-1 binds CCL21 and inhibits CCL21-induced migration of melanoma cells in vitro with sub nM affinity. In addition, Chemotrap-1 blocked migration of melanoma cells towards lymphatic endothelial cells in vitro and in vivo. Finally, Chemotrap-1 strongly reduced lymphatic invasion, tracking and metastasis of CCR7 expressing melanoma cells in vivo. Together, these results show that CCL21 chemokine inhibition by Chemotrap-1 is a potential therapeutic strategy for metastasis, and provide further support for the hypothesis that lymphatic mediated metastasis is a chemokine-dependent process.
doi:10.1158/0008-5472.CAN-10-0175
PMCID: PMC3034641  PMID: 20736366
CCL21; CCR7; metastasis; melanoma; lymphatic
10.  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.
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-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.
Methods.
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.
Results.
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.
Conclusions.
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.
doi:10.1167/iovs.09-4296
PMCID: PMC2910648  PMID: 20237249
11.  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.
Purpose.
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.
Methods.
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.
Results.
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.
Conclusions.
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.
doi:10.1167/iovs.09-4360
PMCID: PMC2910649  PMID: 20237252
12.  Neurotrophin-3 Is a Novel Angiogenic Factor Capable of Therapeutic Neovascularization in a Mouse Model of Limb Ischemia 
Objective
To investigate the novel hypothesis that neurotrophin-3 (NT-3), an established neurotrophic factor that participates in embryonic heart development, promotes blood vessel growth.
Methods and Results
We evaluated the proangiogenic capacity of recombinant NT-3 in vitro and of NT-3 gene transfer in vivo (rat mesenteric angiogenesis assay and mouse normoperfused adductor muscle). Then, we studied whether either transgenic or endogenous NT-3 mediates postischemic neovascularization in a mouse model of limb ischemia. In vitro, NT-3 stimulated endothelial cell survival, proliferation, migration, and network formation on the basement membrane matrix Matrigel. In the mesenteric assay, NT-3 increased the number and size of functional vessels, including vessels covered with mural cells. Consistently, NT-3 overexpression increased muscular capillary and arteriolar densities in either the absence or the presence of ischemia and improved postischemic blood flow recovery in mouse hind limbs. NT-3–induced microvascular responses were accompanied by tropomyosin receptor kinase C (an NT-3 high-affinity receptor) phosphorylation and involved the phosphatidylinositol 3-kinase–Akt kinase–endothelial nitric oxide synthase pathway. Finally, endogenous NT-3 was shown to be essential in native postischemic neovascularization, as demonstrated by using a soluble tropomyosin receptor kinase C receptor domain that neutralizes NT-3.
Conclusion
Our results provide the first insight into the proangiogenic capacity of NT-3 and propose NT-3 as a novel potential agent for the treatment of ischemic disease.
doi:10.1161/ATVBAHA.109.205468
PMCID: PMC2881585  PMID: 20360537
angiogenesis; endothelial cells; limb ischemia; neurotrophin-3; PI3K/Akt/eNOS
13.  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.
doi:10.1080/10739680902997333
PMCID: PMC2929464  PMID: 19521900
VEGF; splicing; angiogenesis
14.  Vascular Endothelial Growth Factors and vascular permeability 
Cardiovascular research  2010;87(2):262-271.
VEGFs (VEGFs) are key regulators of permeability. The principal evidence behind how they increase vascular permeability in vivo and in vitro, and the consequences of that increase, are here addressed. Detailed analysis of the published literature showed that in vivo and in vitro data on VEGF mediated permeability differed in its time course, but had common involvement of many specific signalling pathways, in particular VEGF-Receptor-2 activation, calcium influx through transient receptor potential channels, activation of phospholipase C gamma and downstream activation of nitric oxide synthase. Downstream of endothelial-nitric oxide synthase appears to involve the guanylyl cyclase mediated activation of the rho-rac pathway and subsequent involvement of junctional signalling proteins such as vascular endothelial-cadherin and the tight junctional proteins zona occludens and occludin linked to the actin cytoskeleton. The signalling appears to be co-ordinated through spatial organisation of the cascade into a signalplex, and arguments for why this may be important are considered. Many proteins have been identified to be involved in the regulation of vascular permeability by VEGF, but still the mechanisms through which these are thought to interact to control permeability are dependent on the experimental system, and a synthesis of existing data reveals that in intact vessels the co-ordination of the pathways is still not understood.
doi:10.1093/cvr/cvq105
PMCID: PMC2895541  PMID: 20400620
VEGF; vascular permeability; calcium; capillary; endothelium
15.  Functional distinctions in cytosolic calcium regulation between cells of the glomerular filtration barrier 
Cell Calcium  2010;48(1-3):44-53.
The importance of intracellular calcium ([Ca2+]i) regulation in the glomerular filtration barrier (GFB) has recently been highlighted by mutations in the cation channel TRPC6, resulting in a renal-specific phenotype. We examined the effects of FFA, a tool that can activate TRPC6, on [Ca2+]i in human conditionally immortalised glomerular endothelial cells (ciGEnC) and human podocytes (ciPod) that form the GFB. Changes in [Ca2+]i stimulated by FFA were measured in Fura 2-AM loaded cells. In GEnC, cell activation by FFA was dependent on external Ca2+, yet in ciPod it was not. Depletion of internal Ca2+ stores with thapsigargin did not affect cell activation by FFA in ciGEnC, but inhibited it in ciPod in a nephrin-dependent manner, demonstrated using nephrin deficient (ND) ciPod in conjunction with nephrin rescue experiments. FFA induced [Ca2+]i store release in ciPod, but not in ciGEnC or ND ciPod. In parallel, there were differences in the localisation of overexpressed TRPC6 between ciGEnC and ciPod. Furthermore, co-transfection of nephrin with TRPC6 in HEK293 cells reduced the FFA-induced increase in [Ca2+]i and nephrin clustering altered TRPC6 distribution. In conclusion, cell activation by FFA in podocytes stimulates the opening of a Ca2+ channel, probably TRPC6, in a nephrin-dependent manner with a different activation profile to GEnC.
doi:10.1016/j.ceca.2010.06.005
PMCID: PMC2937225  PMID: 20674014
Flufenamic acid; TRPC6; Glomerular endothelial cells; Podocytes; Calcium
16.  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.
doi:10.1042/BST0371207
PMCID: PMC2882696  PMID: 19909248
angiogenesis; carcinoma sample; Denys-Drash syndrome; human vitreous fluid; rheumatoid arthritis; vasculogenesis
17.  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.
doi:10.2217/fon.09.33
PMCID: PMC2879319  PMID: 19519209
angiogenesis; anti-angiogenic; cancer; eye disease; splicing; VEGF; VEGF165b
18.  Measurement of Angiogenic Phenotype by Use of Two-Dimensional Mesenteric Angiogenesis Assay 
Successful therapeutic angiogenesis requires an understanding of how the milieu of growth factors available combine to form a mature vascular bed. This requires a model in which multiple physiological and cell biological parameters can be identified. The adenoviral-mediated mesenteric angiogenesis assay as described here is ideal for that purpose. Adenoviruses expressing growth factors (vascular endothelial growth factor [VEGF] and angiopoietin 1 [Ang-1]) were injected into the mesenteric fat pad of adult male Wistar rats. The clear, thin, and relatively avascular mesenteric panel was used to measure increased vessel perfusion by intravital microscopy. In addition, high-powered microvessel analysis was carried out by immunostaining of features essential for the study of angiogenesis (endothelium, pericyte, smooth muscle cell area, and proliferation), allowing functional data to be obtained in conjunction with high-power microvessel ultrastructural analysis. A combination of individual growth factors resulted in a distinct vascular phenotype from either factor alone, with all treatments increasing the functional vessel area. VEGF produced shorter, narrow, highly branched, and sprouting vessels with normal pericyte coverage. Ang-1 induced broader, longer neovessels with no apparent increase in branching or sprouting. However, Ang-1-induced blood vessels displayed a significantly higher pericyte ensheathment. Combined treatment resulted in higher perfusion, larger and less-branched vessels, with normal pericyte coverage, suggesting them to be more mature. This model can be used to show that Ang-1 and VEGF use different physiological mechanisms to enhance vascularisation of relatively avascular tissue.
PMCID: PMC2879323  PMID: 19301676
Angiogenesis; angiopoietin 1; pericyte; VEGF
19.  Vascular endothelial growth factors and vascular permeability 
Cardiovascular Research  2010;87(2):262-271.
Vascular endothelial growth factors (VEGFs) are key regulators of permeability. The principal evidence behind how they increase vascular permeability in vivo and in vitro and the consequences of that increase are addressed here. Detailed analysis of the published literature has shown that in vivo and in vitro VEGF-mediated permeability differs in its time course, but has common involvement of many specific signalling pathways, in particular VEGF receptor-2 activation, calcium influx through transient receptor potential channels, activation of phospholipase C gamma and downstream activation of nitric oxide synthase. Pathways downstream of endothelial nitric oxide synthase appear to involve the guanylyl cyclase-mediated activation of the Rho–Rac pathway and subsequent involvement of junctional signalling proteins such as vascular endothelial cadherin and the tight junctional proteins zona occludens and occludin linked to the actin cytoskeleton. The signalling appears to be co-ordinated through spatial organization of the cascade into a signalplex, and arguments for why this may be important are considered. Many proteins have been identified to be involved in the regulation of vascular permeability by VEGF, but still the mechanisms through which these are thought to interact to control permeability are dependent on the experimental system, and a synthesis of existing data reveals that in intact vessels the co-ordination of the pathways is still not understood.
doi:10.1093/cvr/cvq105
PMCID: PMC2895541  PMID: 20400620
VEGF; Vascular permeability; Calcium; Capillary; Endothelium
20.  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.
doi:10.1152/ajplung.00353.2009
PMCID: PMC2886605  PMID: 20228180
ARDS; vascular endothelial growth factor
21.  Critical Role of Tissue Kallikrein in Vessel Formation and Maturation 
Objective
Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function polymorphic variant, rs5515 (R53H), which possesses reduced kinin-forming activity.
Methods and Results
Here, we demonstrate that tissue kallikrein knockout mice (KLK1−/−) show impaired muscle neovascularization in response to hindlimb ischemia. Gene-transfer of wild-type Ad.hKLK1 but not Ad.R53H-hKLK1 was able to rescue this defect. Similarly, in the rat mesenteric assay, Ad.hKLK1 induced a mature neovasculature with increased vessel diameter through kinin-B2 receptor-mediated recruitment of pericytes and vascular smooth muscle cells, whereas Ad.R53H-hKLK1 was ineffective. Moreover, hKLK1 but not R53H-hKLK1 overexpression in the zebrafish induced endothelial precursor cell migration and vascular remodeling. Furthermore, Ad.hKLK1 activates metalloproteinase (MMP) activity in normoperfused muscle and fails to promote reparative neovascularization in ischemic MMP9−/− mice, whereas its proarteriogenic action was preserved in ApoE−/− mice, an atherosclerotic model of impaired angiogenesis.
Conclusions
These results demonstrate the fundamental role of endogenous Tissue Kallikrein in vascular repair and provide novel information on the cellular and molecular mechanisms responsible for the robust arterialization induced by hKLK1 overexpression.
doi:10.1161/ATVBAHA.108.182139
PMCID: PMC2827862  PMID: 19164804
angiogenesis; gene therapy; gene mutations; metalloproteinases; tissue kallikrein
22.  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.
doi:10.1074/jbc.M109.074930
PMCID: PMC2820781  PMID: 19906640
Growth Factors; RNA/Splicing; ASF/SF2; Angiogenesis; SRPK1; VEGF
23.  Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx 
Cardiovascular Research  2009;83(1):24-33.
Aims
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.
Conclusion
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.
doi:10.1093/cvr/cvp093
PMCID: PMC2695703  PMID: 19297368
Permeability; Angiopoietin-1; Glycocalyx; Microvessel; Glomerulus
24.  The Alternatively Spliced Anti-Angiogenic Family of VEGF Isoforms VEGFxxxbin Human Kidney Development 
Nephron. Physiology  2008;110(4):p57-p67.
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 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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1159/000177614
PMCID: PMC2635558  PMID: 19039247
Kidney development; Vascular endothelial growth factor 165b; Angiogenesis
25.  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.
doi:10.1042/CS20080270
PMCID: PMC2635543  PMID: 18826376
vascular endothelial growth factor165b; pre-eclampsia; splice variant; plasma marker; vascular permeability; angiogenesis

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