David O. Bates

VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant Mechanism of Action, In vivo Effect On Angiogenesis and Endogenous Protein Expression

Growth of new blood vessels (angiogenesis), required for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is up-regulated in all known solid tumors but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms have been universally described as proangiogenic cytokines. Here, we show that an endogenous splice variant, VEGF165b, is expressed as protein in normal cells and tissues and is circulating in human plasma. We also present evidence for a sister family of presumably inhibitory splice variants. Moreover, these isoforms are down-regulated in prostate cancer. We also show that VEGF165b binds VEGF receptor 2 with the same affinity as VEGF165 but does not activate it or stimulate downstream signaling pathways. Moreover, it prevents VEGF165-mediated VEGF receptor 2 phosphorylation and signaling in cultured cells. Furthermore, we show, with two different in vivo angiogenesis models, that VEGF165b is not angiogenic and that it inhibits VEGF165-mediated angiogenesis in rabbit cornea and rat mesentery. Finally, we show that VEGF165b expressing tumors grow significantly more slowly than VEGF165-expressing tumors, indicating that a switch in splicing from VEGF165 to VEGF165b can inhibit tumor growth. These results suggest that regulation of VEGF splicing may be a critical switch from an antiangiogenic to a proangiogenic phenotype.

VEGF-A splicing: the key to anti-angiogenic therapeutics?

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.

Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors

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.

Vascular Endothelial Growth Factors and vascular permeability

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.

Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms A NOVEL THERAPEUTIC STRATEGY FOR ANGIOGENESIS

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as 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.

Lymphatic density and metastatic spread in human malignant melanoma

Malignant melanoma (MM), the most common cause of skin cancer deaths, metastasises to regional lymph nodes. In animal models of other cancers, lymphatic growth is associated with metastasis. To assess if lymphatic density (LD) was increased in human MM, and its association with metastasis, we measured LD inside and around archival MM samples (MM, n=21), and compared them with normal dermis (n=11), basal cell carcinoma (BCC, n=6) and Merkel cell carcinoma (MCC), a skin tumour thought to metastasise through a vascular route (MCC, n=6). Lymphatic capillary density (mm−2), as determined by immunohistochemical staining with the lymphatic specific marker LYVE-1, was significantly increased around MM (10.0±2.5 mm−2) compared with normal dermis (2.4±0.9 mm−2), BCC (3.0±0.9 mm−2) and MCC (2.4±1.4 mm−2) (P<0.0001). There was a small decrease in LD inside MM (1.1±0.7 mm−2) compared with normal dermis, but a highly significant decrease in BCC (0.14±0.13) and MCC (0.12±2.4) (P<0.01 Kruskal–Wallis). Astonishingly, LD discriminated between melanomas that subsequently metastasised (12.8±1.6 mm−2) and those that did not (5.4±1.1 mm−2, P<0.01, Mann–Whitney). Lymphatic invasion by tumour cells was seen mainly in MM that metastasised (70% compared with 12% not metastasising, P<0.05 Fishers Exact test). The results show that LD was increased around MMs, and that LD and tumour cell invasion of lymphatics may help to predict metastasis. To this end, a prognostic index was calculated using LD, lymphatic invasion and thickness that clearly discriminated metastatic from nonmetastatic tumours.

Loss of the Endothelial Glycocalyx Links Albuminuria and Vascular Dysfunction

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

Vascular endothelial growth factor (VEGF)-A165b is a weak in vitro agonist for VEGF receptor-2 due to lack of coreceptor binding and deficient regulation of kinase activity.

Vascular endothelial growth factor (VEGF)-A165b is a COOH-terminal splice variant of VEGF-A that has been implicated in negative regulation of angiogenesis. We compared the properties of VEGF-A165b with those of VEGF-A121, VEGF-A145, and VEGF-A165. Induction of tyrosine phosphorylation sites in VEGFR-2 differed between the VEGF ligands as determined by tryptic phosphopeptide mapping and by use of phosphosite-specific antibodies. VEGF-A165b was considerably poorer in inducing phosphorylation of the positive regulatory site Y1052 in VEGFR-2. Whereas this did not affect activation of VEGFR-2 in vitro, we show that VEGF-A165b failed to induce vasculogenesis and sprouting angiogenesis in differentiating embryonic stem cells and vascularization of s.c. Matrigel plugs. In addition, the ability of the different VEGF ligands to induce angiogenesis correlated with their abilities to bind the VEGF coreceptor neuropilin 1 (NRP1). Our data indicate that loss of VEGFR-2/NRP1 complex formation and Y1052 phosphorylation contribute to the lack of angiogenic properties of VEGF-A165b.

Chemokine-mediated migration of melanoma cells towards lymphatics – a mechanism contributing to metastasis

The mechanisms that cause tumors such as melanomas to metastasize into peripheral lymphatic capillaries are poorly defined. Non-mutually-exclusive mechanisms are lymphatic endothelial cell (LEC) chemotaxis and proliferation in response to tumor cells (chemotaxis-lymphangiogenesis hypothesis) or LECs may secrete chemotactic agents that attract cancer cells (chemotactic metastasis hypothesis). Using migration assays, we found evidence supporting both hypotheses. Conditioned medium (CM) from metastatic malignant melanoma (MMM) cell lines attracted LEC migration, consistent with the lymphangiogenesis hypothesis. Conversely, CM from mixed endothelial cells or LECs, but not blood endothelial cells, attracted MMM cells but not non-metastatic melanoma cells, consistent with the chemotactic metastasis hypothesis. MMM cell lines expressed CCR7 receptors for the lymphatic chemokine CCL21 and CCL21 neutralizing antibodies prevented MMM chemotaxis in vitro. To test for chemotactic metastasis in vivo tumor cells were xenotransplanted into nude mice ∼1 cm from an injected LEC depot. Two different MMM grew directionally towards the LECs, whereas non-metastatic melanomas did not. These observations support the hypothesis that MMM cells grow towards regions of high LEC density owing to chemotactic LEC secretions, including CCL21. This chemotactic metastasis may contribute to the close association between metastasizing tumor cells and peri-tumor lymphatic density and promote lymphatic invasion.

Expression of VEGFxxxb, the inhibitory isoforms of VEGF, in malignant melanoma

Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis – the growth of new vessels from preexisting vasculature – is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGFxxxb, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGFxxxb expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGFxxxb staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) P<0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGFxxxb expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.