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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.

IgA nephropathy is the major cause of end-stage renal failure in patients with primary glomerular diseases. Tumor suppressor cylindromatosis (CYLD), the recently identified member of the deubiquitinating enzymes, has been actively involved in regulation of inflammation. This study was undertaken to investigate the CYLD expression profile in IgA nephropathy and identify factors associated with CYLD expression.Forty-one cases of IgA nephropathy were selected. CYLD expression in the kidney biopsy tissue was measured by immunohistochemical staining. Relevant clinical and pathological data were analyzed, and Logistic regression analysis was carried out to identify factors associated with CYLD expression.CYLD was specifically expressed in renal tubular epithelial cells in 70% of the studied patients with IgA nephropathy. All patients with positive CYLD staining had proteinuria, while only 72.7% of patients with negative CYLD had proteinuria (P = 0.003). Among studied proteinuric patients, those with positive CYLD had significantly less tubulo-interstitial lesions and higher estimated glomerular filtration rate (eGFR) levels when compared with those patients showed negative CYLD results. Logistic regression analysis indicated that the urinary protein excretion and eGFR were identified as predictors for the CYLD expression.CYLD is expressed in renal tubular epithelial cells and appears to be associated negatively with tubulointerstitial lesions, however, its exact functional role remains to be clarified in further experiments.

Despite production by podocytes of the proangiogenic molecule vascular endothelial growth factor-A (VEGF), the glomeruli are not sites of angiogenesis. We recently described mRNA expression of an inhibitory splice variant of VEGF (VEGF165b) in normal kidney (Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. Cancer Res 62: 4123–4131, 2002). Available anti-VEGF antibodies do not distinguish stimulatory from inhibitory VEGF families. To assess the production of VEGF165(stimulatory) and VEGF165b (inhibitory) isoforms by human podocytes, we examined both primary cultured and conditionally immortalized human podocytes using family- and isoform-specific RT-PCR. In addition, VEGF protein production was analyzed in podocytes, using isoform-specific double-strand small-interference RNAs (siRNA). RT-PCR demonstrated the production of VEGF189mRNA by podocytes of both phenotypes. In contrast, on differentiation there was a splicing change from VEGF165to VEGF165b mRNA. In addition, VEGF protein in the supernatant of conditionally immortalized, differentiated podocytes was reduced by VEGF165b siRNA to 20 ± 11% of the level of mock-transfected cells ( P < 0.01). No reduction was seen with mismatch siRNA. Moreover, there was no reduction in VEGF protein concentration in the supernatant of primary cultured, dedifferentiated human podocytes (109 ± 8% of mismatch siRNA, P > 0.1). In conclusion, differentiated but not dedifferentiated human podocytes secrete significant amounts of VEGF165b protein. It is possible that this may explain the paradox of high VEGF production in the glomerulus but no angiogenesis. Furthermore, the existence of this splicing switch in relation to podocyte phenotype suggests that alternative splicing of the VEGF pre-RNA is a regulated process that is open to manipulation and therefore could be a target for novel cancer therapies.

Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF) is the most potent growth factor of tumor neovasculature, has been shown to be up-regulated in every tumor studied thus far, and is correlated with tumor stage and progression. To determine whether specific VEGF splice variants were differentially expressed in renal cell carcinomas, 18 polar tumor samples were analyzed by reverse transcription-PCR using primers designed to differentiate between VEGF splice variants. Control tissue was derived from the opposite normal pole. An amplicon of length consistent with the previously described variant VEGF(148) was found in normal kidney tissue. Subsequent sequencing revealed a new VEGF isoform formed by differential splicing from the end of exon 7 into the 3' untranslated region of the mRNA. Cloning of this transcript showed that translation would result in a 165-amino acid peptide with an alternative terminal 6 amino acids, followed by a stop codon. We have termed this new isoform VEGF165b. This isoform was present in 17 of 18 normal kidney samples but only 4 of 18 cases from matched malignant tissue. VEGF165b was therefore expressed in a significantly higher proportion of normal tissue than malignant tissue from the same patients (P0.001). To determine the functional significance of this new isoform, we expressed the full-length protein in a heterologous expression system. Conditioned medium containing this isoform significantly and dose dependently inhibited VEGF165-mediated proliferation, migration of endothelial cells, and vasodilatation of mesenteric arteries. This novel isoform VEGF165b is therefore an endogenous inhibitory form of VEGF that is down-regulated in renal tumors and, therefore, may be anti-angiogenesis.