Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis.

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.

The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man.

Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.

VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.

Features of syndrome X develop in transgenic rats expressing a non-insulin responsive phosphoenolpyruvate carboxykinase gene.

AIMS/HYPOTHESIS: Obesity, glucose intolerance, dyslipidaemia and hypertension are a cluster of disorders (syndrome X) affecting many people. It has been hypothesised that these abnormalities are caused by insulin resistance, but definitive proof is lacking. We have developed transgenic rats in which the rate-limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is non-insulin responsive. The aim of our study was to investigate whether syndrome X develops in these animals and if a high-fat diet interacts with this genetic defect. METHODS: Chow-fed transgenic and control rats aged 1, 3, 6 and 17 months and a subgroup of transgenic and control rats fed chow plus cafeteria foods for 6 months were examined for features of syndrome X. RESULTS: At 3 months, transgenic rats had fasting and postprandial hyperinsulinaemia, mild obesity (in abdominal and, to a lesser extent, peripheral regions) and fasting hypercholesterolaemia. Hypertriglyceridaemia was evident after 6 months while hyperglycaemia was apparent at 17 months. Hypertension had not developed by 17 months. The effect of a high-fat diet on insulin, glucose, body weight and body fat was more dramatic than the effect of the transgene alone while the effect of a high-fat diet on cholesterol and triglyceride was similar to the transgene. This illustrates that a high-fat diet is a potent catalyst for many abnormalities associated with syndrome X. There was no evidence of an additive effect of the high-fat diet plus transgene. CONCLUSION/INTERPRETATION: Therefore rats genetically-engineered with a non-insulin responsive gluconeogenic enzyme develop several aspects of syndrome X, supporting the hypothesis that insulin resistance initiates this cluster of disorders.