Glucokinase-activating GCKR polymorphisms increase plasma levels of triglycerides and free fatty acids, but do not elevate cardiovascular risk in the Ludwigshafen Risk and Cardiovascular Health Study.

Two strongly correlated polymorphisms located within the gene of the glucokinase regulator protein (GKRP), rs780094 and rs1260326, are associated with increased plasma triglyceride levels and provide a genetic model for the long-term activation of hepatic glucokinase. Because pharmacological glucokinase activators are evaluated for the treatment of diabetes, the aim of the study was to assess if these polymorphisms could provide evidence for an increased cardiovascular risk of long-term glucokinase activation. Therefore, these polymorphisms were tested in 3 500 patients of the Ludwigshafen Risk and Cardiovascular Health study, which was designed to assess cardiovascular risk factors. The two variants were associated with a significant increase of both plasma triglycerides (p<0.0001) and VLDL triglyceride levels (p<0.0001). Plasma free fatty acid concentrations were also significantly elevated (p<0.0078). LDL and HDL cholesterol levels were unchanged. No association was found with respect to coronary stenosis, myocardial infarction, left ventricular wall hypertrophy, and hypertension. In conclusion, long-term genetic glucokinase activation by the GKRP polymorphisms was not associated with an increased cardiovascular risk in the study population.

Comparative gene-expression analysis.

The study of differences in gene-expression patterns is one of the most promising approaches for understanding mechanisms of differentiation and development. In addition, the identification of disease-related target molecules opens new avenues for rational pharmaceutical intervention. Recent technical advances and improvements are accelerating the analysis of gene-expression profiles at the transcript level. The knowledge and comprehension of currently applied methods is one of the central criteria for an efficient and successful gene-screening approach.

Transcriptional regulation of the Ras-related protein TC21/R-Ras2 in endothelial cells.

Applying an RNA display strategy to identify genes of autocrine activated endothelial cells, we identified among others the Ras-related protein TC21/R-Ras2 as a differentially expressed gene of bovine aortic endothelial cells (BAEC). Migrating BAEC express prominently upregulated steady state levels of TC21/R-Ras2 mRNA (Northern blot, in situ hybridization) and protein (Western blot). Growth factor stimulation identified TC21/R-Ras2 as aFGF, bFGF, and EGF inducible molecule of BAEC. Exposure to actinomycin D revealed a half life time of TC21/R-Ras2 mRNA of > 2 h. These results strongly suggest that transcriptional regulation of Ras molecules contributes to their signal transduction capacity and a possible role of TC21/R-Ras2 in the signal transduction of autocrine activated endothelial cells.

The activin-binding protein follistatin regulates autocrine endothelial cell activity and induces angiogenesis.

Increasing evidence suggests that autocrine endothelial cell activity contributes significantly to the angiogenic cascade once the endothelial cells are initially activated by exogenous stimuli. We have employed the differential RNA-display technique to identify endothelial cell genes that are expressed under autocrine control as a result of the cells' release from growth arrest. Among the differentially expressed genes was the activin-binding and- neutralizing glycoprotein follistatin (FS), which was expressed by migrating endothelial cells and down-regulated once the cells had reached growth arrest. Cytokine exposure identified FS as a basic fibroblast growth factor (bFGF)-inducible gene. In contrast, activin-beta A, an inhibitor of endothelial cell proliferation, was constitutively expressed by migrating and resting endothelial cells. Exogenous recombinant FS induced proliferation of human umbilical vein endothelial cells and low bFGF-expressing bovine aortic endothelial cells. In vivo, FS was moderately angiogenic in the rabbit cornea. However, FS implantation in the cornea in combination with subcritical concentrations of bFGF induced a strong angiogenic response. The data demonstrate that FS by itself and particularly in synergy with bFGF induces angiogenesis. Furthermore, differential expression by endothelial cells suggests a critical role of the FS/activin-beta A system in regulating autocrine endothelial cell activity.

Rapid identification of differentially expressed endothelial cell genes by RNA display.

Endothelial cells line the inside of all blood vessels forming a structurally and functionally highly heterogenous population of cells. Here we describe the application of the differential RNA display technique to the analysis of the heterogeneity of endothelial cells. Multiple fragment cDNAs from quiescent resting and from activated migrating endothelial cells were amplified by RT-PCR using random 10mer 5' primers and T11XY 3' primers. Labelled amplification products were displayed on a sequencing gel. Expression patterns of more than 5000 bands of the two cell populations were approximately 98% identical. Of the differentially expressed bands, 26 fragment cDNAs were reamplified, sequenced, and used as probes for Northern blots. Approximately 50% of the analyzed fragment cDNAs could be confirmed as being differentially expressed by Northern blot analysis. Among the differentially expressed cDNAs was follistatin, which was exclusively expressed by migrating and not by quiescent arrested endothelial cells. Stimulation by exogenous bFGF, however, induced follistatin expression in arrested endothelial cells. These experiments support the use of the differential RNA display technique as a rapid cloning strategy for the identification of differentially expressed genes and suggest a role of the follistatin/activin system in the autocrine control of endothelial cell growth and differentiation.

Differentiation of endothelial cells: analysis of the constitutive and activated endothelial cell phenotypes.

Endothelial cells line the inside of all blood vessels, forming a structurally and functionally heterogeneous population of cells. Their complexity and diversity has long been recognized, yet very little is known about the molecules and regulatory mechanisms that mediate the heterogeneity of different endothelial cell populations. The constitutive organ- and microenvironment-specific phenotype of endothelial cells controls internal body compartmentation, regulating the trafficking of circulating cells to distinct vascular beds. In contrast, surface molecules associated with the activated cytokine-inducible endothelial phenotype play a critical role in pathological conditions including inflammation, tumor angiogenesis, and wound healing. Differentiation of the endothelial cell phenotypes appears to follow similar mechanisms to the differentiation of hematopoietic cells, with the exception that endothelial cells maintain transdifferentiating competence. The present review offers a scheme of endothelial cell differentiation and discusses the possible applications of differentially expressed endothelial cell molecules as targets for directed therapeutic intervention.