Increased cytochrome P4502E1 expression and altered hydroxyeicosatetraenoic acid formation mediate diabetic vascular dysfunction: rescue by guanylyl-cyclase activation.

OBJECTIVE: We investigated the mechanisms underlying vascular endothelial and contractile dysfunction in diabetes as well as the effect of HMR1766, a novel nitric oxide (NO)-independent activator of soluble guanylyl cyclase (sGC). RESEARCH DESIGN AND METHODS: Two weeks after induction of diabetes by streptozotocin, Wistar rats received either placebo or HMR1766 (10 mg/kg twice daily) for another 2 weeks; thereafter, vascular function was assessed. RESULTS: Endothelial function and contractile responses were significantly impaired, while vascular superoxide formation was increased in the aortae from diabetic versus healthy control rats. Using RNA microarrays, cytochrome P4502E1 (CYP2E1) was identified as the highest upregulated gene in diabetic aorta. CYP2E1 protein was significantly increased (16-fold) by diabetes, leading to a reduction in levels of the potent vasoconstrictor 20-hydroxy-eicosatetraenoic acid (20-HETE). Induction of CYP2E1 expression in healthy rats using isoniazide mimicked the diabetic noncontractile vascular response while preincubation of aortae from STZ-diabetic rats in vitro with 20-HETE rescued contractile function. Chronic treatment with the sGC activator HMR1766 improved NO sensitivity and endothelial function, reduced CYP2E1 expression and superoxide formation, enhanced 20-HETE levels, and reversed the contractile deficit observed in the diabetic rats that received placebo. CONCLUSIONS: Upregulation of CYP2E1 is essentially involved in diabetic vascular dysfunction. Chronic treatment with the sGC activator HMR1766 reduced oxidative stress, decreased CYP2E1 levels, and normalized vasomotor function in diabetic rats.

Use of functional highly purified human platelets for the identification of new proteins of the IPP signaling pathway.

INTRODUCTION: Identification of the full content of platelet proteins and their mRNAs would be helpful for further studies of human platelet function. For this purpose, proteomic as well as transcriptomic methods (SAGE and qRT-PCR) can be utilized, but the purity of the platelet samples studied is crucial. Here we report the development of a new, effective, and efficient technique for purification of human platelets from washed apheresis platelet concentrates and whole blood. MATERIALS AND METHODS: Methods used are a combination of differential and gradient centrifugation steps. The level of purification was determined by nephelometry, FACS, and PCR. RESULTS: We could show that even the P2Y purinoceptor 12 (P2Y(12)) receptor, which undergoes rapid homologous desensitization, was still functional after the purification procedure. The presence of PINCH (particularly interesting new Cys-His protein) and alpha-parvin, which constitute the IPP (ILK-PINCH-parvin) complex together with the integrin-linked kinase (ILK), has been predicted in platelets by proteomic analysis. We could confirm this observation with our purified platelets. Detection of these proteins is an example of the application of this purification protocol that can be used for the verification of proteins postulated by high-throughput studies. CONCLUSIONS: The procedure for obtaining purified platelets described here provides an essential, much-needed tool for the comprehensive investigation of platelet proteins and functions.

Analysis of SAGE data in human platelets: features of the transcriptome in an anucleate cell.

A comprehensive SAGE (serial analysis of gene expression) library of purified human platelets was established. Twenty-five thousand (25,000) tags were sequenced, and after removal of mitochondrial tags, 12,609 (51%) non-mitochondrial-derived tags remained, corresponding to 2,300 different transcripts with expression levels of up to 30,000 tags per million. This new, highly purified SAGE library of platelets is enriched in specific transcripts. The complexity in terms of tag distribution is similar to cells that are still able to replenish their mRNA pool by transcription. We show that our SAGE data are consistent with recently published microarray data but show further details of the platelet transcriptome, including (i) longer UTR regions and more stable folding in the enriched mRNAs, (ii) biologically interesting new candidate mRNAs that show regulatory elements, including elements for RNA stabilization or for translational control, and (iii) significant enrichment of specific, highly transcribed mRNAs compared to a battery of SAGE libraries from other tissues. Among several regulatory mRNA elements known to be involved in mRNA localization and translational control, CPE elements are in particular enriched in the platelet transcriptome. mRNAs previously reported to be translationally regulated were found to be present in the library and were validated by real-time PCR. Furthermore, specific molecular functions such as signal transduction activity were found to be significantly enriched in the platelet transcriptome. These findings emphasize the richness and diversity of the platelet transcriptome.