The endothelial transcriptome in diabetic environment

Diabetic nephropathy is one of the leading causes of end-stage renal disease. The events leading to the well known diabetic changes in the kidney have not yet been completely described. This is due to the extremely complicated structure and interaction patterns in the glomerulus between cells (glomerular endothelial cells -EC-, mesangial cells, and specialized epithelial cells called podocytes), the glomerular basement membrane composed of different types of matrix proteins and environmental factors such as blood flow-velocity. The goal of the proposed study is to define genes potentially involved in the development of diabetic kidney disease. Our approach is to look at the consequences of interactions between advanced glycation endproducts (AGE) with EC. The term "advanced glycation" describes a series of post-translational modifications of lipids, nucleic acids and proteins, which is triggered by a non-enzymatic condensation reaction of circulating glucose with amino groups, e.g. in the diabetic environment. As any other protein matrix proteins as well, such as fibronectin, are irreversibly modified by glucose in diabetic individuals and interact with endothelial AGE-receptors. As mentioned before, environmental factors such as blood flow play an important role in endothelial cell biology and make it impossible to derive results from the usual experimental settings in the lab. Shear stress originates from the flow of blood at the endothelial cell apical surface. The main factors determining shear stress are mean blood flow rate, fluid viscosity, and vessel`s radius, of which the vessel radius is the most important. Shear stress will be significantly higher in smaller vessels, e.g. glomerular capillaries. Today, there is growing evidence that shear stress produces a series of changes in EC behaviour. In this study we will use an advanced molecular biology technique namely "serial analysis of gene expression -SAGE-". This method allows us to list and compare transcripts in cells or tissues in a quantitative fashion. In this study lists of expressed genes in glomerular EC under shear stress cultured on normal (fibronectin) - versus "diabetic"-matrix (advanced glycation endproduct-modified fibronectin) will be obtained and compared. Shear stress will be used on one hand to define the transcripts involved in response of EC to shear stress and on the other hand to mimic the in vivo environment of the endothelium. The comparison of these lists will enable us to make a more precise predication about genes responsible for diabetic changes in the glomerular endothelium.

The endothelial cells make up the innermost cell layer of all blood vessels. They are the only cells that are exposed to the mechanical force of the blood flow. This force is called "shear stress" and is dependent on vessel radius, flow velocity and viscosity. High shear stress is involved in a series of endothelial mechanisms including vessel or collateral formation, endothelial differentiation, as well as pathological changes leading to endothelial dysfunction. Yet, a detailed list of endothelial transcripts regulated in response to high shear stress has not been described thus far. A similar list for differentially expressed endothelial transcripts in diabetic environment has never been generated either. In this project we defined the culture of endothelial cells on irreversibly glycosylated fibronectin as the diabetic environment. Such glycosylation leads to advanced glycosylation endproducts (AGE); in this case AGE-fibronectin. In this project we generated quantitative transcript lists by means of serial analysis of gene expression (SAGE) in three groups of microvascular endothelial cells that were grown in fibers to imitate capillaries. SAGE was the method of choice because it enables quantitative comparison of frequencies of already known and unknown transcripts between distinct cell populations and each list is called a SAGE library. Each of our three libraries was submitted and accepted at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/geo) and was assigned to a unique number (GSM): a. Microvascular endothelial cells grown on native fibronectin under high shear stress (17 dyn/cm2 ) for 10 days: 30, 615 transcripts (GSM32266) b. Microvascular endothelial cells grown on native fibronectin under low shear stress (0.7 dyn/cm2 ) for 10 days: 31, 141 transcripts (GSM41248) c. Microvascular endothelial cellsgrown on AGE-FN under high shear stress (17 dyn/cm2 ) for 10 days: 30, 870 transcripts (GSM45608) The comparison between these 3 libraries using a software developed for this project identified transcripts which appear to be specific for microvascular endothelial cells under high shear stress or cultured on AGE-FN. Experiments confirming their in vivo expression are underway.