Molecular Mechanisms of Human Diabetic Microangiopathy

All forms of diabetes mellitus systemically affect the patient`s microvasculature, involving the small vessels of kidney, retina and peripheral nerves of 40-60% of long-term diabetic patients. Histopathologically, blood capillaries show a diffuse thickening of their basement membranes as the common feature in all organs. Endothelial cells show metabolic alterations, which suggest their involvement in local diabetic pathology. Thus, it is of importance to compare the gene expression patterns of endothelial cells from capillaries of diabetic and non- diabetic patients, to (i) identify pathways specifically expressed in diabetics that (ii) could provide potential therapeutic targets. Inaccessibility of clinically affected organs precluded direct studies of the molecular basis of diabetic microangiopathy satisfactorily to date. Similar changes of microvessels (i.e. thickening of their basement membranes) have, however, been reported frequently in the dermal layer of skin of diabetics, which can be obtained more easily. Their isolation, however, generated mixed endothelial cell populations, e.g. the differentiation between endothelial cells of blood and lymph vessels has not been possible so far. We have discovered a way to circumvent this problem by the use of the plasma membrane protein podoplanin as a specific marker for lymphatic vascular endothelial cells, and we have succeeded in clean sepa-ration of blood and lymph vessel endothelial cell populations using this marker molecule. We plan to investigate the molecular alterations of diabetic microangiopathy: We will prepare microvascular endothelial cells from dermatom split skin samples of diabetic and non-diabetic patients, using standard enzymatic-mechanic tissue dissociation, and subsequent immunophenotypic separation by FACS, which is preferable to MACS in this setting. Without intermediate tissue culture we will prepare and hybridize linearly amplified mRNA against oligonucleotide chips in order to screen for alterations on the RNA level. We will analyze the resulting expression profiles of diabetics and non-diabetics by appropriate and established bioinformatic procedures. Using native, highly pure blood microvascular endothelial cell populations is crucial, as currently only a few, arguably critical or not diabetes-specific pathways are known, that do not offer useful clues for specific therapeutic intervention. It is our aim to identify pathogenetically relevant pathways in diabetic endothelium.