Abstract 971: Quantification of Endothelial MCP-1 Expression in Diabetic Patients by a Novel Methodology: Human Endothelial Biopsy Coupled With Real-Time PCR Analysis
Background: In-vitro and animal studies suggest that monocyte chemoattractant protein-1 (MCP-1) has a pivotal role in the pathogenesis of diabetic vasculopathy. Hyperglycemia promotes MCP-1 expression in cultured endothelial cells (ECs). Limited availability of endothelial tissue is a major constraint when studying the cellular mechanisms of diabetic vasculopathy in humans. We developed a novel method, venous endothelial biopsy coupled with Real-Time PCR, to quantify MCP-1 expression in pts with diabetic vasculopathy and hyperglycemia.
Methods: Venous ECs were harvested in 5 pts with Type 2 DM, macrovascular complications and elevated HbA1c, and 8 healthy subjects. ECs were collected from a superficial forearm vein using 5 guide wires sequentially inserted through a 20-gauge angiocath. ECs were purified using magnetic beads coated with CD146 antibodies. mRNA was linearly amplified using a RiboAmp HS RNA Amplification Kit, and then subjected to Real-Time PCR.
Results: 500 to 2,000 ECs were collected. Linearity of RNA amplification was validated by Real-Time PCR using RNA from 1,000 HUVECs before and after amplification. Agilent Bioanalyzer confirmed purity and integrity of amplified RNA from human biopsies. MCP-1 mRNA expression was 5.3 fold higher in DM pts when compared to healthy (ΔCt 8.5±.2.3 vs. 10.9±.2.4, P=0.1) (figure⇓).
Conclusions: Endothelial biopsy coupled with Real-Time PCR is an innovative technique to monitor vascular inflammation, and ultimately to gauge response to drug therapy and behavior modifications in DM pts. Our preliminary results suggest that MCP-1 expression is increased in ECs of pts with diabetic vasculopathy and sustained hyperglycemia.