Microvascular endothelial response to cocaethylene exposure: morphological and molecular observations
Cocaethylene (CE) is an active metabolite of cocaine and ethanol and is a toxicant of physiological relevance due to the high rate of cocaine and ethanol co-exposure (~80%) in cocaine abusers. It has prolonged action and increased potency on known physiological targets relative to the effect of cocaine. Since pathology in cocaine abusers is typically chronic and systemic, and CE persists in the body three to five times longer than cocaine, a link between CE and systemic disease in cocaine abusers was proposed. Consequently, this dissertation contains the studies that were used to test the hypothesis that the microvascular endothelium is a target tissue that is central in the pathogenic mechanism of cocaine-associated systemic disease, and that endothelial injury after CE exposure would result in dysregulation and altered barrier function due to changes in intracellular second messengers and signaling. To test this hypothesis, an in vitro model of CE exposure in human dermal microvascular endothelial cells (HMEC-1) was developed. Four Aims were designed to compartmentalize various components of the endothelial response to CE. The Aims included an array of methods to address cellular toxicity and dysfunction, including classical cytotoxicity and viability assays (Aim One), microscopic and electrical analyses of monolayer integrity (Aim Two), molecular analysis of second messengers, signaling molecule phosphorylation, and transcription factor DNA binding activity (Aims Three and Four). Aim One experiments demonstrated a lack of overt endothelial cytotoxicity caused by CE. Aim Two morphological analysis of endothelial intercellular borders and barrier integrity showed that CE exposure in the endothelial monolayers resulted in increased permeability, and hence a decrease in barrier integrity. These changes were observed temporally with alterations in cytosolic and total cellular free calcium ion (Aim Three), inositol 1,4,5 trisphosphate, and phosphorylated p38 mitogen-activated protein kinase concentrations, as well as changes in DNA binding activity and dimer composition of nuclear factor-kappaB (Aim Four). The observed changes suggest a distinct alteration of endothelial cell and monolayer function consistent with increased vascular permeability in vivo. Potential pathological outcomes of such effects include inflammation, vasculitis, systemic disease, and organ failure.