Endothelial dysfunction in atherosclerosis and type 2 diabetes : clinical and molecular studies of the role of endothelin and arginase

Abstract

Background: Atherosclerosis is a modern world scourge and the number of people worldwide diagnosed with type 2 diabetes (T2D) is on the rise, causing much morbidity and mortality. Endothelial dysfunction (ED) is an early sign of atherosclerosis and contributes to vascular complications in T2D. Endothelin-1 (ET-1) and arginase are two potent inhibitors of endothelium-derived nitric oxide (NO) and are known to cause ED. Short term studies have shown that blocking ET-1 can improve ED in T2D but no longer term studies have been performed. In addition, it remains unclear which ET receptor subtypes are beneficial to block. Arginase is an enzyme that causes ED and oxidative stress by metabolizing the NO substrate L-arginine. The role of arginase in human atherosclerosis and its regulation by ET-1 is unknown. The aim of the project was to investigate the role of ET-1 for vascular dysfunction in atherosclerosis and T2D, to evaluate the efficacy of ET receptor blockade to improve endothelial function as well as investigating the possible interaction between ET-1 and arginase in atherosclerosis.

Materials and methods: In study I we investigated whether treatment with bosentan, an oral ETA/ETB receptor blocker could improve endothelial function in patients with T2D and microvascular complications. Endothelial function was tested with Endo-PAT (peripheral artery tonometry) and FMD (flow mediated vasodilatation). In study II, the effect of selective ETA receptor blockade vs. combined ETA/ETB receptor blockade on endothelial function was investigated in patients with coronary artery disease (CAD) and T2D. In study III we investigated the extent of endothelial injury by measuring the number of endothelial progenitor cells (EPC) in the patients of Study I. The method used to isolate the EPC was flow cytometry by staining for cells positive for CD133, CD34 and KDR. In study IV the biobank of carotid endarterectomies (BiKE) was used to determine the gene and protein expression of ET-1 and arginase in the atherosclerotic plaque, and to investigate the functional interaction between ET-1 and arginase activity in endothelial cells and macrophages.

Results: Study I demonstrated that 4 weeks oral treatment with the dual ETA/ETB receptor blocker bosentan improved peripheral microvascular endothelial function in patients with T2D and microalbuminuria. In study II it was demonstrated that both selective ETA and dual ETA/ETB receptor blockade markedly improved endothelial function in patients with CAD and T2D. However, the addition of ETB receptor blockade did not improve endothelial function further. Among patients with T2D and vascular disease, high plasma levels of ET-1 were associated with a higher number of EPC. The recruitment of EPC did not seem to be regulated via ET receptor activation since treatment with a dual ETA/ETB receptor blockade did not affect circulating EPC numbers. Study IV demonstrated co-localization of ET-1 and arginase in human atherosclerotic plaques. ET-1 stimulated arginase expression and activity in endothelial cells as well as formation of reactive oxygen species in macrophages via an arginase-dependent mechanism.

Conclusion: Longer term treatment with a dual ETA/ETB receptor blocker improves endothelial function which may indicate a role in the treatment of microvascular complications of T2D. Both selective ETA and dual ETA/ETB receptor blockade improved endothelial function in a first head to head comparison in this patient category. Important interactions exist between the ET-1 pathway and arginase in human atherosclerotic plaques. Our data suggest that a part of the effect of ET-1 on NO and ROS production is through upregulation of arginase.