Research Grants 1997-1998

In Vitro Evaluation of Pharmacological Interventions Aimed to Prevent Atherosclerosis

Rita B. Alevriadou, PhD
Johns Hopkins University, Baltimore, Maryland

Studies of animals with artificially-induced hypercholesterolaemia have shown arteries that contain retracted endothelial cells (ECs), which provide the sites for underlying macrophages and for the formation of mural platelet thrombi. The cellular events that occur in vessels of hypercholesterolaemic animals are exactly mirrored by those observed in human atherosclerotic coronary arteries in hearts removed in transplant operations. Early events may be summarized as follows:

High concentrations of native low-density-lipoprotein (LDL) promote the adhesion of blood monocytes to the ECs, suggesting that the latter undergo activation upon such exposure. Oxygen radicals from activated ECs oxidize LDL (oxLDL). Oxidized LDL (oxLDL):
- Greatly increases adherence and migration of monocytes and lymphocytes into the subendothelial space, by inducing formation of adhesive EC-surface glycoproteins (eg. vascular cell adhesion molecule, VCAM-1), secretion of chemoattractants (eg. monocyte chemotactic protein-1, MCP-1) and inhibition of nitric oxide (NO) release.
- Initiates platelet interactions with ECs and with subendothelial proteins exposed in the gaps between injured ECs, by inducing a procoagulant EC activity, via expression of surface proteins (eg. tissue factor, TF) leading to thrombin generation, and via inhibition of NO release.

Using intravital microscopy in animals, different groups have demonstrated that antioxidants and NO donors separately reduced the oxLDL-induced leukocyte adherence. in vitro studies of atherogenesis (initiation of atherosclerosis) have been limited to exposing cultured ECs to minimally-modified LDL (MM-LDL), assaying for the EC proadhesive/procoagulant activities, and microscopically observing and measuring the number of monocytes suspended in culture media that adhere to LDL-treated EC monolayers. Recently, an in vitro study duplicated the in vivo finding of the beneficial effect of antioxidants on monocyte adhesion to simulated ECs. We propose to systematically evaluate the merits of pharmacological interventions that target key contributors of the early atherosclerotic process, using an in vitro model that allows us to assess the effect of MM-LDL on both the leukocyte and platelet adherence to endothelium.

Specifically, we will:

Develop an in vitro model of MM-LDL toxicity to human aortic ECs (HAECs). We will verify the proadhesive and procoagulant status of cultured HAECs, before and after MM-LDL exposure, by measuring reactive oxygen species generation (spectrophotometrically), NO and MCP-1 release (nitrate/nitrate assay and Western blot respectively) and formation of surface of TF (protein expression and activity assay). We will then perfuse heparinized human whole blood with both fluorescently-labeled leukocytes and platelets over HAEC monolayers, treated or not with MM-LDL, and employ epifluorescence video microscopy and digital image processing to quantify the extent of leukocyte and platelet deposition on and between ECs.
Use the model to compare the following agents that potentially protect ECs from atherogenesis:
- Antioxidants HAECs will be pretreated withh either alpha-tocopherl or probucol before stimulation with MM-LDL, and the effect of antioxidants on leukocyte and platelet interactions from flowing whole blood with cultured and HAECs will be quantified.
- NO donors Inhibition of NO is shown to increase MCP-1 expression and monocyte chemotactic activity. NO is also an inhibitor of platelet aggregation. Hence, incubation with NO donors, sodium nitroprusside or SIN-1, should restore the antiatherogenic EC properties.
- Thrombin inhibitors Administration of TF pathway inhibitor (rTFPI) that inhibits thrombin generation will be tested.

This study aims to develop an in vitro assay for the better assessment of lipoprotein toxicity to vascular endothelium, since it will closely mimic the in vivo interactions between flowing blood cells and ECs. The assay has the potential to reduce the testing of antiatherogenic drugs in cholesterol-fed animals by pointing to the most promising treatment.