Nicotine Induces Mononuclear Leukocyte Adhesion and Expression of Adhesion Molecules,VCAM and ICAM, in Endothelial Cells In Vitro

Abstract

The pathology of atherosclerotic cardiovascular disease (ASCVD) has been characterized as an inflammatory response to vessel injury. The initial steps of this response involve mononuclear leukocyte (MNL) attachment and infiltration into the vessel wall. Leukocyte adhesion is potentiated by expression of cellular adhesion molecules. Vascular cell adhesion molecule-1 (VCAM) and intracellular adhesion molecule-1 (ICAM) are markers of cellular activation and have the ability to attach leukocytes to the endothelium, which is an initial event in the inflammatory response in the vessel wall. Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM) on plastic coverslips and grown until cells were 75% confluent. Free base nicotine (FBN) was diluted in EGM to a concentration of 10⁻⁸ M and added to experimental cells. At 3 hr, coverslips were removed and fixed. Immunohistochemical staining (IHCS) was performed using a monoclonal antibody to human ICAM and VCAM. Digital image analysis (DIA) was performed to quantify the expression of ICAM and VCAM. An intensity stain index (ISI) measuring area and intensity of stain/total cellular area was determined. Additional HUVEC grown in a similar manner were either exposed to 10⁻⁸ M FBN in EGM or EGM control for 4 hr, then were exposed to MNL suspension for 10 min. Coverslips were removed, rinsed, and fixed. Hematoxylin and eosin staining was performed and cells examined under light microscopy. Leukocyte number per high power field (HPF) was counted and compared to controls. Data were analyzed using analysis of variants (ANOVA) and Student’s t-test. Differences were considered significant if p < 0.05. ICAM and VCAM expression was absent in control cells. Nicotine exposure at 3 hr induced expression of VCAM (ISI = 30.85 ± 0.77) and to a lesser extent ICAM (ISI = 16.6 ± 1.39) (p < 0.001). MNL adhesion was markedly increased in cells exposed to nicotine (79.4 ± 16.9/HPF) when compared to control cells (1.8 ± 0.91/HPF) exposed to MNL (p < 0.01). These data show nicotine’s ability to activate HUVEC as evidenced by induction of ICAM and VCAM expression in vitro. The biological effects of these adhesion molecules are demonstrated by a marked increase in MNL adhesion to HUVEC as demonstrated by leukocyte adhesion assay (LAA). MNL adhesion and subsequent migration into the intima, if occurring in vivo, may be a vital step in the pathogenesis of ASCVD associated with nicotine exposure.