Tobacco Smoke and Elevated Shear Stress Enhance Classical Pathway Complement Activation on Human Endothelial Cells.
Authors: Yin Wei , Kim Jiyeon , Weksler Babette B. , Ghebrehiwet Berhane , Peerschke Ellinor I.B. 
Published: April 2020

Cigarette smoke and hemodynamic stress contribute to inflammatory processes associated with the development of atherosclerosis. Endothelial cells are intimately involved in vascular pathology. We recently demonstrated classical pathway complement activation on human umbilical vein endothelial cells (HUVEC) and on endothelial cell lines including human bone marrow microvascular endothelial cells (BMEC). Complement components have been identified in atherosclerotic lesions, and complement derived inflammatory mediators likely play a role in vascular injury. Endothelial cells express several complement receptors, including gC1qR/p33. gC1qR/p33 recognizes the globular domain of the first component of complement, C1q, and directly activates the classical pathway. The present study was designed to explore the effects of two known atherosclerotic risk factors, tobacco smoke and shear stress, on classical pathway complement activation on endothelial cells. BMEC were grown to confluence on type I collagen, and treated for 24 hours with tobacco smoke extract (0.015 or 0.03 puffs/ml) from 2R4F research cigarettes. BMEC monolayers, in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% human plasma (anticoagulated with 0.32 % sodium citrate), were exposed to constant shear stress at 18 dynes/cm2 for 1 hour at room temperature using a cone and plate shearing device (courtesy of Dr. David Varon, BDR Technologies Ltd., Israel). BMEC monolayers were subsequently fixed with 0.5% glutaraldehyde. C1q dependent C4 activation (C4d deposition) was quantified on the cell surface using an ELISA approach. BMEC activation was characterized using endothelial cell intracellular adhesion molecule 1 (ICAM-1, CD54). Compared to background (0.26±0.13, O.D. 405 nm, n=5), low baseline levels of C4d deposition were noted on unstimulated BMEC (0.37±0.15, O.D. 405 nm, n=5, P&lt;0.05). However, moderately increased C4d deposition was observed after treatment with tobacco smoke (0.03 puffs/ml) or exposure to shear stress (Table). Moreover, combined effects from tobacco smoke (0.03 puffs/ml) and shear stress resulted in an ~ 2-fold increase in C4d deposition. This was accompanied by a 50% increase in surface gC1qR/p33 and a 33% increase in ICAM-1 expression (Table). Tobacco smoke at 0.015 puffs/ml with or without shear stress had no effect on BMEC activation or C4d deposition. Similar results were obtained with HUVEC. In summary, these data provide further insight into the atherogenic effects of tobacco smoke by suggesting that high dose tobacco smoke in combination with elevated shear stress leads to enhanced endothelial cell surface classical pathway complement activation.

Table. C4d deposition, gC1qR/p33 and ICAM-1 expression on BMEC treated with tobacco smoke and shear stress. C4d* gC1qR/p33* ICAM-1* * Results are expressed relative to baseline. P values were obtained by ANOVA. NA: Baseline 1 1 1 Tobacco smoke 0.03 puffs/ml 1.25±0.2 (n=5, P&lt;0.05) NA NA Shear 18 dynes/cm2 1.45±0.4 (n=5, P&lt;0.05) NA NA Tobacco smoke plus shear 1.92±0.5 (n=5, P&lt;0.05) 1.51±0.4 (n=4, P&lt;0.05) 1.33±0.2 (n=4, P&lt;0.05)