Elastin Damage and Vascular Smooth Muscle Cell Alterations Are the Initial Changes in a Novel Model of Tobacco Smoke-induced Aortic Injury

Abstract

Tobacco smoke exposure is a major risk factor for aortic aneurysm development. However, the initial aortic response to tobacco smoke, preceding aneurysm formation, is not well understood. We developed a novel model to examine early effect of solubilized tobacco smoke (STS) on the thoracic and abdominal aorta of mice as well as on cultured human aortic smooth muscle cells (HASMC). Nineteen Male C57BL6 mice received an infusion of STS or vehicle via osmotic minipumps for 2 weeks. Six mice were treated with vehicle-containing pumps, eight mice received STS pumps in the peritoneal position and five mice received STS pumps in the subcutaneous position. The thoracic and abdominal aortas were assessed for elastic lamellar damage, smooth muscle cell response, and infiltration of inflammatory cells. Elastin breaks were counted by two independent investigators and differences between groups were assessed using a two-tailed Student t-test. Cultured HASMC were exposed to STS after growth in differentiation media and assessed for α-smooth muscle actin (αSMA) and Mac-2 expression using immunofluorescence and western blot analysis. STS exposure caused increased numbers of breaks in the elastic lamellae of the thoracic and abdominal aorta (Fig 1; P < .001). Elastin breaks occurred more frequently in the abdominal aorta than the thoracic aorta (3.7 breaks per cross-section vs 2.3 breaks per cross-section; P < .001). In vivo, STS exposure caused microdissections of the aorta and a phenotypic switch in vascular smooth muscle cells characterized by downregulation of αSMA (Fig 2) and expression of Mac-2. Cultured HASMC exposed to solubilized tobacco smoke demonstrated loss of αSMA compared to HASMC not exposed to STS. Western blot analysis confirmed the loss of αSMA in HASMC exposed to tobacco smoke. HASMC exposed to STS demonstrated altered, rounded morphology within 2 days of tobacco smoke exposure, correlating with the αSMA loss. In initial experiments, Mac-2 expression was not different between HASMC exposed to STS and those exposed to vehicle. We demonstrate a new model for investigating the initial structural and cellular aortic changes in response to tobacco smoke exposure. Our findings suggest that STS exposure causes aortic elastic lamellar damage and phenotypic changes in VSMC, involving αSMA downregulation and possible Mac-2 upregulation. The VSMC phenotypic changes indicate a loss of normal contractile phenotype and expression of inflammatory markers. Utilizing this model, the mechanism of early tobacco smoke-induced aortic injury and strategies for early detection and prevention of aortic injury prior to clinical disease development can be investigated.Fig 2Immunofluorescence staining of aortic microdissection showing Mac-2 expression (bright green) and α-smooth muscle actin (αSMA) expression (red). The area of microdissection (*) shows increased Mac-2 expression (thick arrow, right) and decreased αSMA expression (thick arrow, left) compared to normal area or aorta showing no Mac-2 expression and normal αSMA expression (thin arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT)