Abstract 103: Cross-Talk Between Macrophages, Smooth Muscle Cells and Endothelial Cells in Response to Cigarette Smoke: The Effects on MMP2 and -9

Abstract

Introduction: Abdominal Aortic Aneurysm (AAA) development is linked to smoking and is characterized by extracellular matrix remodeling by MMP2 and -9. We hypothesized that MMP2 and -9 secretions and regulation in response to cigarette smoke is modulated by interactions between resident cells within the aorta, namely, aortic smooth muscles, endothelial cells, and infiltrating macrophages and could influence AAA formation and/or progression. Methods: Aqueous extract of cigarette smoke (AEC) was applied to male rat aortic smooth muscle (RASMC), endothelial (RAEC) or RAW cells in serum free medium (SFM). After 24h the cells were washed, fresh SFM was added and collected after 24h. This conditioned media (CM) was applied to the cells for 24h, again washed, fresh SFM added, and then incubated for 72h, at which point the media was saved for MMP2 and -9 analysis by zymography while the cellular protein was used to determine the ratio (pS/pJ) of phospho-Stat3 (pStat3) and phospho-Jak2 (pJak2) by western blot. Results: Unstimulated CM from RAW cells increased MMP2 by 24% (p=0.0357), MMP9 by 282.1% (p=0.0004) and pS/pJ by 136.5% in RASMC. AEC-CM from RAW and RAEC cells increased MMP9 by 200.5% (p=0.0004) and 17.1% (p=0.0301) respectively in RASMC and corresponding increases in pS/pJ (305.7% and 227.6%, respectively). AEC-CM derived from RAW cells induced RAEC to produce moderate amounts of MMP2 (16.5%, p=0.0140) and MMP9 (29.5%, p=0.0207) and a 137.0% increase in pS/pJ. RAW cells receiving unstimulated CM from RASMC and RAEC cells produced significant amounts of MMP9 (127.7%, p=0.0026 and 236.8%, p=0.0020, respectively) and increased levels of pS/pJ ratios (45.2% and 1282.8%, respectively). Moreover, AEC-CM from RASMC and RAEC induced significant production of MMP9 from RAW cells (154.8%, p=0.0001 and 162.4%, p=0.0022, respectively) and increase in pS/pJ ratios (1348.2%, and 1493.9%, respectively) over baseline. Conclusion: This is the first in vitro study demonstrating that cigarette smoke results in differential interactions between aortic smooth muscle cells, endothelial cells, and macrophage lineage cells resulting in enhanced levels of MMP2, MMP9, pStat3 and pJak2. These interactions may play an important role in AAA formation under in vivo conditions.