Phenotypes of vascular smooth muscle sense bacteria differently: implications for tlrs in vascular health

Shaun McMaster, Mark Paul-Clark, *Al-Rehan Dhanji, Shiranee Sriskandan, *Timothy D. Warner and Jane A. Mitchell. Cardiothoracic Pharmacology, Critical Care, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK. *The William Harvey Research Institute, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London EC1M 6BQ.

Vascular smooth muscle cells constitute the main cell type of the vessel wall. In a healthy vessel these cells have little or no secretory function. However, when vessels are inflamed, vascular smooth muscle express vasoactive genes and release large amounts of vasoactive hormones including nitric oxide (NO). The vessel wall is comprised of different phenotypes of vascular smooth muscle, of which one broad differentiation is between contractile and epithelioid. The relative role of different phenotypes of vascular smooth muscle in health and disease is not well understood. Infection of the vessel wall with bacteria is now a recognised risk factor for vascular disease and acute cardiovascular events. Thus, in the current study we have compared the ability of different phenotypes of vascular smooth muscle cells to ‘sense’ pathogen activated molecular patterns (PAMPs) including those associated with Gram negative E. Coli (which activates Toll like receptor (TLR4) and Gram positive S. aureus (which activates TLR2; Jimenez et al., 2005).

Rat vascular smooth muscle cells were cultured by explant in DMEM containing 10% FCS, using standard techniques. Epithelioid or contractile (spindle shaped) cells were identified and clonal cultures of epithelioid and contractile phenotypes were grown from single cell populations. For comparison, primary cultures of rat aortic smooth muscle cells (RASMs; which contain mixed populations of the different smooth muscle cell phenotypes) and J774 murine macrophages were used. Cells were incubated with bacteria or selective PAMPs for 24 or 48 hours. Medium was then removed and nitrite measured (using the Griess reaction) as and index of NOSII activity.

At 24 hours, under control culture conditions NO release by vascular smooth muscle was <5μ M. E. coli (108 CFU/ml) induced NO release from primary cultures of RASMs (15±3 μM), epithelioid (22±1 μM) and contractile (18±1 μM) phenotypes of vascular smooth muscle. S. aureus (3x10 8 CFU/ml) induced NO release from RASMs (19±2 μM) and from the contractile phenotype (12±0.1 μM) of vascular smooth muscle cells, but not from the epithelioid phenotype. When TLR selective PAMPs (lipopolysaccharide, LPS from E. Coli , 10 μg/ml; PAM3CSK4, 1 μg/ml; FSL-1, 1 m g/ml; which activate TLR4, TLR2/TLR1 and TLR2/TLR6 respectively) were added to cells, they had no effect on NO release by any of the vascular smooth muscle cells tested. J774 macrophages released NO in response to E. Coli (22±1 μM) or LPS (28±1 m M) but not to S. aureus.

These results show that bacterial PAMPs are sensed differently by vascular smooth muscle cells of different phenotypes as well as macrophages. These results have implications for our understanding of how infection may modulate vascular inflammation.

Jimenez, R et al.,Proc Natl Acad Sci U S A. 2005 Mar 22; 102(12):4637-42.

Funded by the British Heart Foundation and the European Union.