Platelets and smooth muscle cells affecting the differentiation of monocytes.

BACKGROUND: Atherosclerosis is characterised by the formation of plaques. Monocytes play a pivotal role in plaque development as they differentiate into foam cells, a component of the lipid core whilst smooth muscle cells (SMC) are the principal cell identified in the cap. Recently, the ability of monocytes to differentiate into a myriad of other cell types has been reported. In lieu of these findings the ability of monocytes to differentiate into SMCs/smooth muscle (SM)-like cells was investigated. METHOD AND RESULTS: Human monocytes were co-cultured with platelets or human coronary aortic SMCs and then analysed to assess their differentiation into SMCs/SM-like cells. The differentiated cells expressed a number of SMC markers and genes as determined by immunofluorescence staining and quantitative polymerase chain reaction (qPCR). CD array analysis identified marker expression profiles that discriminated them from monocytes, macrophages and foam cells as well as the expression of markers which overlapped with fibroblast and mesenchymal cells. Electron microscopy studies identified microfilaments and increased amounts of rough endoplasmic reticulum indicative of the SM- like cells, fibroblasts. CONCLUSIONS: In the appropriate environmental conditions, monocytes can differentiate into SM-like cells potentially contributing to cap formation and plaque stability. Thus, monocytes may play a dual role in the development of plaque formation and ultimately atherosclerosis

Immunofluoresence staining of M+SMCs, M+Ps and Monocytes on day 14 of culture.

<p><b><i>M+SMCs</i>:</b> A) CD34 (red), B) Procollagen I (green), C) the expression of double staining of CD34 (red cell surface) and procollagen I (green cytoplasm). D) αSM actin and E) calponin (both green). <b><i>M+P:</i></b> F) CD34 (red), G) Procollagen I (green), H) positive expression of both CD34 and procollagen I (orange). I) αSM actin and J) calponin (both green). <b><i>Monocytes:</i></b> lack of expression of αSMC actin and calponin (K & L respectively). <b><i>Positive controls:</i></b> M) CD34 (red), N) Procollagen I (green), O) αSMC actin (green), P) Calponin (green). <b><i>Isotype controls:</i></b> (Q) rabbit IgG, (R) mouse IgG and (S) rat IgG. Nuclei of the cells are blue. Scale bars 100 µm.</p

CD surface markers expressed by each cell type.

<p>The table records all the markers which were significantly (P<0.001) expressed by each cell type listed in the horizontal axis (bold font) when compared to the cell types listed in the vertical axis (italics font) with their associated 95% confidence intervals (CI). For example, monocytes expressed the marker CD182, 53.26 fold significantly higher than macrophages.</p

Primers used for Real Time PCR.

<p>Primers used for Real Time PCR.</p

Electron microscopy analysis of M+SMCs and M+Ps.

<p><b><i>M+SMCs</i>.</b> Figure A) Day 7 of culture containing ribosomes (arrows) (B). Figure C) Day 10 of culture with microfilaments* surrounded by lysosomes (Ly) (D) and present closer to the nucleus (E) <b><i>M+Ps</i></b> Figure F) Day 7 culture containing microfilaments* (G). H) Day 10 culture containing microfilaments* surrounded by monoribosomes (arrows) (I). Figure J) Day 5, containing rough endoplasmic reticulum (rER) with ribosomes attached (small electron dense beads) (arrows) and day 14 cells showing rER with average length of 1.9 µm (Figure K). <b><i>Monocytes</i></b> Figure L) Day 14 culture with the presence of autophagy (A) and a nucleus (N).</p

List of antibodies used for staining. (N/A = no designation for clone).

<p>List of antibodies used for staining. (N/A =  no designation for clone).</p

Monocytes co-cultured with HcaSMCs (M+SMCs) and platelets (M+Ps) induce the expression of smooth muscle cell markers analysed by qPCR.

<p>A) M+SMCs (n = 3) and B) M+Ps (n = 3) were compared with cultured monocytes for the expression of αSM actin, collagen I, calponin and CD34. Data were normalised to the expression of GAPDH. Statistically significant * denotes P-value<0.05.</p