Concordant upregulation of type II-TGF-β-receptor, the cyclin-dependent kinases inhibitor P27Kip1 and cyclin E in human atherosclerotic tissue: implications for lesion cellularity Original Research Article

Atherosclerosis is a ‘response-to-injury’ process associated with chronic inflammation, tissue repair and a considerable cell turnover. These growth-related processes are controlled by the ‘cell cycle clock’ which is composed of cyclin-dependent kinases (Cdks), their activating subunits, the cyclins, and by inhibitors of Cdks (Ckis). P27 is a Cki which associates with cyclin A–Cdk2, cyclin D–Cdk4 and with cyclin E (CE)–Cdk2 complexes thereby abrogating their catalytic activity leading to potent inhibition of late G1 to S-phase transition. Furthermore, TGF-β1 mRNA and immunoreactivity are locally increased in atherosclerotic lesions. Since TGF-β1 growth suppressive function in the late G1 phase may be mediated by p27, blocking the catalytic activity of CE-Cdk2 complexes, via the stimulation of TGF-β-RI and TGF-β-RII, we investigated the topographical association between TGF-β-RI, TGF-β-RII, P27Kip1 and CE by immunohistochemistry in coronary artery segments without atherosclerosis and carotid atheromatous plaques of 11 patients undergoing carotid endarterectomy. P27-immunoreactivity was present in 11/11 atherosclerotic (92.7±3.3% of the cells) and 5/5 control (80.9±3.7% of the cells; P<0.002 versus control) specimens and localized to nuclei of macrophages (CD68-positive), vascular smooth muscle cells (α-actin positive), T-lymphocytes (CD3-positive) as well as to the nuclei of endothelial cells. In the atherosclerotic tissue, TGF-β-RI and TGF-β-RII-immunoreactivity was present in 11/11 specimens and localized to inflammatory cells and to cells with VSMC-like-morphology. TGF-β-RI-immunoreactivity was present in 87.4±5.3% (controls 75.3±7.48%; n.s.) and TGF-β-RII-immunoreactivity was present in 83.7±6.8% (controls 39.5±7.3%; P<0.002) of the cells. Double immunolabeling, and investigation of serial sections revealed co-expression of TGF-β-RI and TGF-β-RII in virtually all cells positive for P27. In the atherosclerotic specimens, CE-immunoreactivity was present in all specimens in macrophages (CD68-positive), vascular smooth muscle cells (α-actin positive) and in endothelial cells in 12.58±13.58% of the nuclei whereas in the controls CE staining was restricted to 0.19±0.43% of the cells (P<0.001). Importantly, as shown by immunofluorescent double-labeling, we found cells expressing P27 that were simultaneously positive for CE. In summary, the present study provides evidence that TGF-β1 present in human atherosclerotic tissue may mediate its growth suppressive activity also by p27, blocking the activity of CE–Cdk2 complexes. Quantitative analysis revealed that TGF-β-RII, p27 and CE are concordantly upregulated in the atherosclerotic tissue with chronic inflammation, supporting the view that TGF-β1, p27 and CE may play an important role in the processes associated with chronic inflammation and cell turnover in advanced human atherosclerotic plaques. Taken together, these results provide a possible link between the chronic inflammation associated with advanced atherosclerosis, the effects of extracellular growth factors and cell cycle control.