Etosis, rather than apoptosis or cell proliferation, typifies thrombus progression – An immunohistochemical study of coronary aspirates

Figures

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  Fig. 1. Cell death and cell proliferation in coronary thrombus. Positive staining of CRP (A-D), Casp3 (E-H), CitH3 (I-L) and Ki67 (M-P) visualized in Perma Blue and counterstained with Feulgen pink to identify necrosis (A-D), apoptosis (E-H), etosis (I-L) and proliferation (M-P), respectively, in different stages of thrombus progression: fresh (A, E, I and M), lytic (B, F, J and N), organized (C, G, K and O); and in plaque components within thrombus (D, H, L and P). CRP also stained non-necrotic areas in fresh (A) and organized (C) thrombus, therefore, is not a reliable marker for necrosis. Apoptosis and etosis mostly occurred in lytic (B and E, respectively), whereas cell proliferation in organized thrombus (O). Scale bar in A and B: 25 µm.

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  Fig. 2. Apoptosis, etosis and proliferation rates in different stages of thrombus progression. Rates of apoptosis, etosis and proliferation were measured as a percentage of total number of immunopositive pixels of Casp3(A), CitH3(B) and Ki67(C), respectively, versus total number of Feulgen positive pixels in the selected region of interest in fresh (FR), lytic (LY), early (EO) and late organized (LO). The highest rates of etosis and apoptosis were found in lytic while the lowest in organized thrombi (*p < 0.05, significant to LO). In contrast, rate of proliferation was higher in organized than in fresh and lytic thrombi.

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  Fig. 3. Cellular sources of apoptosis in coronary thrombus. Representative images of apoptotic cells in coronary thrombus (top to bottom): granulocytes (A-D), macrophages (E-H), platelets (I-L), smooth muscle cells/SMC (M-P) and endothelial cells (Q-T). Boxed areas in HE stains (A, E, I, M and Q) show the areas of interest for higher magnification of double immunostaining Casp3 (C, G, K, O and S) antibodies with cell specific antibodies: anti- CD15 (B), CD68 (F), CD61 (J), αSMA (N) and CD34 (R) as well as of false-colour images to show co-localization of the positive cells (in red) with Casp3+ (in green) as apoptotic cells (D, H, L, P and T in yellow). Scale bar in HE overview (A): 100 µm and in high power detail (B and F): 25 µm; (J, N and R): 10 µm.

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  Fig. 4. Sources of proliferating cells in coronary thrombus. Representative images of cells undergoing proliferation in coronary thrombus (top to bottom): macrophages (A-D), smooth muscle cells/SMC (E-H), and endothelial cells (I-L). Boxed areas in HE stains (A, E, I) show the areas of interest for higher magnification of double immunostaining Ki67 (C, G, K) antibodies with cell specific antibodies: anti- CD68 (B), αSMA (F) and CD34 (J), as well as of false-colour images to show co-localization of the positive cells (in red) with Ki67+ (in green) as proliferating cells (D, H and L, in yellow). Scale bar in HE overviews (A and I): 100 µm and in high power detail (B and F): 25 µm.