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Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated, Ca2+-dependent exocytosis of WPBs critically depends on their proper targeting to the plasma membrane, but the mechanism of WPB-plasma membrane tethering prior to fusion is not well characterized. Here we describe a method to visualize and analyze WPB tethering and fusion in living human umbilical vein endothelial cells (HUVEC) by total internal reflection fluorescence (TIRF) microscopy. This method is based on automated object detection and allowed us to identify components of the tethering complex of WPBs and to monitor their dynamics in space and time. An important tethering factor identified by this means was Munc13-4 that was shown to interact with S100A10 residing in a complex with plasma membrane-bound annexin A2.

We previously reported that reducing the expression of the gap junction protein connexin (Cx)43 in mice restricts intimal thickening formation after acute vascular injury by limiting the inflammatory response and the proliferation and migration of smooth muscle cells (SMCs) toward the damaged site. SMC populations isolated from porcine coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening, suggesting that they participate in the restenotic process. Here, we further investigate the relationship between connexin expression and SMC phenotypes using porcine coronary artery SMCs. Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was downregulated and Cx43 was markedly upregulated in stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. In R-SMCs, Cx43 expression was increased and Cx40 was absent. We confirmed that S-SMCs treated with platelet-derived growth factor-BB acquire an R phenotype. This was accompanied by an upregulation of Cx43 and a loss of Cx40. Importantly, platelet-derived growth factor-BB–induced S-to-R phenotypic change was prevented by a reduction of Cx43 expression with antisense, ie, S-SMCs retained their typical elongated appearance and the expression of α-smooth muscle actin, a well-known SMC differentiation marker, whereas the expression of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in S-SMCs prevents platelet-derived growth factor-BB–induced S-to-R modulation. This suggests that Cx43 may be an additional target for local delivery strategies aimed at reducing restenosis.

We studied the effects of cytostatic drugs on porcine coronary artery spindle-shaped (S) and rhomboid (R) smooth muscle cell (SMC) biological activities related to intimal thickening (IT) formation.Imatinib, and to a lesser extent curcumin, decreased proliferation of S- and R-SMCs and migratory and urokinase activities of R-SMCs more efficiently compared with cyclosporine plus rapamycin. Imatinib increased the expression of α-smooth muscle actin in both SMC populations and that of smoothelin in S-SMCs. It decreased S100A4 expression in R-SMCs.By promoting SMC quiescence and differentiation imatinib and curcumin may represent valid candidates for restenosis preventive and therapeutic strategies.

Purpose To investigate whether the peeled internal limiting membrane (ILM) contains cellular retinal cell fragments, and to learn more about their possible origin. Design Experimental study. Methods ILM peeled from ten eyes during vitrectomy by infracyanine green (ICG) was studied immunohistochemically using the markers: GFAP, S-100, and vimentin. Five ILM specimens were from eyes with diabetic macular edema (DME), two from eyes with a macular hole, and three from eyes with persisting macular edema after retinal detachment surgery. Results In eight of the ten ILM specimens, we found GFAP-positive staining, indicating the presence of remnants of footplates from Muller cells or glial cells. Two ILM specimens were positive for S-100, indicating the presence of neural cells or ganglion cells. Conclusions ILM peeled from the retina during vitrectomy using ICG may contain remnants of Muller cell footplates, neural cells, and ganglion cells.

The p63 gene encodes six protein isoforms. The transactivating isoforms have similar actions with p53, while the N-isoforms inhibit transcription activation by p53 and transactivating isoforms. p63 is expressed in stratified epithelia and in basal cells of the prostate and salivary glands. In mammary epithelium p63 has been shown to be expressed only in the myoepithelial layer. In the present study we investigated the immunohistochemical expression of p63, in benign and malignant breast lesions, and compared it with known myoepithelial cell markers. Our material consisted of 140 benign and 126 malignant breast lesions. We used the antibodies anti-p63, anti-a-smooth muscle actin, anti-S-100 protein and anti-cytokeratin 14. In all benign lesions, p63 immunoreactivity was noted in the myoepithelial cell layer surrounding the luminal epithelial cells. A less continuous peripheral rim of myoepithelial cells was also highlighted with p63- staining in all situ carcinomas. All invasive breast carcinomas were devoided of peripheral p63 staining. Interestingly, strong nuclear p63 immunoreactivity was noted in a small fraction (5-15%) of epithelial cells in all cases of papillomatosis, in 62.5% of in situ ductal papillary-type carcinomas and in 33.3% of invasive papillary carcinomas. Comparable staining was observed with S-100. The stromal cells were unreactive to p63. Our findings suggest that p63 is a sensitive and specific myoepithelial marker, and may be included in immunohistochemical panels aiming to identify myoepithelial cells in problematic breast lesions. Regarding papillary neoplasms, it is possible that tumor cells acquire and exhibit at least in part a myoepithelial differentiation program.