[Cytohistologic and immunohistochemical characteristics of the aortic intima and media in coarctation of the aorta of the adult type]. / Citohistoloske i imunohistohemijske odlike intime i medije aorte u koarktaciji adultnog tipa.

INTRODUCTION: Classically, coarctation of the aorta has been divided into infantile and adult forms. The vascular malformation responsible for coarctation is a defect of the vessel intima and media giving rise to a prominent posterior infolding ("the posterior shelf") which, in some cases, may extend around the entire circumference of the aorta. Histological examination of the coarcted aortic segment discloses intimal and medial lesion consisting of thickened ridges that protrude posteriorly into the aortic lumen. Intimal proliferation and disruption of elastic tissue may occur in adult type. OBJECTIVE: The smooth muscle cells phenotype in the aortic intimal thickening, presence of inflammatory cells and contents of the intimal and medial pseudocysts were investigated. MATERIAL AND METHODS: The samples of coarctation segments excised at surgery from 10 patients aged from 2 to 13 years were examined. For light microscopy, the specimens were dehydrated in graded ethanol (70-100%), cleared in xylol and embedded in paraffin. Sections of 5 microm thick were cut on Leica SM 2000R and Leica Reinhart Austria microtome and stained with orcein and Alcian blue-PAS at pH 1.0 and pH 2.5. Immunocytochemical staining was performed on 5 microm sections from formaldehyde-fixed paraffin-embedded blocks, using a labeled streptavidin-biotin method with an LSAB kit (Dako). Sections were deparaffinized and rehydrated. After microwave treatment of 21 minutes in citrate buffer pH 6.0, endogenous peroxidase activity was blocked with 3% H2O2 for 15 minutes. The sections were first incubated with the primary antibody for 60 minutes (alpha-smooth muscle actin-alpha-SMA, vimentin, desmin, myosin haevy chains-MHC, CD3, CD45, S-100 and Proliferating Cell Nuclear Antigen-PCNA), then with biotinylated link antibody and finally with peroxidase-labeled streptavidin. Slides were counter-stained with hematoxylin, washed in water and mounted. For electron microscopy, the primary fixative consisted of 2.5% glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer (pH 7.4) for 24 h at 4 degrees C. The specimens were postfixed for 1 h at 4 degrees C in 1% osmium tetroxide in 0.1 M cacodylate buffer and 4.8% uranyl acetate for 24 h at 4 degrees C. The samples were dehydrated in graded ethanol (70-100%) and embedded in Epon 812. The samples were cut with a diamond knife on an LKB Ultratome. Ultra-thin sections were stained with 2% uranyl acetate and alkaline lead citrate. RESULTS: All samples had focal intimal thickening on the posterior aortic wall, with accumulation of mucins which were stained with Alcian blue-PAS on pH 1.0, followed by prominent hypocellularity. Rare smooth muscle cells (SMC) showed immunoreactivity on alpha-SMA and vimentin, but not on desmin, MHC or CD3 and CD45. A large number of cells in apoptosis was noticed in the inner media on the posterior wall. On the anteromedial wall, a large number of PCNA- and S-100- positive cells was noted in the inner media while one layer of MHC- and desmin-positive cells was noted in the outer media. The elastic lamellae were focally disrupted by pools which were stained with Alcian blue-PAS at pH 1.0. DISCUSSION: In all examined samples, the immunocytochemical and TEM results revealed the presence of dedifferentiated smooth muscle cells which express alpha-SMA and vimentin, with a lack of expression of desmin and MHC. Results of this study also showed the reduction of cell number in the intima and media, followed by apoptotic smooth muscle cells in the inner media of the posterior wall and the absence of inflammatory cells. Such finding suggests that apoptosis but not necrosis may be the mechanism of reduction of cell number. The presence of smooth muscle cell proliferation in the inner media of the anteromedial wall and one layer of differentiated SMC in the outer part may lead us to suppose that changes of media (including dedifferentiation of the cells and disruption of elastic tissue) appear from inner to outer part and from posterior to anteromedial wall. The presence of pseudocysts which are stained with Alcian blue-PAS at pH 1.0 show large amount of mucins in elastic fibers. CONCLUSION: The intimal thickening on the posterior aortic wall is composed of small number of dedifferentiated smooth muscle cells (SMC). Some of these cells are in apoptosis. On the anteromedial wall, the intima and media are composed of proliferated SMC and small number of SMC which exhibit contractile phenotype. In all parts of the aortic wall, there is a large number of pseudocysts with large amount of mucins, without presence of inflammatory cells.