Studies on the histogenesis of myxomatous tissue of human coronary lesions.

Myxomatous tissue is a characteristic component of human coronary artery lesions, found more often in restenotic lesions. It represents a bulky accumulation of stellate-shaped cells of unknown histogenesis that are embedded in a loose stroma. We analyzed 64 atherectomy specimens containing substantial amounts of myxomatous tissue by using immunohistochemistry, in situ hybridization, and electron microscopy techniques. Stellate cells represented a heterogeneous population, sharing features of smooth muscle cells (SMCs), macrophages, as well as antigen-presenting dendritic cells. Like quiescent medial SMCs, the stellate cells in all specimens expressed high levels of SM alpha-actin message and protein and showed heterogeneity with respect to heavy-chain myosin, SM22, desmin, and vimentin. Ultrastructurally, stellate cells resembled SMCs, with some peculiarities that distinguish them from both differentiated and dedifferentiated SMCs. In contrast to quiescent SMCs, the stellate cells expressed high levels of acidic fibroblast growth factor mRNA and protein similar to cells of monocyte/macrophage lineage. However, stellate cells did not express the marker of mature macrophages, HAM56, and were heterogeneous with respect to CD68. Moreover, unlike SMCs, the stellate cells bore some of the major phenotypic markers of dendritic cells: they were S100-positive and showed various reactivity with respect to CD1a and human leukocyte antigen (HLA)-DR. Invasion of myxomatous tissue with CD45RO-positive T lymphocytes was correlated with strong expression of CD1a in these specimens. Stellate cells also expressed a pericyte marker, high-molecular-weight melanoma-associated antigen. We conclude that stellate cells of myxomatous tissue represent a specific phenotype of mesenchymal cells (possibly pericytes), which is activated to express some markers of antigen-presenting cells. These findings suggest involvement of the stellate cells in a local immune response.

Oxidized LDL mediates the release of fibroblast growth factor-1.

Fibroblast growth factor-1 (FGF-1) and lipoproteins play an important role in atherogenesis. In the present study, we explored a possible mechanism by which abnormal lipid metabolism could be linked to the proliferative aspects of the disease. We tested oxidized LDL (oxLDL) as a possible pathophysiological mediator of the release of FGF-1, using FGF-1-transfected mouse NIH 3T3 cells and FGF-1-transfected rabbit smooth muscle cells, and compared it with the release caused by elevated temperature. Immunoblot analysis showed that oxLDL induced the release of FGF-1 in a concentration-dependent manner from 10 to 100 micrograms/mL. The effect correlated with the extent of oxidative modification of LDL and was maximal within 4 hours of exposure of cells to oxLDL. In contrast to the temperature stress-induced FGF-1 secretion pathway, FGF-1 released in response to oxLDL (1) appeared in the conditioned medium as a monomer, (2) appeared independently of the presence of either actinomycin D or cycloheximide, and (3) was neither enhanced nor inhibited by brefeldin A. We did not detect cell loss, significant morphological changes, changes in growth characteristics, or other indications of lethal toxicity in oxLDL-treated cells. Although the level of lactate dehydrogenase activity was elevated after oxLDL exposure, the calculations showed that > 90% of the FGF-1 was released by viable cells. We propose that oxLDL-induced FGF-1 release is mediated by sublethal and apparently transient changes in cell membrane permeability. In the environment of an atherosclerotic lesion, oxLDL-induced FGF-1 release may be among the mediators of endothelial and smooth muscle cell proliferation.

Proliferative response of smooth muscle cells in hypertension.

A significant increase (up to 20% from about 10% in normals) in the number of smooth muscle cells (SMC) with tetraploid DNA content was found in the media and intima of human hypertensive aorta. A similar process was detected during normal human vessel aging. It was found that SMC from normal human aorta and normotensive rat aorta, which were able to incorporate 3H-thymidine, had diminished proliferative potency and a tendency to polyploidization in primary culture. We failed to detect a similar phenomenon in SMC obtained from aorta from spontaneously hypertensive rats. It was found that 10 mumol/L of norepinephrine significantly increased (by approximately twofold) the frequency of true polyploid cells in a subculture of rat aortic SMC. The effect of norepinephrine was blocked only by simultaneous action of alpha- and beta-adrenoreceptor antagonists. SMC polyploidization was also stimulated by simultaneous application of two direct activators of the second messenger systems, forskolin and phorbol-12-myristate-13-acetate. Thus, a subpopulation of SMC prone to polyploidization exists in normal vessels, and norepinephrine may be one of the mediators of the "hypertensive" response of vessel wall SMC, which probably occurs due to the synergism of two second messenger systems.

A 90-kDa surface antigen of immature smooth muscle cells is ICAM-1.

A monoclonal antibody, designated 10F3, that reacts with an antigen of molecular mass 90,000 Da has been developed by immunization of BALB/c mice with smooth muscle cells in long-term culture. The cells were originally isolated from fetal human aorta. The 10F3 was identified as an antibody that reacts with the ICAM-1 molecule. ICAM-1 is a mesenchymal antigen that is lost during differentiation of cells other than endothelium but is reexpressed by the intimal cells of vessels involved in atherogenesis.

Morphogenesis of intimal thickening in nonspecific aortoarteritis.

Hyperplastic intima in nonspecific aortoarteritis was investigated immunomorphologically and by means of electronmicroscopic autoradiography. The cellular composition of the involved vascular wall was studied by the method of alcohol-alkaline dissociation. The prevalence of elongated cells with side processes and stellate cells in thickened intima was shown. These cells are characterized by essentially intensive RNA synthesis and, according to the electron microscopic data, can be regarded as pericytes. A significant amount of capillaries and precapillaries in thickened intima was visualized. Distribution of laminin and type IV collagen coincided with localization of the basal membrane of the vasa vasorum and with that of microvessels growing in the intima. Considered together, these data suggest that pericytes that surround a capillary/precapillary network may play a significant role in the morphogenesis of vascular transformation in nonspecific aortoarteritis intimal thickening.

Noradrenaline induces the polyploidization of smooth muscle cells: the synergism of second messengers.

The effect of noradrenaline (NA) on DNA replication of cultured smooth muscle cells (SMC) isolated from rat aorta was examined. It was found that 10 microM NA significantly increased (approximately by twofold) the frequency of tetraploid cells. Cultivation of 4C cells isolated by flow cytometric cell sorting revealed that they were true polyploid cells. This receptor-mediated effect of NA was blocked only by simultaneous action of alpha- and beta-adrenoreceptor antagonists. SMC polyploidization was also stimulated by simultaneous application of direct activators of "second messenger" systems forskolin and phorbolmyristate-acetate. Thus, NA may be one of mediators of the "hypertensive" response of vessel wall SMC, which probably occurs due to synergism of two second messenger systems.

A 90-kd surface antigen from a subpopulation of smooth muscle cells from human atherosclerotic lesions.

A monoclonal antibody, designated 10F3, that reacts with an antigen with a molecular weight of 90,000 daltons has been obtained after immunization of BALB/c mice with long-term cultured smooth muscle cells (SMC) originally isolated from fetal human aorta (fSMC). In adults the antigen is present on venous, arterial and capillary endothelial cells of heart, kidney, liver, spleen, intestine, skin, uterus, placenta, and arteries only, as shown by immunohistochemical investigation using the PAP technique. The antigen 10F3 is also present on the mesenchymal cells of human fetal tissues (7 and 18-week-old fetuses) and on SMC of 7-week-old fetal aorta, and a subpopulation of cells reacting with 10F3 antibody also has been found in atherosclerotic intima. Double staining using 10F3 antibody and muscle actin-specific monoclonal antibody HHF-35 showed that the antigen-positive cells are smooth muscle cells. In primary culture of adult SMC, antigen-positive cells were detected 2 days after seeding (about 90% positive in medial and intimal cultures). It is suggested that 10F3 is a mesenchymal antigen that, lost during differentiation by cells other than endothelium, but expressed again by the SMC involved in atherogenesis.

The expression of specific surface antigen of smooth muscle cells is related to proliferation.

Monoclonal antibody L1 has been obtained after immunization of BALB/c mice with long-term cultured smooth muscle cells (SMC) originally isolated from rat aortic media. Antibody L1 recognizes only the surface antigen of cultured SMC and does not react with other cultured rat cell types. It has been shown that in primary culture of SMC the L1-positive cells appear on the 2nd to 3rd day and their proportion increases up to the 7th day up to 40% in DMEM supplemented with 10% of fetal calf serum (FCS), up to 25% in DMEM with 5% of rat whole-blood serum, but up to only 5% in DMEM with 5% rat plasma-derived serum. These results are in agreement with data on [14C]thymidine incorporation and on flow cytometry. Using FACS II, the SMC were sorted into subpopulations on the 4th and 8th days of primary culture according to the intensity of their specific immunofluorescence. It has been found that the DNA profile in intensively labelled cells corresponds to that in an intensively proliferating population of cells. These findings suggest that antigen L1 appears to be the specific marker of modulated SMC entering the cell cycle.

Proliferation kinetics of aortic smooth muscle cell populations: comparison of normotensive and spontaneously hypertensive rats.

An in vitro autoradiographic study of the proliferation of smooth muscle cells (SMC) from the aorta of normotensive and spontaneously hypertensive rats has been made. It was found, in primary culture, that SMC of spontaneously hypertensive rats entered proliferation at 2-2.5 days later than those from normotensive animals. As revealed by their very intensive labelling, a subpopulation of SMC with a high turnover rate was found in primary culture. In freshly isolated SMC from normotensive rat aorta, a subpopulation in S phase was detected, but we failed to detect it in aortae from spontaneously hypertensive rats. A difference in proliferative behaviour was also observed in subcultures of SMC from rats of both strains.