Proteoglycan biosynthesis is required in BC3H1 myogenic cells for modulation of vascular smooth muscle alpha-actin gene expression in response to microenvironmental signals.

Induction of vascular smooth muscle (VSM) alpha-actin mRNA expression during cytodifferentiation of mouse BC3H1 myogenic cells coincides with the accumulation of cell surface- and extracellular matrix-associated sulfated proteoglycans. Inhibition of proteoglycan biosynthesis in myogenic cells using an artificial beta-D-xyloside glycosaminoglycan acceptor was accompanied by a reduction in cell surface/extracellular matrix proteoglycans and VSM alpha-actin mRNA expression while enhancing the secretion of free chondroitin sulfate/dermatan sulfate and heparan sulfate glycosaminoglycans into the culture medium. Maximum inhibition of VSM alpha-actin mRNA expression required that proteoglycan biosynthesis be blocked during the early phase of cytodifferentiation when myoblasts were fully confluent and quiescent. The inhibitory effect of beta-D-xyloside on alpha-actin mRNA expression resulted from attenuation at both the transcriptional and post-transcriptional control points. Sustained proteoglycan biosynthesis was required for induction of VSM alpha-actin mRNA in quiescent myoblasts in response to cytodifferentiation-permissive, substrate-associated macromolecules (SAM) or upon exposure to soluble serum factors capable of transiently stimulating VSM alpha-actin gene transcription. The results suggested that efficient myoblast cytodifferentiation and modulation of VSM alpha-actin mRNA levels depended on intact cell surface proteoglycans to convey signals generated as a consequence of cellular interaction with substrate components and serum factors.

Clonal mapping of the human aorta. Relationship of monoclonal characteristics, lesion thickness, and age in normal intima and atherosclerotic lesions.

The surfaces of 8 aortas from women heterozygous for G-6-PD isoenzymes were mapped for an examination of the relationships of monoclonality, lesion type, lesion thickness, and age of the patient. The percent B isoenzyme value of samples of normal intima (n = 315), fatty steak (n = 68), or fibrous plaque (n = 64) was used to define monoclonality, expressed as the [Z] score, the number of standard deviations from the mean percent B isoenzyme of samples of underlying media. Intimal thickness increased significantly with type of lesion, such that intima less than fatty streak less than fibrous plaque, and with the age of the patient. The percentage of monoclonal portions also increased with lesion type, such that 1% of samples of normal intima were monoclonal, compared with 4.4% of fatty streaks and 12.5% of fibrous plaques (P less than 0.005). Monoclonality increased with intimal thickness when normal intima, fatty streaks, and fibrous plaques were combined (P = 0.0001). When examined separately, normal intima showed a direct correlation between monoclonality and intimal thickness. In contrast, the monoclonality of fatty streaks was inversely associated with thickness (P = 0.016) and the monoclonality of fibrous plaques not related to thickness. When entered into a multiple regression model, lesion type and age, but not lesion thickness, significantly predicted monoclonality. The lack of association of intimal thickness with monoclonality suggests that it is the type of lesion that determines monoclonality and not merely its thickness. This implies that mechanisms other than clonal selection are responsible for the monoclonal characteristics of human atherosclerotic lesions.

The clonal characteristics of human aortic intima. Comparison with fatty streaks and normal media.

The clonal characteristics of normal-appearing but thickened aortic intima were studied by the use of the isoenzymes of glucose-6-phosphate dehydrogenase (G-6-PD) as cellular markers in females heterozygous for this X-linked enzyme. Isoenzyme patterns of 133 samples of intima were compared with those of 237 samples of underlying media and with those of 58 fatty streaks dissected from the same aortas. The proportion of samples of intima and fatty streaks with monoclonal or intermediate characteristics was the same, but both had more monoclonal or intermediate samples than underlying media (P less than 0.05). However, samples of intima showed a central clustering tendency of isoenzyme values similar to that of underlying media, while values from fatty streaks showed a bimodal distribution suggesting the presence of cell populations in the process of becoming monoclonal. The data suggest that clonal proliferation may begin in normal-appearing intima but that it progresses through a fatty streak stage before proceeding to the monoclonal fibrous plaque.

Polyunsaturated fatty acid accumulation in the lipids of cultured fibroblasts and smooth muscle cells.

The lipid content per cell of cells in tissue culture depended on the cell type. Fibroblasts derived from human neonatal foreskin contained less triglyceride and phospholipid and more cholesteryl ester than smooth muscle cells derived from guinea pig aorta. When fibroblasts and smooth muscle cells were challenged with 120 microM polyunsaturated fatty acid, the fibroblasts accumulated much less fatty acid than smooth muscle cells. Fibroblasts and smooth muscle cells challenged with either 8, 11, 14-eicosatrienoic acid, 5, 8, 11, 14-eicosatetraenoic acid, or 7, 10, 13, 16-docosatetraenoic acid accumulated the polyunsaturated fatty acid, its microsomal chain elongation products, and its extramicrosomal retroconversion products in cellular lipids. A new retroconversion sequence was observed, 8, 11, 14-eicosatrienoic acid to 6, 9, 12-octadecatrienoic acid. Microsomal desaturation products did not accumulate. The total fatty acid content of the phospholipid fraction was unchanged in cells challenged with a fatty acid. The polyunsaturated fatty acid and its derivatives exchanged with fatty acyl groups in the cellular phospholipid fraction. These fatty acyl groups were transferred to the triglyceride fraction and the total cellular content of each fatty acid was conserved. The total fatty acid content of the triglyceride fraction was markedly increased in cells challenged with a fatty acid. The polyunsaturated fatty acid and its derivatives accumulated in the triglyceride fraction. The triglyceride fraction contained an unusual triacyl derivative of the polyunsaturated fatty acid. These data support the hypothesis that microsomal fatty acyl-CoA intermediates are shunted into neutral lipid droplets when cells stimulated to accumulate lipid.

Clonal characteristics of cutaneous scars and implications for atherogenesis.

Atherosclerotic plaques in man have monoclonal characteristics. One possible explanation for this observation is that clonal selection occurs as cells proliferate at sites of intimal injury. To test whether such clonal selection could occur during the formation of an intimal "scar", the clonal characteristics of human cutaneous scars were studied with the use of glucose-6-phosphate dehydrogenase (G-6-PD) as a cellular marker. Scars from 7 black women heterozygous for the A and B isoenzymes of G-6-PD were divided into 870 portions consisting of contiguous normal skin, scar margin, outer scar, and inner scar. Portions were further divided into surface and deeper portions of scar. In 4 of the 7 cases, significant differences were observed between the mean percentage of total activity in the B isoenzyme band in portions of scar and portions of skin. Significant differences between surface and deeper portions of both skin and scar were also observed in 5 cases. The variance of isoenzyme values from portions of scars was not significantly greater than that within portions of skin. It is concluded that the results are consistent with clonal selection occurring during the healing of skin wounds. However, the magnitude of the difference (no greater than a 5.7% B isoenzyme difference) is far too small to even partially explain the marked differences in isoenzyme patterns observed between atherosclerotic plaques and uninvolved aortic wall. The results do not support the idea that the monoclonality of human atherosclerotic plaques is due to clonal selection following the healing of an intimal injury.

Evidence for two populations of fatty streaks with different roles in the atherogenic process.

Isoenzymes of glucose-6-phosphate dehydrogenase, used as cellular markers, showed that a minority of fatty streaks have clonal characteristics intermediate between those of normal arterial wall and those of fibrous plaques, suggesting that such streaks are evolving into fibrous plaques. "Intermediate" fatty streaks are found to cluster in patients in the middle-age years, suggesting that few fatty streaks evolve into plaques in the advanced years. It is suggested that two populations of fatty streaks exist: one destined, perhaps by its location or risk factors, to develop into fibrous plaques; and a second which remains as fatty streaks and never goes on to become fibrous plaques. This hypothesis helps to explain some of the evidence disclaiming the fatty streak as a forerunner of the atherosclerotic plaque.

Clonal characteristics in layers of human atherosclerotic plaques. A study of the selection hypothesis of monoclonality.

Two hypotheses have been proposed to explain the origin of monoclonal cell populations within human atherosclerotic plaques. The first of these proposes a mutational origin: the second suggests that the single clone of cells with the greatest proliferative advantage is selected following repetitive intimal injury. If the selection hypothesis is true, monoclonality should be observed more frequently in the layer of plaque most recently formed. Glucose-6-phosphate dehydrogenase (G-6-PD) isoenzymes were used as cellular markers in aortas of females heterozygous for the A and B isoenzymes. Ten plaques were divided into 45 portions, each of which was subdivided into upper layer, lower layer, and underlying media. No predominance of monoclonality was observed in the upper or lower layers of plaque, with 53% of samples from each layer being monoclonal. In all, 73% of portions of plaque contained at least one monoclonal layer. The layers tended to resemble each other in their clonal characteristics, with 60% of portions having layers with the same clonal characteristics. A significant correlation between isoenzyme distributions in upper and lower layers of the same portion was observed. No consistent trends in isoenzyme distribution in the three layers of each portion were observed. The results are interpreted as providing no evidence for clonal selection as the mechanism by which human atherosclerotic plaques become monoclonal.

Clonal markers in the study of the origin and growth of human atherosclerotic lesions.

The X-linked enzyme, glucose-6-phosphate dehydrogenase (G-6-PD) was used as a cellular marker to study the clonal characteristics of human atherosclerotic lesions from females heterozygous for G-6-PD isoenzymes. Portions of uninvolved aortic wall contained both isoenzyme types (A and B), and their isoenzyme patterns were used to establish criteria for polyclonal lesions. Portions of uterine leiomyomas contained predominantly one isoenzyme type (either all A or all B) and their isoenzyme patterns were used to establish criteria for monoclonal lesions. These techniques were used to address three questions concerning atherogenesis. First, evidence for the monoclonal origin of fibrous-capped plaques was provided by the findings that small plaques had G-6-PD isoenzyme distributions similar to those of leimyomas; that in large plaques with multiple portions assayed for G-6-PD, a large proportion (25 of 26, 96%) of plaques had monoclonal characteristics; and that multiple monoclonal portions were present in the same plaque. Second, the role of the fatty streak as a precursor of fibrous plaques was supported by the demonstration that a proportion (11 of 66, 16.7%) of fatty streaks contained isoenzyme patterns intermediate between those of polyclonal uninvolved aortic wall and monoclonal leiomyomas. Increased cellularity of fatty streaks correlated with increased deviation of isoenzyme pattern toward monoclonality. Third, the assay of portions of both small and large plaques provided no evidence for clonal selection as plaques increase in size.