Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta.

Smooth muscle cells with 4C (double diploid) DNA content have been found in major arteries. The proportion of 4C cells increases with normal aging and with hypertension. These cells may represent a state of arrest at the G2 phase of the cell cycle or may be examples of true tetraploidy. Flow cytometric cell sorting was used to isolate 4C smooth muscle cells from the rat aorta, and the cells were cultured. Flow cytometry, Feulgen microdensitometry, and karyotyping of the progeny of the 4C cells established the presence of true tetraploid cells. These findings demonstrate the presence of reproductively viable tetraploid cells in a normal mammalian tissue.

Collagen polymorphism: characterization of molecules with the chain composition (alpha 1 (3)03 in human tissues.

Collagen moleculess with the chain comizposition [alpha1(III)](3), have been isolated from pepsin-solubilized collagen of dermis, aorta, and leiomlyoma of the uterus by differential salt precipitation. On denaturation, approximately 90 percent of this collagen is recovered as a gamma component (300,000 daltons). Reduction and alkylation of the high-molecular-weight component yields alpha1(III) chains (95,000 daltons). In addition to containing cysteine, alpha1(III) chains exhibit several other compositional differences when compared to alpha1(I), alpha1(II), or alpha2 chains from human tissues.

Arterial wall metabolism in experimental hypertension of coarctation of the aorta of short duration.

Coarctation of the mid-thoracic aorata was surgically produced in mongrel dogs which were sacrificed from 4-12 wk after the operation. As compared to the findings in control animals, the sodium, chloride, and water content of the hypetensive portion of the coarcted thoracic aorta was significantly elevated, whereas the electrolyte and water content of the relatively normotensive portion of the coarcted aorta was normal. The sodium, potassium, and water content of the pulmonary artery, skeletal muscle, and cardiac muscle of the coarcted dog was not altered. These observations suggest that an elevated arterial pressure may influence the electrolyte and water composition of the arteries. The arterial pressure also may influence the content and synthesis of acid mucopolysaccharides (MPS) in the arteries since the content of sulfated MPS and the incorporation of injected radiosulfate into sulfated MPS were significantly increased in the hypertensive portion of the coarcted thoracic aorta but were significantly reduced in the relatively normotensive ("hypotensive") portion of the coarcted aorta. The observed increase in MPS may have been a factor directly responsible for the increase in the sodium content of the hypertensive aorta since MPS can act as polyelectrolytes and bind cations. Although the arterial pressure may influence certain metabolic functions in the arteries, it did not appear to have a direct effect on the arterial lipids since the lipid content of the hypertensive and of the relatively normotensive portions of the coarcted aorta were comparable to the values found in the normal aorta.

Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells.

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.

The structural characterization of proteoglycans of cultured aortic smooth muscle cells and arterial wall of the pig.

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (rho av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (rho av = 1.60 g/ml) fraction and three fifths (rho av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83-90%) with lower proportions of heparan sulfate (5-11%) and hyaluronic acid (3-6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11-13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with Kav 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column Vo and the rest at a Kav of 0.34. The majority of subunit macromolecules eluted at the Vo fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4-7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.

Monoclonal DLR1a/104G antibody recognizing peroxidized lipoproteins in atherosclerotic lesions.

Monoclonal DLR1a/104G antibody which recognizes peroxidized lipoproteins was raised. Mice were immunized with the float-up fraction of the atherosclerotic arterial homogenate from WHHL rabbits. Sensitized spleen cells were fused with myeloma cells (P3/U1). Hybridoma clones were selected using peroxidized LDL prepared by CuSO4-catalyzed peroxidation and native LDL as positive and negative standards, respectively. The monoclonal DLR1a/104G antibody was highly reactive with peroxidized LDL, slightly with LDL modified with malondialdehyde, but not significantly with acetyl- or native LDL. The antigenicity in the case of peroxidized LDL did not decrease on extraction with hexane/isopropanol (3:2). The antigenicity coincided with the fluorescence (E350, F430) of the protein fraction of LDL peroxidized with CuSO4. These results suggest that an antigenic determinant exists in atherosclerotic lesions which is the same as that for lipoproteins peroxidized with CuSO4.

Desmosines in aneurysms of the ascending aorta (annulo-aortic ectasia).

Amino acid chromatography was used for determination of the elastin-specific amino acids desmosine and isodesmosine in acid hydrolyzates of intima-medial samples taken intraoperatively from aneurysms of human ascending aorta. Elastin concentration of the specimens was also estimated by hot alkali extraction followed by nitrogen determination of the extracted material and the insoluble residue. All patients studied had annulo-aortic ectasia i.e., dilatation of the aortic annulus and the ascending aorta. Two patients with the Marfan syndrome had low aortic elastin concentration determined by both methods. A third Marfan syndrome patient, youngest of the three, also had a slightly reduced concentration of elastin in the aorta. Aortic samples were studied from five patients who did not have the classical Marfan syndrome. Two patients of those five had decreased aortic elastin concentration. The change in elastin concentration was accompanied by high hydroxyproline/proline or hydroxylysine/lysine ratios which indicates that the proteins of the aneurysmatic aortic wall contained more collagen than the proteins of the control aortic wall. These findings point to a change in the structure or metabolism of elastin in the aortic wall in the Marfan syndrome and at least in some other patients with annulo-aortic ectasia.

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta.

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.

Further studies on a highly purified glycoprotein from the intimal region of procine aorta.

Highly purified glycoprotein from the intimal region of porcine aorta was isolated with minor modifications of the procedure described previously. The molecular weight of the glycoprotein as determined by sedimentation equilibrium method either in presence of 0.1 M NaCl or 6 M guanidine-HCl containing beta-mercaptoethanol was 72 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native glycoprotein and its S-carboxyamidomethyl derivative at different acrylamide concentrations showed no difference in the molecular weight indicating the absence of subunits. Attempts to determine the identity of the amino-terminal acid by a dansylation technique indicated that the amino group is not free. The carboxy-terminal amino acid was found to be serine after treatment of the glycoprotein with carboxypeptidase A. The glycoprotein did not contain an alkali-labile (O-glycosidic) carbohydrate-protein linkage as tested by the beta-elimination reaction. The release of monosaccharides from the glycoprotein as a function of time was studied employing mild acid hydrolysis (0.5 M HCl, 80 degrees C) and also by the use of neuraminidase, alpha-D-and beta-D-glucosidases and beta-D-N-acetylglucosaminidase. From the observations of the release of monosaccharides and analogy with standard features determined by other investigators on soluble aortic glycoproteins, a prediction has been made as to the general features of the carbohydrate moiety of the glycoprotein.

Biochemical investigation of possible lesions in human aorta that predispose to dissecting aneurysms: pyridinoline crosslinks.

Pyridinoline, a collagen specific covalent crosslink, was quantified in acid hydrolysates of human aorta using a non-equilibrium inhibition ELISA. The study was based on specimens from seven cases of aortic dissection and from seven control subjects whose death was unrelated to thoracic aortic dissection. There were no significant differences in the amounts or concentrations of pyridinoline in aortas with dissecting aneurysms compared with normal tissue, thus excluding the possibility of a causative relation between the degree of pyridinoline crosslinking of collagen molecules and dissection of the thoracic aorta. In all cases, however, the number of pyridinoline crosslinks per molecule of collagen in the ascending aorta and arch approached the theoretical maximum for lysyl derivatives and was as high as that present in cartilage. Thus in this region of the vessel pyridinoline represents the major stabilising crosslink of collagen. In contrast, the number of pyridinoline crosslinks per collagen molecule decreased maximally by a factor of 10 between the arch and the proximal regions of the descending thoracic aorta. This suggests a possible correlation between the rigidity of collagen fibres and the forces exerted on the aortic wall during diastole and systole.

Reversibility of arterial and venous changes in renal hypertensive rats.

The effect of reversal of hypertension on vascular function and composition was investigated in renal-hypertensive rats. The study comprised three groups of rats: 1) 21 male Sprague-Dawley rats with one-kidney, one clip Goldblatt hypertension of 9 weeks' duration; 2) 20 rats with one-kidney, one clip hypertension that underwent removal of renal artery clip (were "unclipped") at 6 weeks of hypertension, 3 weeks prior to the study; and 3) sham-operated normotensive control rats. Venous pressure-volume and arterial pressure-flow relationships were measured at maximal vasodilation (sodium nitroprusside and papaverine) in the denervated, pump-perfused vascular beds of the hindquarters of rats. Anatomically defined segments of the aorta and of the vena cava were removed from rats for water, sodium, and potassium analysis. Hypertension was completely reversed in the "unclipped" rats. Compared to values obtained in normotensive control rats, the water concentration of the aorta and of the vena cava, the potassium concentration of the aorta, and the sodium concentration of the vena cava were increased (p less than 0.05) in rats with one-kidney, one clip hypertension. These changes were reversed in the "unclipped" rats. In contrast, the shift of the venous pressure-volume and of the arterial pressure-flow curves toward the pressure axis at maximal vasodilation in hypertensive rats (p less than 0.02) persisted following reversal of hypertension in the "unclipped" rats (p less than 0.05). In chronic, one-kidney renovascular hypertension, the contribution of vascular wall "water-logging" to increased structural arterial resistance and decreased structural venous capacity appears to be minor.

1,2-diacylglycerol content in thoracic aorta of spontaneously hypertensive rats.

Phosphoinositide metabolism participates in the control of cell calcium homeostasis. Because a notable neutral lipid (1,2-diacylglycerol) is generated from phosphoinositide hydrolysis and is assumed to be a secondary messenger, we determined 1,2-diacylglycerol content and its fatty acid profiles in the thoracic aorta of spontaneously hypertensive rats (SHR) and compared it with those of normotensive Wistar-Kyoto (WKY) rats. After the aorta was exposed to 10(-5) M norepinephrine as a stimulant, 1,2-diacylglycerol content in SHR was significantly higher by 33% than in WKY rats at 4 weeks of age, whereas there was no difference in 1,2-diacylglycerol content between the two strains at 20 weeks of age. Before norepinephrine stimulation, there was no significant difference in 1,2-diacylglycerol level between the two strains at 4 weeks of age. Analysis on a gas chromatograph showed that 1,2-diacylglycerol was composed of similar molecular species of fatty acids in aortas obtained from SHR and WKY rats. On the other hand, the cholesterol content of aortas was higher in SHR than in WKY rats at 20 weeks of age, whereas the difference at 4 weeks was not significant. Phosphatidylcholine, phosphatidylethanolamine, and triglyceride showed no significant difference between the two strains. It is concluded that norepinephrine-induced 1,2-diacylglycerol production increases in the thoracic aorta of SHR before the development of hypertension.

The gene for the atrial natriuretic factor is expressed in the aortic arch.

The gene for atrial natriuretic factor is expressed within the adventitial cells of the rat aortic arch. Atrial natriuretic factor transcripts, similar in overall size (1100-1200 nucleotides) and 5'-termini to those found in the atria, were identified in the arch. Much lower levels (approximately 10-20%) of these transcripts were present in distal thoracic aorta. Atrial natriuretic factor peptide was localized by immunocytochemistry to the adventitia of the arch in regions thought to harbor the aortic baroreceptors. These data suggest a previously unsuspected role for the peptide in regulating systemic blood pressure through the baroreceptor reflex.

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.

Quantitative HPLC analysis of human plaque proteins in coronary and thoracic aorta arteries.

Quantitative HPLC analysis of saline-soluble proteins obtained from human coronary and thoracic aorta plaque and from whole internal mammary artery were performed. Protein extracts were characterized by anion exchange and reverse-phase HPLC and the integrated chromatographs revealed significant differences in both peak retention times and areas for protein species from coronary artery compared to thoracic aorta artery plaque. Coronary artery plaque proteins possessed a high degree of cationic charge and polarity compared to those present in thoracic aorta plaque and normal mammary artery. This suggests that specific protein markers may be expressed in plaque of different anatomical origin, and that the processing of protein may be distinct to plaque sites. In contrast, characterization of molecular weight by gel electrophoresis resolved no major differences between plaque types. These findings indicate that proteins in human plaque lesions of different anatomical origin can be resolved by HPLC methodology and that they exhibit different charge and polarity. Such an HPLC approach may prove useful in the quantitative identification and ultimate isolation of specific protein markers present in plaque during atherogenesis, and in the study of mechanisms of protein involvement in plaque formation.

Aortic glycopeptide sialic acid, hexose and hexosamine in a genetically selected (WC-2) strain of atherosclerosis-susceptible pigeon.

The aortic content of glycopeptide sialic acid, hexosamine and hexose was studied in a genetically selected strain of White Carneau pigeons (WC-2) with significantly more severe atherosclerosis than randomly bred White Carneau pigeons (RBWC). Pigeons were fed an atherogenic diet for 3 months and examined to determine differences in content of glycopeptide-sugars between WC-2 and RBWC, changes with the progression of atherosclerosis and the relationship of aortic cholesterol to glycopeptide-sugar content. In animals with mainly normal aorta (cholesterol content of 0.2-0.3 mg/cm2-aorta) sialic acid was significantly lower in WC-2 pigeons. The progression of atherosclerosis was associated with increased aortic glycopeptide sialic acid (r = 0.78; p less than 0.05) in WC-2 pigeons whereas an inverse relationship was suggested in RBWC pigeons. In WC-2, but not RBWC pigeons, significant positive relationships were seen for aortic glycopeptide hexosamine and aortic cholesterol and for aortic glycopeptide hexose and aortic cholesterol. The findings implicate a possible role of aortic glycoproteins in either the initiation or modulation of atherosclerosis of the WC-2 pigeon.

Limitations of the lipid state hypothesis for atherosclerosis are revealed by X-ray diffraction measurements.

The lipid state hypothesis proposes that liquid crystalline states of cholesteryl esters play a role in the development and persistence of the fatty streak lesions characteristic of atherosclerosis. We have tested several corollaries suggested by this hypothesis and find that the ensemble of droplets in atherosclerotic tissue are predominantly in the isotropic (fluid) state at 37.0 degrees C. Furthermore, the liquid-crystalline state transition behavior of these droplets is not influenced significantly by the distribution of component cholesteryl ester species. There are no significant correlations between the transition behavior of the droplets and the age, sex, or race of the subjects from which tissue samples were taken. These results show that the lipid state hypothesis is weak, and that the origin and persistence of fatty streak lesions in humans is probably dominated by other factors.

Crystals in atherosclerotic lesions: real or artefact?

Atherosclerotic lesions were obtained from man during surgery and from cholesterol-fed rabbits. They were maintained at about 37 degrees C during handling. Smears were prepared on glass slides and these were examined microscopically at 37 degrees C. Solid rhomboidal or thick needle-like crystals were present at 37 degrees C but increased in numbers or in size or both on cooling. Staining studies and measurement of melting point (133-153 degrees C) suggested that such crystals are composed largely of free cholesterol or a related sterol. Liquid crystals exhibiting conic focal (Maltese cross) anisotropism were present at 37 degrees C and did not appreciably increase in either size or numbers on cooling. Staining studies and their resistance to digitonin suggested that these Maltese cross crystals are largely esterified cholesterol. Thin needle-like crystals arranged like feathers or in rosettes were seen in smears of adipose tissue and were attributed to triglycerides.

Characterization of the phenotype of smooth muscle cells in human fetal aorta on the basis of ultrastructure, immunofluorescence, and the composition of cytoskeletal and cytocontractile proteins.

The phenotype of smooth muscle cells (SMCs) in the aortic media of 7 human fetuses (14-20 weeks of gestation) was examined with transmission electron microscopy, immunofluorescence microscopy, and gel electrophoresis of the cytoskeletal and cytocontractile proteins. Ultrastructurally, virtually all medial cells were identified as SMCs having a poorly differentiated phenotype with a cytoplasm rich in rough endoplasmic reticulum and organelles, and with only a few myofilaments. All medial cells stained intensely with antibodies to vimentin, but only in a 20-week-old fetus could we find a few SMCs staining with antibodies to desmin. Nor was desmin detectable with SDS gel electrophoresis followed by immunoblotting, while clear bands corresponding to vimentin, myosin, and actin were present. In isoelectric focusing and two-dimensional gel electrophoresis beta-actin was the most prominent of the 3 actin isoforms in all cases. The present results show that SMCs in the media of fetal human aorta have a poorly differentiated phenotype, which morphologically and biochemically resembles that previously described in the aorta of fetal and newborn rat, in the arterial intima after endothelial injury, in atherosclerotic lesions, and after spontaneous modulation of medial SMCs in culture.

Proteoglycan distribution in the intima and media of the aortas of young and aging rabbits: an ultrastructural study.

Aortas from normal healthy rabbits, approx. 3 months old, were examined by light and transmission electron microscopy. The proteoglycan of the extracellular matrix, which was stained by ruthenium red and appeared as granules by transmission electron microscopy, was quantitated morphometrically in the intima and the superficial media. The intima included areas which were thickened and which contained connective tissue, including proteoglycan, and some smooth muscle cells. In the thickened intima there was a greater proportion of extracellular space which was occupied by proteoglycan, and the proteoglycan was present in higher concentration than in the media. In the aortas of rabbits, approx. 2 years old, the extent of intimal thickening and the concentration of proteoglycan increased in the thickened intima but there was no evidence of extracellular lipid deposition. The endothelial basement membrane contained small proteoglycan granules (heparan sulphate) which decreased in concentration in older animals. It is possible that the accumulation of proteoglycan in the thickened intima increases the susceptibility of the intima to accumulate lipid following an additional stimulus, such as hyperlipaemia, in the initial stages of atherosclerosis.