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.

Collagen in human aorta. Changes in the type III/I ratio and concentration of the reducible crosslink, dehydrohydroxylysinonorleucine in ascending aorta from healthy subjects of different age and patients with annulo-aortic ectasia.

The type III/I + III collagen ratio was studied in intima-medial samples of ascending aortas obtained from patients with the Marfan syndrome or other annulo-aortic ectasia (dilatation of the ascending aorta) and from control subjects, using electrophoretic analysis of cyanogen bromide peptides. The [3H]borohydride-reduced crosslinks of collagens were analysed by ion-exchange chromatography. Type III/I + III collagen ratios were twice as high in adult aortas as those found in skin samples of the same age. This ratio was lower in fetal and very young aortic samples and in 6-8 out of 12 pathological aortas (including one sample from a Marfan patient) when compared with adult controls. In contrast, the type III/I + III collagen ratio was high in fetal or very young skin and the values obtained from several patients did not differ from those of the control skin samples. In one pathological aorta out of six studied, the concentration of the reducible crosslink, dehydrohydroxylysinonorleucine, was higher than in controls, suggesting increased collagen synthesis or impaired maturation of collagen. These changes point to altered collagen metabolism in aortas of patients with annulo-aortic ectasia.

High density lipoproteinemia due to vigorous physical work inhibits the incorporation of [3H]thymidine and the synthesis of glycosaminoglycans by human aortic smooth muscle cells in culture.

The effect of sera from normolipidemic men engaging in normal physical activity and from high density lipoproteinemic lumberjacks engaging in vigorous physical activity on the incorporation of [3H]thymidine and the synthesis of glycosaminoglycans by human aortic smooth muscle cells in culture was measured. At high concentration (15%) high density lipoproteinemic serum inhibited significantly (P less than 0.001) the incorporation of thymidine. The serum inhibited the synthesis of glycosaminoglycans at all concentrations tested (1--15%) with the most marked inhibition at 10 and 15%. At lower concentrations (1--5%) the inhibition was more pronounced for sulphated glycosaminoglycans than for hyaluronic acid. The inhibition was of the same magnitude for the subclasses (chondroitin, dermatan and heparan sulphates) of sulphated glycosaminoglycans studied. High density lipoproteinemia due to vigorous physical work is postulated as a protective factor against the early biochemical reactions of arterial smooth muscle cells in the development of atherosclerosis.

Type IIA hyperlipoproteinemic sera decrease the synthesis of hyaluronic acid by cultured human aortic smooth muscle cells.

The synthesis of sulphated glucosaminoglycans (S-GAGs) and hyaluronic acid (HA) by cultured human aortic smooth muscle cells (SMC) was measured in the presence of sera from type IIA hyperlipoproteinemic patients (7 males and 6 females) and their age- and sex-matched controls. Experiments with pooled sera showed that type IIA and control sera differed in their effects on the synthesis of GAGs at high serum concentrations with preincubation times of 24 and 48 h. Therefore, individual sera were tested at 15% with 24 h preincubation. Type IIA sera decreased the synthesis of HA by 21% (P less than 0.001) but had no significant effect on sulphated GAGs. The ratio of sulphated GAGs to Ha was higher in the presence of type IIA sera than control sera. The incorporation of thymidine, the number of cells and the amount of DNA did not differ in cultures incubated with type IIA and control sera. Early human and experimental atherosclerotic lesions are known to have an increased sulphated GAGs to HA ratio. Our results suggest that one explantation for the association between type IIA hyperlipoproteinemia and an increased risk for atherosclerosis is the disturbance in the proportions of hyaluronic acid and sulphated GAGs synthesized by arterial SMC.

Massive cutaneous hyalinosis. A newly recognized disease.

A disease characterized by massive tumorous cutaneous hyalinosis has been studied histologically, immunologically, and biochemically. The precipitated hyalin material differed from amyloid in being Congo-red-negative and ultrastructurally nonfibrillary. In lipoid proteinosis, massive hyalin deposits have not been encountered and the clinical course is distinct from the course of massive cutaneous hyalinosis. The clinical and histologic pictures of both adult and juvenile forms of colloid milium differed from that found in our patient, although the colloid milium in adult form is ultrastructurally also nonfibrillary like the hyalin from our patient. A strong humoral immune response to components of the cytoskeleton of fibroblasts and especially to keratin was found in our patient.

A rapid assay to measure collagen synthesis in cell cultures.

Limited pepsin digestion and precipitation of resistant parts of proteins with perchloric acid on glass-fiber filters has been used as a rapid way to determine the radioactive collagen secreted into fibroblast culture media. The specificity of the pepsin cleavage was tested by digesting [14C]- or [3H]proline- and [3H]tyrosine-labeled procollagens. Radioactivities obtained with this method were comparable with those obtained with collagenase digestions or hydroxyproline determinations. Dialysis of the samples is avoided and the radioactive collagen can thus be determined from the small medium samples obtained from microtest plates. The method was used to localize a collagen synthesis-increasing factor in preparative isoelectric focusing of microphage culture media.

Lysyl oxidase activity and synthesis of desmosines in cultured human aortic cells and skin fibroblasts: comparison of cell lines from control subjects and patients with the Marfan syndrome or other annulo-aortic ectasia.

The activity of lysyl oxidase, the cross-linking enzyme of elastin and collagen, was measured in culture media of human skin fibroblasts, human aortic medial smooth muscle cells (SMCs) and adventitial fibroblasts using [3H]lysine-labelled elastin substrate. In addition, biosynthesis of isodesmosine and desmosine, the cross-linking amino acids of elastin, was studied by metabolic labelling with [14C]lysine and subsequent amino acid chromatography of protein hydrolysates. Lysyl oxidase activity in culture media of skin fibroblasts and aortic smooth muscle cells increased with the growth of the cell population and was at the highest level in cultures of high cell density. Lysyl oxidase activity in the aortic cell cultures was about three times that of skin fibroblasts. Aortic smooth muscle cells synthesized at least 100 times more desmosines than skin or adventitial fibroblasts. No differences were observed in lysyl oxidase activity and synthesis of desmosines between aortic smooth muscle cells or skin fibroblasts from patients with the Marfan syndrome or other annulo-aortic ectasia (dilatation of the ascending aorta) and the corresponding controls.

Effect of changes in inspired oxygen tension on wound metabolism.

This work was prompted by earlier findings of the beneficial effect of increased oxygen supply on wound healing. Enzyme activities in the limiting step of glycolysis, citric acid cycle and pentose phosphate cycle were determined in cellulose sponge implants of rats chronically, breathing 12% O(2), air or 55% O(2.) Respiratory gas tensions and concentrations of pyruvate and lactate were measured in wound fluid aspirated from the implants. Significant portions of repair tissue exist in conditions of extremely low oxygen tension. Probably because all added oxygen is readily consumed, the wound fluid PO(2) increased only slightly in hyperoxic environment. The wound PCO(2) increased in parallel with the inspired PO(2), probably due to enhanced production of carbon dioxide. Hyperoxia shifted the wound metabolism from anaerobic towards aerobic glycolysis. This occurred concurrently with activation of citric acid cycle. Succinic dehydrogenase, a linking enzyme between citric acid cycle and electron transfer chain, also increased with increasing oxygen tension. This oxygen-induced metabolical change has been previously observed in many other tissues.

Identification of glutamine as a hepatic factor which influence the synthesis of collagen by freshly isolated fibroblasts.

Earlier we have found that 35,000 g supernatants of liver homogenates from both normal and hypercholesterolaemic rats stimulate the synthesis of collagen by freshly isolated fibroblasts. Supernatants from the fatty livers of hypercholesterolaemic rats showed greater stimulation. In the present study we fractionated the 35,000 g supernatants using gel filtration, ion-exchange chromatography, paper electrophoresis and paper chromatography. One of the stimulating factors turned out to be glutamine. However, the concentration of glutamine was the same in normal and fatty livers suggesting that glutamine is not responsible for the greater stimulating activity found in the 35,000 g supernatants from fatty livers. Authentic glutamine increased the synthesis of collagen by 40% at a concentration of 40 mumol/l but inhibited it 80% at 4 mmol/l, which is widely used in cell culture media. There, the concentration of glutamine should be controlled carefully in tests for collagen synthesis in vitro.

Energy metabolism of experimental wounds at various oxygen environments.

Energy metabolism of healing tissue was studied in experimental wounds of rats chronically breathing 11% O2, air or 55% O2. Increasing oxygen supply elevated both PO2 and PCO2 in the wound tissue. At the early phases of healing hypoxic wounds contained less DNA than normoxic or hyperoxic tissues. In hypoxia the accumulation of wound collagen was clearly retarded. Furthermore, tissue taken from wounds healing in hypoxic environments and tested ex vivo in air showed decreased capacity for glucose utilization, lactate production and oxygen consumption. Concentrations of AMP, ADP and ATP in repair tissue increased as healing progressed. The more oxygen available the higher the amounts of ADP and ATP. The AMP content was not affected by changes in local oxygen tension. These results support the earlier concept that the supply of oxygen in healing tissue may be rate-limitimg. Reduction of available oxygen either by systemic hypoxia or by increased diffusion distance impedes healing.

Collagen synthesis in cells cultured from v. Recklinghausen's neurofibromatosis.

Subcutaneous tumors of a patient with v. Recklinghausen's neurofibromatosis contained about 31% collagen calculated on the basis of lipid-free dry weight. Slices of the tumors synthesized collagen at a rate (4.7-8.5% from total protein) which was higher than that of the skin slices (2.8-5.9%). Neurofibromatosis cells were cultured from tumors of two patients. They synthesized relatively much more collagen than cultures of skin fibroblasts of the same patient or of healthy age-matched control persons. The second patient's cultures were studied in detail. The cell densities of these cultures were higher and expressed more variation than the densities of control skin fibroblasts. Ion exchange cellulose chromatograms, SDS-polyacrylamide gel electrophoresis and 3-hydroxyproline analysis of the radioactive proteins made by the cultures indicate that most of the collagenous proteins resembled type I collagen. High proliferative capacity and high collagen synthesis of selected neurofibromatosis cells explains the growth of solid tumors.

Elastin and collagen in the aortic wall: changes in the Marfan syndrome and annuloaortic ectasia.

Elastin and collagen concentrations were determined in intimal-medial samples of ascending aortas from healthy controls of different ages and from 20 patients with annuloaortic ectasia (AAE). Five patients had the Marfan syndrome. In controls the highest elastin concentrations (estimated from desmosine concentrations or insoluble residues after hot-alkali extraction) were found in children. During aging until 60 years, elastin concentration decreased when determined by the hot-alkali extraction method while desmosine concentration changed less. Aorta samples from the Marfan-syndrome patients showed a great variation of elastin concentration from total lack to normal values. Samples from the other AAE patients could be divided into two groups. One contained clearly less elastin and more collagen than the controls whereas in the other group this difference was less marked. Histological examination of the aortic wall of the first group also showed marked fibrosis accompanied by severe elastin fragmentation and acellularity. From the 15 non-Marfan patients 14 were men. By means of clinical examination these patients could also be divided into "familial" and "nonfamilial" groups, because increased diameter of the aortic root was found in relatives of almost half of the patients. However, there were no differences in elastin and collagen concentrations between the familial and nonfamilial cases. As well, no correlation was found between biochemical findings and diameters of the aortic roots. These results point to altered elastin and/or collagen metabolism in the aortic wall of AAE patients.

The borohydride-reducible compounds of human aortic elastin. Demonstration of a new cyclic amino acid in alkali hydrolysate, and changes with age and in patients with annulo-aortic ectasia including one with Marfan syndrome.

Human aortic elastin reduced with [3H]borohydride was analysed by ion-exchange chromatography after alkali or acid hydrolysis. Alkali hydrolysates of elastins contained a radioactive peak that was eluted between proline and leucine. This peak was not present in foetal elastin, but its proportion increased steadily during aging. Aortic samples from patients with annulo-aortic ectasia (aneurysm of the ascending aorta), including one with classical Marfan syndrome, contained less elastin (CNBr-insoluble material) than did the age-matched controls. The proportion of radioactivity in the new peak of all these aortas was low when compared with age-matched controls. Gas-chromatographic/mass-spectrometric analysis suggested that it contained a cyclic derivative of a hydrated aldol-condensation product. The concentration of the cross-link precursors, lysine aldehyde and aldol-condensation product (estimated from the acid-hydrolysis product 6-chloronorleucine and the acid-degradation product of reduced aldol-condensation product) was high in very young aortas but remained quite stable after childhood. No differences were observed in cross-link profiles of acid hydrolysates between pathological and control aortas. A low proportion of radioactivity in the new peak may indicate the presence of young or immature elastin in the pathological aortas.

Effect of sera from hyperlipidemic subjects and high-density lipoproteinemic runners on the synthesis of DNA and glycosaminoglycans by cultured human aortic smooth muscle cells.

Effects of hyperlipidemic, normolipidemic and high-density-lipoproteinemic (HD lipoproteinemic) sera from active runners were studied on cultured human aortic smooth muscle cells. The synthesis of DNA, hyaluronic acid and sulphated glycosaminoglycans (GAGs) in the presence of the various sera was measured by the incorporation of [3H]-thymidine and [3H]-glucosamine. The HD lipoproteinemic sera from runners stimulated the synthesis of DNA and sulphated GAGs less than normolipidemic and hyperlipidemic sera. The hyperlipidemic sera stimulated the synthesis of DNA slightly more than the other sera, but only after a 24 h preincubation. Accordingly, the concentration of HDL-cholesterol in serum was negatively correlated with the synthesis of DNA (r = -0.77, P less than 0.01) and sulphated glycosaminoglycans (r = -0.81, P less than 0.01). The sulphated GAGs/hyaluronate ratio was smaller in the presence of HD lipoproteinemic serum as compared with the other sera. The proliferation of aortic smooth muscle cells and the rate at which they synthesize sulphated GAGs have been considered important during the initiation of atherosclerosis in vivo. The present results suggest that sera having differences in the relative amounts of various lipoprotein fractions differ significantly in their influence on both of these arterial smooth muscle cell functions in vitro.

Serum lipoprotein composition, hormones, and the synthesis of glycosaminoglycans by human aortic smooth muscle cells.

The synthesis of hyaluronic acid (HA) and sulphated glycosaminoglycans (S-GAGs) by cultured human aortic smooth muscle cells (SMC) was measured in the presence of human sera with high LDL-cholesterol concentration (type IIA hyperlipoproteinemic patients) or high HDL-cholesterol concentration (marathon runners and lumberjacks). In addition, the effect of some hormones on GAG-synthesis was studied. As compared to normolipidemic controls, type IIA hyperlipoproteinemic sera decreased markedly the synthesis of HA without affecting the synthesis of S-GAGs. On the contrary, sera with high HDL-cholesterol concentration decreased the synthesis of S-GAGs causing a decrease in S-GAGs/HA ratio. Cortisol in concentrations slightly above the physiological level inhibited the synthesis of HA and increased the S-GAGs/HA ratio. Insulin, and interestingly also sex hormones (estradiol, progesterone, testosterone) had no clear effect on GAG-synthesis. It is known that there is an increased S-GAGs/HA ratio in early atherosclerotic lesions. Our results suggest that the determination of this ratio in SMC cultures provides an useful method of studying the atherogenity and antiatherogenity of various sera and hormones.

Xanthoma tendinosum in a normolipemic ectodermal dysplasia patient.

A female patient with congenital ectodermal dysplasia is described, who developed xanthoma tendinosum in her hands simulating rheumatoid arthritis. The serum lipids of this patient were normal. Both ectodermal dysplasia and xanthoma tendinosum in a normolipemic patient are rather uncommon and to our knowledge have not been previously reported in one and the same patient.