Extracellular matrix.

A succinct overview of recent results on the biochemistry of extracellular matrix (ECM) is presented. The rapid expansion of this discipline over the best decades renders impossible to give an even approximately complete coverage of matrix biology. Some selected results concerning the four major families of macromolecules composing the ECM, that is, collagens (14 types described), elastin(s), proteoglycans and structural glycoproteins (especially fibronectin) are described. Special attention is directed to a crucial aspect of matrix biology: cell-matrix interactions. A number of cell membrane receptors were recently described mediating the two way information flow from the cells to the matrix via the 'programme' of ECM synthesis coded in the genome and unfolding during differentiation and from the ECM to the cells through the membrane receptors which contact the cytoskeleton. One of them at least, the elastin receptor was shown to be linked through a G-protein-phospholipase C-IP3 mediated relay to the regulation of intracellular calcium. Modifications of the ECM will therefore influence cell behaviour. Derangements of this informational feed back mechanisms appear to be involved in most age-related connective tissue diseases.

Changes in the physical behavior of low density lipoprotein in the presence of glycosaminoglycans and high density lipoprotein.

Temperature dependent techniques - differential scanning calorimetry (DSC), polarizing microscopy - were used to study the properties of human serum low density lipoprotein (LDL) and its glycosaminoglycan (GAG) complexes, and to investigate the influence of the addition of high density lipoprotein (HDL) to the complex system. In the LDL molecule a reversible endothermic transition took place with its peak at 33 degrees C. Cholesteryl esters within the LDL core existed as an isotropic solution above this temperature (i.e. around body temperature), and in the form of smectic liquid crystals below it. When LDL was converted in vitro into GAG-LDL complexes by the addition of chondroitin-6-sulfate, dermatan sulfate, heparin or heparan sulfate, the DSC curves showed an evaluation of the transition temperature: the peak values in these samples were found at 40 degrees C and under the polarizing microscope a birefringence developed, typical of smectic liquid crystals. In chondroitin-4-sulfate-LDL complexes no alteration of the physical structure of the LDL molecule could be demonstrated. HDL decreased the transition temperature of GAG-LDL complexes and the disappearance of birefringence indicated that in the presence of HDL the lipids within the GAG-LDL complexes existed in a liquid phase at a temperature in the vicinity of body temperature.

Plasma high density lipoproteins in coronary, cerebral and peripheral vascular disease. The influence of various risk factors.

High density lipoprotein (HDL) cholesterol and the HDL/total cholesterol ratio have been measured in 440 patients with coronary, cerebral or peripheral vascular disease and in 440 matched controls. The patients were subdivided into sex- and age-groups and according to physical activity, smoking, hypertension and non-insulin-dependent and insulin-dependent diabetes mellitus. The average HDL cholesterol level was significantly decreased in all the three groups of localization of ischaemic vascular disease (IVD). Plasma HDL concentration in men was lower than in women in every age-group. Lowest values were measured in patients with cerebral vascular diseases. From among the risk factors supposed to be related to IVD, lack of physical exercise resulted in a decrease of HDL cholesterol and HDL/total cholesterol values. In all the three localizations of IVD cigarette smokers had lower HDL levels than non-smokers. The influence of hypertension on serum HDL concentration was not unidirectional. The coexistence of non-insulin-dependent diabetes and IVD resulted in decreased lipid parameters. The sera of insulin-dependent diabetics had higher HDL contents and higher HDL/total cholesterol ratios than those of non-diabetics in all the three localizations of the vascular disease in men and in women suffering from peripheral vascular disease.

Elastase-type activity, elastase inhibitory capacity, lipids and lipoproteins in the sera of patients with ischemic vascular disease.

Elastase-type activity and elastase inhibitory capacity were determined in the sera of 130 atherosclerotic patients, suffering from ischemic vascular disease (IVD) localized at various sites, and of 60 control subjects. The concentrations of serum lipoprotein constituents (triglycerides, total cholesterol, HDL cholesterol, apo-A, apo-B) have also been investigated. HDL cholesterol and apo-A levels were decreased at every site on IVD. Plasma HDL concentration in men was lower than that in women. There was a sex-related difference in the elastase-type activity of the sera: within every atherosclerotic group, elastase-type activity in women was significantly higher than in men. Elastase-type activity did not appear to vary with age and did not show any correlation with the concentration of serum lipoprotein constituents. Pancreatic elastase inhibitory capacity of sera was significantly elevated in the sera of atherosclerotic patients. There was a significant negative correlation between the inhibitory capacity and the HDL cholesterol and apo-A content of sera, respectively. Inhibitory capacity did not show sex-, or age-related variation. There was a significant positive correlation between elastase-type activity and elastase inhibitory capacity measured in control sera. In the sera of atherosclerotic patients this correlation could not be established.

Elastase-type enzymes and their relation to blood lipids in atherosclerotic patients.

Serum elastase-type activity, elastase inhibitory capacity and their relation to lipids were examined in 140 male patients with ischemic vascular disease (coronary, cerebral, peripheral) and in 60 control subjects. In a further 24 patients with acute myocardial infarction elastase activity, inhibitory capacity and lipids during the course of the illness have also been investigated. Serum elastase-type activity was found to be significantly lower and inhibitory capacity significantly higher in the groups of patients than in the controls. HDL- and HDL2-cholesterol as well as apo A concentration showed significant negative correlation with elastase inhibitory capacity both in atherosclerotic and in control subjects. During the course of myocardial infarction a significant elevation of serum elastase-type activity could be observed at the end of the first week; serum triglyceride levels increased, HDL- and HDL2-concentrations decreased significantly in the first 3 weeks, than gradually approached the initial values. In the patients with an elevation of serum elastase-like activity by more than 30% in the first week, there was a significantly higher elevation of serum GOT and LDH1 and a greater occurrence of transmural (Q) infarction than in those with a smaller variation of elastase-like activity.

Factor, present in plasma, inhibiting the interaction of low density lipoprotein with arterial proteoglycan.

The complex formation of LDL with arterial proteoglycan or glycosaminoglycan was quantitated as precipitation of LDL-cholesterol after incubation in a low ionic strength buffer containing CaCl2 and MgCl2. It was found that human plasma or serum contains a factor which inhibits this complex formation. Upon density gradient centrifugation this factor was found at d greater than 1.24 g/ml. It could be further purified from the d greater than 1.24 g/ml fraction by affinity chromatography on heparin-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. The content of inhibitory activity was found to vary over a 4-fold range in sera of apparently healthy persons.

Effect of glycosaminoglycans and divalent cations on the thermal properties of dipalmitoylphosphatidylcholine.

The effect of glycosaminoglycans (GAG) and divalent cations on the thermal properties of dipalmitoyl-phosphatidylcholine (DPPC)-water systems was examined in order to model some interactions taking place on low density lipoprotein (LDL) surfaces. The thermal properties of these systems were measured by differential scanning calorimetry (DSC). According to the results, all three glycosaminoglycans used (chondroitin-4-sulfate, chondroitin-6-sulfate and heparin) were effective but to a different extent. Calcium ions enhance the interaction more than magnesium ions, probably because divalent cations form bridges between the negatively charged groups of GAGs and the headgroups of lipids. It is conceivable that similar processes might occur in the case of LDL.

The interaction of human plasma low density lipoproteins with glycosamino-glycans: influence of the chemical composition.

Human plasma of 5 normolipemic individuals was incubated for 24 hr at 37 C in the presence or in the absence of lecithin:cholesterol acyltransferase (LCAT)-inhibitors. Plasma stored at 4 C served as a control. The low density lipoprotein (LDL) fractions of the samples were isolated and investigated with respect to changes in chemical composition and complexing activity with glycosamino glycans (GAG). Incubation of plasma in the presence of LCAT inhibitors caused a significant increase of LDL triglycerides at the expense of cholesteryl esters. Incubation with active LCAT not only changed the core but also the surface constituents (decrease in phospholipids and in free cholesterol). The amount of GAG bound per mg of LDL was not uniformly changed in samples incubated after LCAT inhibition. LDL isolated from plasma incubated in the presence of LCAT, on the other hand, showed a significant reduction in GAG binding. The ratio of free cholesterol:GAG in the complex was most significantly reduced in LCAT-modified LDL. There was in addition a highly significant correlation between the LDL:GAG ratio in the complex and the free cholesterol and phospholipid content of the LDL samples. It is concluded that alterations in surface lipid constituents of LDL strongly affect their interaction with sulfated polysaccharides, an effect which may be relevant also in vivo for the interaction of LDL with cell surfaces and intercellular matrices.

Interaction between fibronectin, proteoglycans and lipoproteins.

The effect of purified human plasma fibronectin on LDL-GAG and LDL-PG complex formation was studied. Fibronectin added to LDL or to GAG or even to preformed LDL-GAG-Ca2+ complexes could inhibit complex formation and dissociated preformed complexes. Similar results were obtained with total serum instead of purified LDL: 1.2 mg fibronectin added to 1.0 mg LDL-cholesterol completely inhibited insoluble complex formation in the presence of Ca2+ between LDL and GAGs or LDL and PGs purified from aorta, whatever the order of mixing of the macromolecules. When fibronectin was added to preformed PG-LDL complexes however dissociation was less complete than with preformed LDL-GAG complexes (60% dissociation instead of 100% at similar concentration ratios). It appears therefore that the protein and GAG portions of PGs may not interact at the same sites of LDL and competition by fibronectin would be more efficient at the GAG binding site. Fibronectin could also dissociate LDL-heparin complexes formed on heparin-Sepharose affinity columns. As PG-LDL complexes were isolated from atherosclerotic plaques and fibronectin was also shown to increase in plaque area and exhibit opsonic-like functions, the above findings may well have physiopathological significance.

Physicochemical studies of two major subpopulations of low density lipoproteins by differential scanning calorimetry and n.m.r. spectroscopy.

Two subpopulations, layer 2 (density 1.025-1.029 g/ml) and layer 3 (density 1.032-1.043 g/ml) of low density lipoproteins (LDL) were isolated from fresh human plasma of normal lipidaemic subjects by density gradient ultracentrifugation. Chemical analyses demonstrated the ratios of triglyceride/cholesterol ester decreased with increasing densities of subfractions. These subfractions together with triglyceride-rich lipoproteins (layer 1, density less than 1.019 g/ml) were subjected to physicochemical studies by differential scanning calorimetry (d.s.c.) and nuclear magnetic resonance (n.m.r.) spectroscopy. The average transition temperature (Tt) of layer 2 was 34.20 +/- 0.83 degrees C and that of layer 3 was 37.25 +/- 0.35 degrees C. In addition, many of the layer 3, but not layer 2 and layer 1, samples showed structural alteration and gave rise to an average Tt of 39.18 +/- 1.24 degrees C. The structural alteration could be detected with polarizing light microscopy showing birefringent spherulites at body temperature. The peak Tt values obtained by d.s.c. were in good agreement with those by n.m.r. spectroscopy. These results demonstrate the physicochemical heterogeneity within the LDL density region and suggest that layer 3 subpopulation is much more labile than the others.

Changes in the cartilage proteoglycans in relation to age and osteoarthrosis.

Changes in the extracellular components of various connective tissues with time and continuous use, as well as within experimentally-induced and human pathological conditions, were studied by morphological, chemical and biochemical methods. Osteoarthrotic lesions were produced surgically by implantation of polyethylene sheets, by continuous compression of the knee joint, and by intraarticular papain injection. Results of the model experiments showed that, irrespective of the method used for the production of experimental cartilage lesions, the alterations are strikingly similar. Within connective tissues a gradual deterioration takes place, as measured by marked differences in the histological and histochemical patterns. The degradative changes occur primarily in the matrix, resulting in a significant decrease in proteoglycan content. Under conditions of stress, in young animals, chondrocytes seem to revert to the chondroblastic state, showing mitotic figures, and are capable of producing matrix much more rapidly than is normally seen. This increased activity might be regarded as a nonspecific "feed-back" response of cells, leading to a reparative normalization of the various destructive tissue alterations.

[Serum elastinolytic activity and blood lipids in various clinical forms of arteriosclerosis and in acute myocardial infarct]. / Szérum elasztinolitikus aktivitás és vérlipidek az arteriosclerosis különbözö klinikai formáiban és acut myocardialis infarctusban.

Serum elastase-type activity, elastase inhibitory capacity and their relation to lipids were examined in 140 male patients with ischemic vascular disease (coronary, cerebral, peripheral) and in 60 control subjects. In further 24 patients with acute myocardial infarction dynamics of elastase activity, inhibitory capacity and of lipids during the course of the illness have also been investigated. Serum elastase-type activity was found to be significantly lower, inhibitory capacity significantly higher in the groups of patients than in the controls. HDL- and HDL2 cholesterol and apo-A concentrations showed significant negative correlations with elastase inhibitory capacity both in atherosclerotic and in control subjects. During the course of myocardial infarction a significant elevation of serum elastase-type activity could be observed at the end of the first week; serum triglyceride levels increased, HDL- and HDL2 concentrations decreased significantly in the first 3 weeks, then gradually approached the initial values. In the subgroup of patients with an elevation of serum elastase-like activity by more than 30% in the first week, there was a significantly higher elevation of serum GOT and LDH1 and a greater occurrence of transmural (Q) infarction than in those with a smaller variation of elastase-like activity.