Misbahuddin Zafar Alavi

Distribution of glycosaminoglycans in the intima of human aortas: changes in atherosclerosis and diabetes mellitus

SummaryArterial glycosaminoglycans are considered to be important in atherogenesis due to their ability to trap lipid inside the vessel wall and to influence cellular migration and proliferation. Atherosclerotic lesions have displayed an altered glycosaminoglycan content and distribution. Diabetes is a recognized risk factor for atherosclerosis, but no information is available on the arterial glycosaminoglycans in human diabetes. We examined glycosaminoglycans in normal and atherosclerotic intima of non-diabetic and Type 2 (non-insulin-dependent) diabetic patients. Intima was stripped from autopsy samples of thoracic aortas; normal and plaque areas were separated. Glycosaminoglycans were isolated by delipidation, proteolytic digestion, and precipitation and characterized by quantitation of total glycosaminoglycan and evaluation of glycosaminoglycan distribution by electrophoresis and densitometry. Results indicate a significant decrease in total glycosaminoglycan and significant changes in their distribution in atherosclerotic plaques: a relative decrease in heparan sulphate, a relative increase in dermatan sulphate and thus a decrease in the ratio of heparan sulphate to dermatan sulphate. A similar but less marked change in the ratio was found in normal intima of diabetic subjects, while in their plaques this change was more pronounced. This suggests that changes in arterial glycosaminoglycans (especially the ratio of heparan sulphate to dermatan sulphate) precede the development of lesions in diabetes and may be important in atherogenesis.

Lipoprotein-proteoglycan complexes from injured rabbit aortas accelerate lipoprotein uptake by arterial smooth muscle cells

Lipoprotein-proteoglycan (LP-PG) complexes are taken up more avidly by macrophages and smooth muscle cells (SMCs) than native lipoproteins (LPs). The enhanced uptake may contribute to lipid accumulation and foam cell formation during atherogenesis. Endothelial injury is known to alter proteoglycan (PG) synthesis and distribution in the neointima developed in response to injury. The present study examines the uptake and degradation of LP-PG complexes, derived from PG of injured aortas by arterial SMCs. Rabbit apo-B lipoprotein (LP), including VLDL, IDL and LDL was isolated by ultracentrifugation and coupled with PG extracted from normal aortas (NPG) or with PG from injured aortas (IPG). Rabbit aortic SMCs were cultured from intima-media explants, incubated with 125I-LP, 125I-LP-NPG or 125I-LP-IPG for 20 h at 37 degrees C. LP binding, internalization and degradation were markedly increased (P < 0.001) for LP-NPG and LP-IPG over native LP. Competition experiments indicated that more than 50% of the LP-PG complexes were taken up by the apo-B/E receptor pathway. Phagocytosis was the second important route of uptake of these complexes, whereas the scavenger receptor played a minor part in the uptake and degradation of LP-PG complexes. Data from this study indicate that LP-PG complexes accelerate LP uptake and degradation by SMC more than native LP. Therefore, these complexes may contribute to lipid accumulation by SMC, thus generating foam cells. Furthermore, LP-PG complexes prepared from PG of injured aortas are more effective in lipid accumulation than LP-PG complexes from PG of normal aortas.

Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in rabbit aortic neointima after selective de-endothelialization

Altered TIMP-1 synthesis in the arterial wall may be important for the balance between metalloproteinases and their inhibitors, and thus contribute to dysregulated extracellular matrix metabolism in atherosclerotic lesions. To examine this, we cloned the rabbit TIMP-1 gene from aortic neointima, developed in response to a balloon-catheter induced de-endothelialization. The apparent homology of cDNA with TIMP-1 genes from several sources suggested that it is a rabbit form of TIMP-1. We examined the recombinant rabbit TIMP-1 expression in Escherichia coli using the pTrxFus expression system and the synthesis of the resulting soluble protein was confirmed by immunostaining with anti-TIMP-1. The TIMP-1 concentration in normal and de-endothelialized rabbit aortas was compared using Northern blot, Western blot and mRNA in situ hybridization techniques. We observed a significant increase of TIMP-1 expression in neointimal SMCs at both nucleic acid and protein levels, suggesting a role of TIMP-1 in injury-induced atherogenesis.

Expression of syndecan-1 in rabbit neointima following de-endothelialization by a balloon catheter

Enrichment of proteoglycans is prominent in early atherogenesis, contributing not only to SMC migration and proliferation, but also to low density lipoprotein retention. A family of integral cell membrane proteoglycans termed syndecans has recently been recognized. Among syndecans, syndecan-1, the first isolated member, has received most research attention. In this study, we examined the expression of syndecan-1 in rabbit aorta and aortic neointima, developed in response to a balloon catheter-induced de-endothelialization. The tissues were processed for Northern blot analysis, in situ hybridization, immunohistochemical staining and immunoblotting. Our results indicate that in normal aorta, the signal for syndecan-1 is weak. However, arterial injury induces syndecan-1 expression at both mRNA and protein levels. The presence of syndecan-1 in the neointimal tissue is persistent, prominent even at the 12th week after injury. Syndecan positive cells are distributed in the whole layer of the neointima, but are not visible in the underlying media. The presence of syndecan-1 in arterial neointima suggests a novel means of mediating interactions between neointimal cells and various agents, including extracellular matrix components, growth factors and lipoproteins.

Isolation of lipoprotein–proteoglycan complexes from balloon catheter deendothelialized aortas and the uptake of these complexes by blood monocyte–derived macrophages

&NA; Lipoprotein‐Proteoglycan (LP‐PG) complexes from the neointima, developed in response to injury, were studied to examine their ability to stimulate lipid accumulation in blood monocyte‐derived macrophages (BMDM). LP‐PG complexes were extracted from intimal‐medial tissues from normal and balloon catheter deendothelialized aortas of normocholesterolemic rabbits, in 0.16 M NaCl for 24 h at 4°C. The extract was purified through an antiapo‐B affinity column. Adsorbed material dissociated with 4 M Gu‐HCl buffer was analyzed for lipoproteins (LP) and glycosaminoglycans (GAG). Results demonstrated that LP‐PG complexes consisted of apo‐B associated with chondroitin sulfate and hyaluronic acid. BMDM were incubated with 125I‐LP, 125I‐LP‐NPG (from normal aortas) or 125I‐LP‐IPG (from injured aortas) for 20 h at 37°C. LP binding, internalization and degradation was markedly increased for LP‐NPG and LP‐IPG over native LP. Phagocytosis appeared to be the primary route of uptake of LP‐PG complexes. Competition experiments indicated that about 40% of the uptake of LP‐PG complexes is mediated by the apo‐B/E receptor pathway. The scavenger receptor played a minor part in the uptake of LP‐PG complexes. Data from this study indicate that LP‐PG complexes are present in normal and injured aortas of normocholesterolemic rabbits and these complexes accelerate LP uptake by BMDM more than native LP. Therefore, LP‐PG complexes may contribute to lipid accumulation by BMDM, thus generating foam cells. Furthermore, LP‐PG complexes prepared from PG of injured aortas are more effective in lipid accumulation that LP‐PG complexes from PG of normal aortas.

In situ ultrastructural characterization of chondroitin sulfate proteoglycans in normal rabbit aorta.

We used a monoclonal antibody recognizing chondroitin sulfate (CS) to investigate by immunocytochemistry the characteristics displayed in situ by aortic proteoglycans (PG) containing CS side chains. The antibody specifically precipitated metabolically labeled PG from aortic extracts. Anti-CS specificity was also tested directly on tissue sections and was confirmed by the virtual abolition of immunolabeling on those previously digested with CS-specific enzymes. The overall CS-PG distribution assessed by light microscopy after embedding in Lowicryl KM4 by silver-enhanced immunogold recapitulated that obtained on frozen sections with immunoperoxidase. Extracellular concentrations of CS-PG were very high in the innermost regions of aorta and decreased in the media. The reaction was weak and diffuse in the adventitia. By electron microscopy, the detailed labeling of CS-PG discriminated patterns of organization at both the regional and the molecular level and enabled morphometric estimations. In relation to other components of the extracellular matrix, we found that CS-PG and elastin mutually excluded each other, while two types of CS-PG were differently associated with collagen within media or adventitia. The use of high-resolution immunodetection for the in situ characterization of aortic CS-PG could add specific information relevant to many biological processes in which these molecules have been implicated.

Collagen biosynthesis by neointimal smooth muscle cells cultured from rabbit aortic explants 15 weeks after de-endothelialization

Extracellular matrix (ECM) accumulation in arterial neointima, developed in response to de‐endothelialization, is a prolonged process. In this study, we examined the relationship between increased collagen accumulation and synthetic activity of neointimal smooth muscle cells (SMCs) derived from aortic explants fifteen weeks after balloon catheter injury. Freshly confluent SMCs, derived either from normal aorta or from aortic neointima, were used in this study. The newly synthesized collagen was analysed by measuring [3H]‐proline incorporation; and the mRNA expression for two major types of collagen, collagen type I and type III, was studied by Northern blot analysis. Our results indicated a three fold increase in protein (collagen) synthesis by neointimal SMCs. At the same time, the steady‐state mRNA for procollagen I and procollagen III was elevated five and three times, respectively. These data indicate that persistent synthesis contributes to collagen accumulation in the arterial neointima and both transcriptional and post‐transcriptional regulation take part in this process.

Proteoglycan synthesis by the neointimal smooth muscle cells cultured from rabbit aortic explants following de-endothelialization

Proteoglycans (PGs), the essential component of the extracellular matrix, are implicated in the pathogenesis of atherosclerosis. In an experimental model of injury, PGs accumulate in the neointimal tissue parallel with lipid deposition. However, it is still not clear whether the PG accumulation is from active smooth muscle cell (SMC) production or is a consequence of trapping within neointima covered by endothelium. To study the effect of endothelial injury on PG synthesis, SMCs were cultured from normal aorta (N-SMC), neointima covered by regenerated endothelium (W-SMC) and neointima without endothelium (B-SMC). Using [35S]-Na2SO4, as a precursor in an in vitro incubation, the kinetics of PG synthesis were determined. PG synthesis by all three cell types increases as a function of time. It is significantly higher in the SMCs cultured from endothelium-denuded aortic explants (W- and B-SMC) than N-SMC. This finding indicates that endothelial injury stimulates PG synthesis by SMCs.

Enhanced incorporation of [14C]glucosamine into glycosaminoglycans of aortic neointima of balloon-injured and cholesterol-fed rabbits in vitro

Glycosaminoglycans (GAG), which form the elementary constituent of extracellular matrix proteoglycans (PG), are implicated in the pathogenesis of atherosclerosis, mainly due to their lipoprotein binding capability and their abundance in a developing lesion during atherogenesis. However, the reasons for the increment of GAG content are poorly understood. In the present study, the influence of two well known atherogenic factors on arterial GAG synthesis were examined by estimating the incorporation of [14C]glucosamine into aortic GAG in an in vitro incubation system. Radioactivity associated with GAG was taken to represent their synthesis. GAG synthesis by neointimal tissue of rabbit aortas, 12 weeks following balloon catheter deendothelialization was measured and compared in rabbits fed a normal or 0.25% cholesterol supplemented diet for the preceding 6 weeks. In normolipaemic rabbits synthesis was found to be 12,438 +/- 173, 17,884 +/- 1390 and 15,960 +/- 1355 dpm/mg dry defatted tissue from uninjured (control), deendothelialized (DEA) and reendothelialized (REA) areas of rabbit aortas, respectively. This incorporation of radioactivity was significantly greater in hypercholesterolaemic rabbits corresponding to 13,426 +/- 239, 32,670 +/- 3077 and 27,496 +/- 3287 in the control, DEA and REA, respectively. The results demonstrated a synergistic effect of cholesterol feeding and arterial endothelial denudation in stimulating GAG synthesis. Although GAG synthesis was found to be stimulated by either cholesterol feeding or arterial injury, the stimulation by cholesterol feeding alone was only marginal. Further, results show a much higher retention of newly synthesized GAG by the tissue from REA. This study provided a possible explanation for increased GAG content in a developing proliferative lesion.

Sulfated proteoglycans of rabbit aorta: Selective extraction and alternative method for glycosaminoglycan moiety analysis

Three different solutions containing urea, guanidine hydrochloride, or a detergent mixture were used to extract proteoglycan molecules (PG) metabolically labeled with 35S from normal rabbit aortic tissue. The size distribution of whole sulfated PG and the glycosaminoglycan (GAG) compositions in the three extracts were compared and found to be characteristically determined by the type of solution used for extraction. The spectrum of sulfated PG isolated by each solution was maintained at consecutive extractions of the tissue, even if this was used after another type of solution. The extracts obtained by using the urea- or guanidine-containing solutions contained similar, rather balanced populations of large and small PG, while the detergent-containing buffer was found to preferentially extract smaller, heparan sulfate-rich aortic PG. The selectivity of various extracting solutions could be exploited to obtain preparations enriched in certain types of sulfated PG. On the other hand, one could obtain a larger variety of 35S-labeled PG from the tissue by consecutively using two solutions with different capacities of extraction. The distribution of GAG moieties among PG populations, separated by size chromatography, was investigated by one of the commonly used methods and by a new method. The standard method is based on comparison of the chromatographic profiles of the extract before and after enzymatic digestions, requiring several chromatographic runs for a sample. In the alternative method proposed, the fractions obtained after a single chromatographic separation are adsorbed onto a support membrane. Processing of the whole membrane by GAG-specific, enzymatic treatments allows simultaneous assessment of GAG types in each fraction.(ABSTRACT TRUNCATED AT 250 WORDS)