Precursors of chondroitin sulfate proteoglycan are segregated within a subcompartment of the chondrocyte endoplasmic reticulum

Immunocytochemical methods were used at the levels of light and electron microscopy to examine the intracellular compartments of chondrocytes involved in extracellular matrix biosynthesis. The results of our studies provide morphological evidence for the compartmentalization of secretory proteins in the ER. Precursors of the large chondroitin sulfate proteoglycan (CSPG), the major proteoglycan species produced by chondrocytes, were present in the Golgi complex. In addition, CSPG precursors were localized in specialized regions of the ER. Link protein, a separate gene product which functions to stabilize extracellular aggregates of CSPG monomers with hyaluronic acid, was segregated similarly. In contrast, type II procollagen, another major secretory molecule produced by chondrocytes, was found homogeneously distributed throughout the ER. The CSPG precursor-containing ER compartment exhibits a variable tubulo-vesicular morphology but is invariably recognized as an electronlucent, smooth membrane-bounded region continuous with typical ribosome-studded elements of the rough ER. The observation that this ER structure does not stain with antibodies against resident ER proteins also suggests that the compartment is a specialized region distinct from the main part of the ER. These results support recent studies that consider the ER as a compartmentalized organelle and are discussed in light of the possible implications for proteoglycan biosynthesis and processing.     

Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro.

Versican is an extracellular matrix proteoglycan produced by many cells. Although versican is generally known as a large chondroitin sulfate proteoglycan (CSPG), the smallest splice variant, V3, consists only of the amino- and carboxy-terminal globular domains and is therefore predicted to be a small glycoprotein, lacking CS chains. The large size, negative charge, and ability of versican variants to form pericellular coats with hyaluronan are responsible for many of its effects. V3, lacking the large size and high charge density, but retaining the hyaluronan-binding domain of the larger isoforms, may have different effects on cell phenotype. To determine whether V3 alters cell phenotype, Fisher rat arterial smooth muscle cells (ASMCs), which express the larger CSPG versican splice forms (V0 and V1) were retrovirally transduced with the rat V3 cDNA. Northern analysis for versican RNAs confirmed that cells transduced with V3 retrovirus, but not cells tranduced with the empty vector, expressed RNA of the size expected for V3/neo(r) bicistronic RNA. V3 overexpressing cells were more spread on tissue culture plastic, had a smaller length-to-breadth ratio and were more resistant to release from the culture dish by trypsin. Interference reflection microscopy of sparsely plated cells showed larger areas of close contact between the V3 expressing cells and the coverslip, in comparison to control cells. Focal contacts in the periphery of V3 expressing cells were larger. Growth and migration studies revealed that V3 transduced cells grow slower and migrate a shorter distance in a scratch wound assay. The increased adhesion and the inhibition of migration and proliferation resulting from V3 overexpression are the opposites of the known and predicted effects of the other variants of versican. V3 may exert these effects through changes in pericellular coat formation, either by competing with larger isoforms for hyaluronan-binding, or by altering other components of the pericellular matrix.     

Spatial interrelationships between proteoglycans and extracellular matrix proteins in cell cultures

Immunolabelling of cultured cells for chondroitin-sulfate proteoglycan (CSPG), in conjunction with antibodies to fibronectin, collagen and laminin, revealed the spatial interrelationships between the different matrix components. CSPG was organized in two major forms. Fibronectin-independent dotted patterns of CSPG were detected on the substrate and cell surfaces at early stages after plating. At later stages, however, significant overlapping was found between the two extracellular matrix components. Immunoelectron microscopic examination indicated that the CSPG was organized as granules of varying sizes which were associated with the cell surface, the substrate, or with the periphery of the fibronectin network.     

Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture

Arterial proteoglycans have been implicated in several important physiological processes ranging from lipid metabolism to regulation of smooth muscle cell growth. Vascular smooth muscle (VSM) cells are the major producers of proteoglycans in the medial layer of blood vessels. To study functional consequences of alterations in VSM proteoglycan metabolism we used 4-methylumbelliferyl-beta-D-xyloside to inhibit proteoglycan synthesis in primary and early passage cultures of rat aortic smooth muscle cells. Biochemical analysis of cultures labeled with 35SO4 showed the drug inhibited synthesis of different classes of proteoglycans by 50 to 62%. Inhibition of proteoglycan synthesis resulted in reduced accumulation of extracellular matrix, as shown by immunofluorescent staining with antibodies to chondroitin sulfate, fibronectin, thrombospondin, and laminin. There was also an inhibition of postconfluent (multilayered) growth of the smooth muscle cells, and a change in the morphology of the cells, with no apparent effect on subconfluent growth. In addition, in drug-treated cells there was a reduction in the number of cytoskeletal filaments that contained alpha-actin, the actin subtype synthesized by differentiated VSM cells. This occurred even though the total content of alpha-actin in the cells was not reduced. The effects of the inhibitor on growth and morphology could be reversed by switching the cultures to normal medium and could be prevented by growing the cells on preformed VSM extracellular matrix. These observations suggest the vascular extracellular matrix may play a role in regulating the growth and differentiation of smooth muscle cells.     

Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts.

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.     

Immunostaining of a heterodimeric dermatan sulphate proteoglycan is correlated with smooth muscles and some basement membranes

A heterodimeric 760-kDa dermatan sulphate proteoglycan tentatively named PG-760 was characterized as a product of keratinocytes, endothelial cells, and fibroblasts. The two core proteins of 460 kDa and 300 kDa are linked by disulphide bridges, and both carry one or only very few dermatan sulphate chains. Different antisera against PG-760 were used in the present study to investigate the distribution in selected murine tissues by light and electron microscopy. PG-760 immunostaining was observed in cornea (epithelium including basement membrane, stroma, and Descemet's membrane), skin, mucosa of the small intestine, Engelbreth-Holm-swarm (EHS)-tumour (matrix and cells), and the smooth muscle layers of uterus, small intestine, and blood vessels. No staining was observed in capillaries, striated muscles, and liver parenchyma including the central vein. The expression of PG-760 in EHS-tumour was also demonstrated after extraction with 4M guanidine and partial purification by diethylaminoethyl (DEAE)-chromatography. We conclude that this novel proteoglycan exhibits a unique tissue distribution being a constituent of some but not all basement membranes, of some other extracellular matrices, and additionally, of all investigated smooth muscle layers.     

Chondroitin sulfate proteoglycans from salmon nasal cartilage inhibit angiogenesis

Because cartilage lacks nerves, blood vessels, and lymphatic vessels, it is thought to contain factors that inhibit the growth and development of those tissues. Chondroitin sulfate proteoglycans (CSPGs) are a major extracellular component in cartilage. CSPGs contribute to joint flexibility and regulate extracellular signaling via their attached glycosaminoglycan, chondroitin sulfate (CS). CS and CSPG inhibit axonal regeneration; however, their role in blood vessel formation is largely unknown. To clarify the function of CSPG in blood vessel formation, we tested salmon nasal cartilage proteoglycan (PG), a member of the aggrecan family of CSPG, for endothelial capillary-like tube formation. Treatment with salmon PG inhibited endothelial cell adhesion and in vitro tube formation. The anti-angiogenic activity was derived from CS in the salmon PG but not the core protein. Salmon PG also reduced matrix metalloproteinase expression and inhibited angiogenesis in the chick chorioallantoic membrane. All of these data support an anti-angiogenic role for CSPG in cartilage.     

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.     

Extracellular matrix proteoglycan degradation by human alveolar macrophages and neutrophils

Degradation and restructuring of the elastin fiber network of the lung is a pivotal process in the pathogenesis of emphysema. Alveolar macrophages and neutrophils are probably directly involved in elastin degradation, but they may also indirectly influence elastin structure and function by altering other extracellular matrix components such as proteoglycans. In this study the mechanisms of proteoglycan degradation by human alveolar macrophages and neutrophils have been explored. Macrophages appear to utilize plasminogen in solubilizing 35SO4-labeled proteoglycans in extracellular matrix produced by neonatal rat vascular smooth muscle cells. Proteoglycan degradation by macrophages is significantly augmented in the presence of 1% human serum. In contrast, neutrophils apparently utilize intrinsic proteinases to solubilize extracellular matrix proteoglycans, and serum inhibits proteoglycan degradation by these cells. Persistent inflammation in the terminal airways of cigarette smokers may produce proteoglycan degradation and influence elastin fiber architecture where the earliest physiological and anatomic evidence of emphysema appears.     

Cell-free translation of mRNA encoding an arterial smooth muscle cell proteoglycan core protein

The size and immunological reactivity of the primary gene products of a small non-aggregating dermatan sulfate proteoglycan from bovine and monkey arterial smooth muscle cells were examined after cell-free translation of mRNA. Antisera against the dermatan sulfate proteoglycans from bovine articular cartilage, DSPG II [Rosenberg et al. J. Biol. Chem. 260, 6304 (1985)] and human skin fibroblasts [Glossl et al. J. Biol. Chem. 259, 14144 (1984)] were used to show that the unmodified smooth muscle precursor core protein was immunologically related to both the cartilage and fibroblast core proteins. The size of the precursor core proteins within each species was identical regardless of the tissue source. Comparison of the precursor core proteins synthesized by primate and bovine cells revealed that the bovine core proteins were approximately 1500 Da larger than the primate core proteins as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A similar size difference was observed when the mature core proteins of monkey smooth muscle cells and bovine articular chondrocytes were compared after removal of the glycosaminoglycan chains. These results indicate that arterial smooth muscle cells synthesize a dermatan sulfate proteoglycan whose core protein is similar to, if not the same as, the cartilage and fibroblast dermatan sulfate proteoglycan core proteins. These core proteins may be encoded by the same gene that has diverged in size during speciation.     

Changes in expression and organization of smooth-muscle-specific α-actin during fibronectin-mediated modulation of arterial smooth muscle cell phenotype

The spreading of freshly isolated arterial smooth muscle cells on a substrate of fibronectin is mediated by an integrin receptor on the cell surface. It is associated with organization of actin filaments in stress fibers and marked changes in cell morphology and function, collectively referred to as a transition from a contractile to a synthetic phenotype. To study further how extracellular matrix components affect smooth muscle phenotype, we have analyzed the expression and organization of smooth-muscle-specific alpha-actin in freshly isolated rat aortic smooth muscle cells cultured on a substrate of fibronectin under serum-free conditions. Northern-blot analysis showed that the expression of mRNA for smooth muscle alpha-actin, but not for nonmuscle actin, was strongly repressed during primary culture. On the other hand, the cellular content of alpha-actin was only moderately changed during the same period. Indirect immunofluorescence staining revealed that nonmuscle actin was rapidly organized in stress fibers, which did not stain with a monoclonal antibody against smooth muscle alpha-actin. Filament bundles containing alpha-actin were most prominent in the central parts of the cytoplasm and gradually disappeared as the spreading of the cells progressed. In contrast to the situation with nonmuscle actin, there was no apparent overlap in the staining for alpha-actin and the fibronectin receptor (alpha 5 beta 1), indicating that this receptor interacted with nonmuscle actin during the initial spreading process. Taken together, the results show that the expression and organization of smooth muscle alpha-actin are changed during interaction of the cells with fibronectin early in primary culture. They support the notion that integrin-mediated interactions between extracellular matrix components and arterial smooth muscle cells take part in the control of smooth muscle phenotype.     

Chondroitin sulphate and heparan sulfate proteoglycan are sequentially expressed in the uterine extracellular matrix during early pregnancy in the rat.

Chondroitin sulfate proteoglycan (CSPG) and heparan sulfate proteoglycan (HSPG) are extracellular matrix proteins that regulate cell adhesion, growth, migration, differentiation and gene expression in many systems. In this study, stromal CSPG label was intense within 10 microm of the uterine lumen. From that distance to the myometrium, CSPG was de-expressed. From the time of implantation on Day 6, this pattern was reversed. CSPG was de-expressed from the uterine epithelium to a distance of approximately 10 microm from the uterine lumen. From that region to the myometrium, labeling was homogeneously intense. This finding suggests that CSPG may inhibit attachment and implantation. Heparan sulfate core proteoglycan (perlecan) was increasingly expressed in the uterine epithelium from the time of implantation, commencing in the basement membrane on Day 6 and extending to the apical epithelium and lateral plasma membranes by Day 7. Perlecan thus appears to facilitate trophoblast attachment and implantation. We propose that attachment and implantation is regulated, at least in part, by the selective and sequential expression of CSPG and perlecan.     

Decorin synthesized by arterial smooth muscle cells is retained in fibrin gels and modulates fibrin contraction

Fibrin serves as a provisional extracellular matrix (ECM) for arterial smooth muscle cells (ASMC) after vascular injury, yet little is known about the effect of fibrin on ECM remodeling by these cells. To address this question, monkey ASMC were grown on fibrin gels and tissue culture (TC) plastic, and proteoglycan synthesis and accumulation were assessed by radiolabeling. Initial rates of (35)S-sulfate incorporation into proteoglycans were identical for both groups, but increased proteoglycan accumulation was observed in cultures grown for 48 h on fibrin. This increased accumulation on fibrin was due to reduced proteoglycan turnover and retention within the fibrin gel. Decorin and biglycan constituted 40 and 14% of the total proteoglycan in the fibrin gels, whereas their combined contribution was only 12% in control matrices. To explore whether the retention of decorin in fibrin had any influence on the properties of the fibrin gel, ASMC-mediated fibrin contraction assays were performed. Both de novo synthesis of decorin as well as decorin added during polymerization inhibited the ability of the cells to contract fibrin. In contrast, decorin added exogenously to mature fibrin matrices had no effect on fibrin gel contraction. This study illustrates that decorin derived from ASMC selectively accumulates in fibrin and modifies fibrin architecture and mechanical properties. Such an accumulation may influence wound healing and the thrombotic properties of this provisional pro-atherosclerotic ECM.     

Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells

Platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1) increase [35S]sulfate incorporation into proteoglycan (PG) by monkey arterial smooth muscle cells but have opposite effects on cell proliferation. The combination of these two growth regulatory peptides has an additive effect on PG synthesis but no effects on cell proliferation. The time course of sulfate incorporation after stimulation indicates that both growth factors cause maximal incorporation of sulfate into glycosaminoglycan chains by 12-18 h. The PG that is most affected is a large CSPG (Mr approximately 1.2 x 10(6)) which can be immunoprecipitated by an antibody against versican, a large CSPG synthesized by human skin fibroblasts. The hydrodynamic size of this molecule increases after PDGF and TGF-beta 1 stimulation, but the size of the core glycoprotein (Mr approximately 450,000) remains the same. Treatment with either growth factor leads to an increase in the amount of core glycoprotein for this PG. This increase correlates with an increase in the steady state level of mRNA identified by hybridization to a cDNA encoding versican. The two growth factors also increase the glycosaminoglycan chain length of this PG accounting for the greater hydrodynamic size of the molecule after stimulation. In contrast, PDGF and not TGF-beta 1 changes the composition of the glycosaminoglycan chains attached to this PG by doubling the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate. These results indicate that although both of these growth factors increase the net synthesis of a large versican like CSPG, they differ in their effects on the structure of the glycosaminoglycan chains. These post-translational modifications may relate to the growth state of the cells.     

Antisense oligonucleotides to stromelysin mRNA inhibit injury-induced proliferation of arterial smooth muscle cells.

Smooth muscle cell migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. To make this possible, the smooth muscle cell has to change from a contractile to an activated repair cell with capacity to synthesize DNA and extracellular matrix components. There is now considerable evidence that the extracellular matrix has important functions in modulating the phenotypic properties of smooth muscle cells, but less is known about the role of the matrix metalloproteinases. The present study investigates the role of stromelysin in the modulation of rat aortic smooth muscle cell morphology and function following mechanical injury in vitro and in vivo. Antisense mRNA oligonucleotides were used to investigate the role of stromelysin expression in injury-induced phenotypic modulation and the subsequent migration and proliferation of vascular smooth muscle cells. Cultured rat aortic smooth muscle cells and balloon-injured rat carotid arteries were used as experimental models. Light- and electron microscopy were used to follow changes in smooth muscle cell phenotype and lesion formation and incorporation of 3H-thymidine to detect DNA synthesis. Injury-induced DNA synthesis and migration in vitro were inhibited by 72% and 36%, respectively, by adding stromelysin antisense oligonucleotides to the medium prior to injury. In primary cultures, 67% of the smooth muscle cells treated with stromelysin antisense were retained in a contractile phenotype as judged by analysis of cell fine structure, compared to 15% untreated cells and 40% in cells treated with mismatched oligonucleotides. Examination of the carotid arteries one week after balloon injury likewise demonstrated a larger fraction of contractile cells in the inner parts of the media in vessels treated with antisense oligonucleotides compared to those treated with mismatched oligonucleotides. The neointima was also distinctly thinner in antisense-treated than in mismatched-treated and control arteries at this time. These findings indicate that stromelysin mRNA antisense oligonucleotides inhibited phenotypic modulation of rat arterial smooth muscle cells and so caused a decrease in migration and proliferation and neointima formation in response to vessel wall injury.     

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical study of NG2 chondroitin sulfate proteoglycan expression in the small and large intestines

The intestinal subepithelial myofibroblasts (ISEMFs) are located in the lamina propria under the epithelial cells. ISEMFs are thought to have an important role in protecting and maintaining the integrity of the epithelial cell layer and also in the process of wound healing. In this study, we report that the membrane-bound proteoglycan NG2 is abundantly distributed in the ISEMF layer of the mouse and human intestines. NG2 immunostaining in this layer is distributed with similar intensity from the crypt to villi. NG2 is also immunolocalized along the membranes of smooth muscle cells in the intestinal muscle layer. However, skeletal and cardiac muscles are not immunostained for NG2, demonstrating selective expression of the proteoglycan by smooth muscle cells. Using electron microscopy, NG2 immunoreactivity was strongly observed along the cell membranes of ISEMF, with weak diffusion into the neighboring matrix, indicative of the presence of some “shed” NG2. This first report of NG2 proteoglycan expression by ISEMF provides insights into the nature of the interaction of these cells with extracellular matrix and/or intestinal epithelial cells.     

Somite Chondrogenesis in vitro: Differential Induction by Modified Matrix- a Biochemical and Morphological Study*

The stimulation of somite chondrogenesis by extracellular matrix components was studied by monitering the synthesis of cartilage-specific large proteoglycan aggregates. Chick embryonic sternal proteoglycans were separated into various components: monomers, hyaluronic acid, link protein and glycosaminoglycan side chains. The effects of these components, either individually or in various combinations, on somite chondrogenesis was examined. Proteoglycan monomers, alone or in a mixture with other components, induced chondrogenesis. The other components did not have any stimulating effect of their own. The results of these induction studies were also observed on a Sepharose CL–2B column and correlated using electron microscopy. Stimulation of somites resulted in an increase in the amount of proteoglycan aggregation (material excluded from the column) and was in agreement with the morphological appearance of the matrix in that there was increased accumulation of large proteoglycan granules. A matrix mixture of collagen and proteoglycans showed significant stimulation. When the matrix environment of the somites was altered to be unfavorable to the explants (medium containing hyaluronic acid) there was altered synthesis of cartilage-specific molecules. The results presented in this report strongly suggest that the composition of the extracellular matrix material is critical for somite chondrogenesis.     

Luminal morphology of small arterial vessels in the contracted spleen,studied by scanning electron microscopy of microcorrosion casts

Unique luminal configurations exhibited by small arterial vessels in contracted spleens of dog and cat were studied by means of vascular corrosion casts examined by scanning electron microscopy. Concertina-like pleating was seen in casts of trabecular arteries/arterioles, whereas within lymphatic nodules arteriolar casts lacked pleating and were smooth and uniformly cylindrical (as were all small arterial vessels in distended spleens). Morphological details of arterial vessels observed in histological sections indicated that pleating is not due to contraction of specially arranged vascular smooth muscle but to overall shortening of trabecular arterial vessels, caused by contraction of longitudinal smooth muscle in trabeculae. Another phenomenon observed in casts from contracted spleens was an almost complete "pinching-off" of many arteriolar lumens; histological evidence indicated that this is due to contraction of vascular smooth muscle, which selectively diverts flow away from certain regions of the organ. Also noted was a markedly convoluted, tortuous configuration of arterioles (penicilli) in the red pulp of contracted spleens.