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Biomedical subjects

R L Trelstad

Publications and source records attributed to R L Trelstad.

At least 37 records · Page 2Linked to original sources
PubMed1988

Hormonal modification of epithelial differentiation and expression of cell surface heparan sulfate proteoglycan in the mouse vaginal epithelium. An immunohistochemical and electron microscopic study.

Immunohistochemical staining of a cell surface antigen was evaluated in the adult mouse vaginal epithelium at different stages of the estrous cycle and in response to exogenous sex hormones and endocrine ablation. The antigen is recognized by a monoclonal antibody directed against the core protein of a heparan sulfate-rich proteoglycan from mouse mammary epithelial cells. Vaginal epithelium at estrus showed the most intense staining; cells of the basal and intermediate layers stained, but the more superficial parakeratotic, cornified, and sloughing layers did not. At metestrus and diestrus, immunostaining was limited to basal cells and some deeper intermediate cells. The staining was absent from the more superficial layers which were invaded by leukocytes. At late diestrus and proestrus, staining was primarily in the intermediate cells; staining was absent from parakeratotic and basal cell layers. There was no staining of submucosal cells throughout the estrous cycle. In ovariectomized mice, staining of the epithelium was reduced in intensity. Diethylstilbestrol treatment of ovariectomized mice increased the intensity and extent of epithelial staining and produced a state comparable to that seen at estrous. Administration of a combination of progesterone and estradiol to ovariectomized mice elicited vaginal stratification and mucification, a state comparable to that observed in diestrus in which basal and intermediate layers stained while the apical mucified cells did not. In animals expressing natural or diethylstilbestrol-induced estrus, electron microscopic immunoperoxidase staining revealed the presence of the antigen on the surface of cell processes in the intercellular spaces between vaginal epithelial cells. Cuprolinic blue staining for glycosaminoglycan using the critical electrolyte concentration method demonstrated filamentous structures on the epithelial surface in the same location to that of the antigen. The stained filaments were reduced by treatment with heparitinase, but not with chondroitinase ABC or heparin, suggesting that they contained heparan sulfate glycosaminoglycan. These data suggest that as vaginal epithelial differentiation fluctuates during the estrous cycle in response to changing levels of estrogens and progesterone, expression of a cell surface heparan sulfate proteoglycan undergoes dramatic changes spatially and quantitatively.

Animals↗
PubMed1988

The extracellular matrix is a soluble and solid-phase agonist and receptor.

The components of the extracellular space have well-recognized structural functions. The role of the matrix as a conduit and/or repository for signals, however, has received much less attention. Because we know that cells require contact with the extracellular matrix for their normal function, that intact matrix and/or fragments of matrix in either soluble or solid phase can effect significant changes in cellular physiology, and that matrix components can be integral membrane proteins, we conclude that a major function of the matrix is as a soluble and solid-phase agonist and receptor.

Cell Communication↗
PubMed1987

Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study.

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.

Animals↗
PubMed1986

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes.

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.

Animals↗
PubMed1986

Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation.

The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.

Animals↗
PubMed1986

Morphologic examination of mesenchymal cells in healing wounds of normal and tight skin mice.

The healing process of an open wound as effected by wound contraction is complete by 3 weeks in the normal mouse. In contrast, its onset is delayed by 3 weeks and complete healing requires 6 weeks in the tight skin mouse (TSM), a mutant mouse strain with the autosomal dominant gene for tight skin. Possible mechanisms for this delay were evaluated. The frequency and distribution of myofibroblasts were studied during the 3-week delay in wound contraction by actin staining and electron microscopy. It was determined, by electron microscopy and phalloidin staining, that myofibroblasts were found in high density in noncontracting TSM wounds. Electron microscopy showed, however, that these myofibroblasts were surrounded by a pericellular matrix that separated their surface from adjacent collagen fibers. No pericellular matrix was found around cells in granulation tissue of normal mice. At 3 weeks, as TSM wounds began to contract, the number and intensity of cells stained by phalloidin in this tissue was less than that seen earlier. The pericellular matrix was fragmented at this time, and cell surface and collagen fiber associations were apparent. Finally, at 5 weeks, when wound contraction was well developed in the TSM, only a small area in the center of the healing wound beneath the epidermis contained phalloidin-positive myofibroblasts. Electron-microscopic examination of the residual granulation tissue at this time revealed the complete absence of the pericellular matrix. It is postulated that during the 3-week delay in wound closure, the presence of a localized pericellular matrix prevents the interaction between cells and collagen fibers necessary for the reorganization of collagen. It is also thought that the tightly adherent uninjured skin surrounding the healing wound may cause delayed wound closure. There was no evidence that the absence of myofibroblasts is responsible for delayed wound contraction.

Animals↗
PubMed1985

The fibroblast in morphogenesis and fibrosis: cell topography and surface-related functions.

'Fibroblast' is a generic term for a population of cells responsible for the establishment, maintenance and repair of three-dimensional form in multicellular organisms. These cells are a major, semi-permanent resident of the 'extracellular space' and the fibroblast compartmentalizes this space to various purposes during the formation of collagen fibrils and fibril bundles. The boundaries of these extracellular compartments blend and overlap, but discrete regions can be identified which are involved in collagen fibril assembly and fibril bundle assembly. The formation of these extracellular compartments in both the tendon and cornea results from a series of fusions of membrane-limited structures, beginning with collagen secretory vacuoles fusing with the cell surface to form deep surface recesses within which fibrils assemble; and progressing through the lateral fusion of these recesses with the consequent formation of fibril bundles. The topography of the fibroblast is also specialized for matrix anchorage, for matrix and cell repositioning, and for matrix degradation.

Animals↗
PubMed1985

Fibroblasts create compartments in the extracellular space where collagen polymerizes into fibrils and fibrils associate into bundles.

Morphogenesis in the animal kingdom is closely coupled with the intracellular synthesis and the extracellular deposition of collagen. These morphogenetically important events involve multiple steps which begin in compartments inside the cell and continue in compartments outside the cell. The movement of matrix components from the intracellular compartments to the extracellular compartments is a functional continuum. Studies of embryonic chick tendon and cornea fibroblasts with the high-voltage (1000 kV) transmission electron microscope have provided the beginnings of an understanding of how this functional continuum is related to the structural compartments which the cells create. This series of compartments, both inside and outside the cell, is involved in collagen fibrillogenesis, collagen fibril bundle formation, and tissue morphogenesis.

Animals↗
PubMed1984

Formation of collagen fibrils by enzymic cleavage of precursors of type I collagen in vitro.

Two systems were used to generate collagen fibrils in vitro by enzymic cleavage of intermediates in the conversion of procollagen to collagen. In one system fibrils were generated by using procollagen NH2-terminal proteinase to cleave pNcollagen, the intermediate which contains the NH2-terminal but not the COOH-terminal propeptides found in procollagen. When pNcollagen was incubated with procollagen NH2-terminal proteinase, the NH2-terminal propeptides were enzymically cleaved from the protein, and there was an increase in the turbidity of the solution over and above the turbidity observed with pNcollagen alone. Electron microscope examination of the samples demonstrated that the increase in turbidity was associated with the assembly of collagen fibrils. The fibrils had a mean diameter of 104 nm +/- 51.7 S.D. or about the same as fibrils formed from pNcollagen alone. However, the fibrils formed by enzymic cleavage of pNcollagen had a more distinct gap-overlap pattern and they appeared to be more tightly packed than fibrils of pNcollagen. Varying the concentration of enzyme varied both the rate of enzymic cleavage of the pNcollagen and the rate of fibril assembly, but there was no consistent effect on the diameter or morphology of the fibrils. In the second system, fibrils were generated with a recently described procedure (Miyahara, M., Njieha, F. K., and Prockop, D. J. (1982) J. Biol. Chem. 257, 8442-8448) in which procollagen COOH-terminal proteinase is used to cleave pCcollagen, the intermediate containing the COOH-terminal but not the NH2-terminal propeptides found in procollagen. When incubated with procollagen COOH-terminal proteinase, the COOH-terminal propeptides were cleaved and collagen fibrils assembled. The collagen fibrils were unusually thick with a mean diameter of 1184 nm +/- 291 S.D. The large diameters of the fibrils made it possible to demonstrate by scanning electron microscopy that each fibril was comprised of a bundle of subfibrils packed into a right-handed helix. The fibrils frequently had branch points which appeared to consist of subfibrils which separated from the main axis of the structure. Also, the surface of the fibrils was scalloped at 270- to 300-nm intervals, suggesting that some of the collagen molecules on the surface were in a 4D staggered array. The results suggested the hypothesis that the order in which the NH2-terminal and COOH-terminal propeptides are cleaved in the conversion of procollagen to collagen may provide a mechanism for controlling the diameter, or both the diameter and morphology, of collagen fibrils.

Amino Acid Sequence↗
PubMed1984

Changing patterns of fibronectin, laminin, type IV collagen, and a basement membrane proteoglycan during rat Mullerian duct regression.

Antibodies to type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin were used to study the regression of the rat Mullerian duct. All four of these matrix constituents are located at the perimeter of the Mullerian duct within the ductal basement membrane. As the Mullerian duct regresses, the staining of all of these basement membrane constituents becomes irregular and discontinuous. Fibronectin, which is also present in the interstitium, becomes undetectable in the mesenchyme which condenses around the regressing Mullerian duct. These data indicate that degradation of the extracellular matrix around the male Mullerian duct is a central event in the regression of this structure.

Animals↗
PubMed1984

Regulation of type I collagen fibril assembly by link protein and proteoglycans.

Link protein, a glycoprotein, that is present both in cartilaginous and non-cartilaginous tissues, has previously been shown to bind to collagen and to proteoglycan. Here, we have examined the effects of link protein and proteoglycans, both alone and in combination, on the assembly of type I collagen fibrils in vitro. Link protein alone had no effect on the kinetics of fibril formation or on the size of the fibrils. Link protein, however, modulated the effects of various proteoglycans including those from bone, cartilage, cornea and sclera. Link protein had the most significant effect on fibril assembly in the presence of the low molecular weight bone proteoglycan. Although the bone proteoglycan alone had no effect on fibril formation, the fibrils were wider in the presence of link protein and proteoglycan. Cartilage proteoglycan alone increased the extent of fibril formation and the resultant fibrils were wider in diameter with a complement of incompletely assembled fibrils. In the presence of both link protein and cartilage proteoglycan, the fibrils were fully formed with the characteristic banding pattern. Further, corneal and scleral proteoglycans alone decreased the extent of fibril formation and the width of the fibrils was either unaltered or slightly decreased in the presence of the link protein. Our results indicate that both link protein and tissue-specific proteoglycans may regulate the organization of collagen fibrils in tissues.

Animals↗
PubMed1984

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts.

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.

Animals↗
PubMed1984

Reduction of chronic hypoxic pulmonary hypertension in the rat by beta-aminopropionitrile.

We administered antifibrotic agent beta-aminopropionitrile (BAPN) to rats exposed to 10% O2-90% N2 for 3 wk to prevent excess vascular collagen accumulation. Groups of Sprague-Dawley rats studied were air breathing, hypoxic, and hypoxic treated with BAPN, 150 mg/kg twice daily intraperitoneally. After the 3-wk period, we measured mean right ventricular pressure (RVP), the ratio of weight of right ventricle to left ventricle plus septum (RV/LV + S), and hydroxyproline content of the main pulmonary artery (PA) trunk. Hypoxia increased RVP from 14 to 29 mmHg; RVP was 21 mmHg in hypoxic BAPN-treated animals. Hypoxia increased the RV/LV + S ratio from 0.28 to 0.41; the ratio was 0.32 in hypoxic BAPN-treated animals. Hypoxia increased PA hydroxyproline from 20 to 239 micrograms/artery; hydroxyproline was 179 micrograms/artery in hypoxic BAPN-treated animals. Thus BAPN prevented pulmonary hypertension, right ventricular hypertrophy, and excess vascular collagen produced by hypoxia. We conclude that vascular collagen contributes to the maintenance of chronic hypoxic pulmonary hypertension.

Aminopropionitrile↗
PubMed1984

Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody.

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.

Animals↗
PubMed1984

Zinc chelation and mullerian duct regression.

After observing that zinc chelation by EDTA (ethyldiamine tetraacetic acid) mimics Mullerian inhibiting substance (MIS)-induced regression of the Mullerian duct, a detailed morphological study of the effects of EDTA on the Mullerian duct was undertaken. Urogenital ridges from the 14-day-old fetal rat were incubated in medium containing EDTA for 14, 24, 38, 48, 62, and 72 hours. The tissues were then processed for light and electron microscopy and stained with antibodies to laminin, fibronectin, heparan sulfate, and type-IV collagen. EDTA simulates the regression produced by endogenous MIS, producing early loss of basement membrane integrity, loss of epithelial cell polarity and organization, and loss of recognizable duct integrity by 72 hours. Like MIS, EDTA has no effect on the Wolffian duct. In contrast to MIS-treated Mullerian ducts, however, those incubated with EDTA showed a different sequence of disappearance of basement membrane components, absence of the specific periductal mesenchymal condensation seen with MIS, and absence of epithelial cell migration into the mesenchyme. Zinc chelation may be part of a series of mechanistic steps involved in Mullerian duct regression.

Animals↗
PubMed1983

Epidermolysis bullosa dystrophica recessive fibroblasts altered behavior within a collagen matrix.

Normal human fibroblasts incorporated into a collagen lattice reduce the size of that lattice over a period of time. Lattice size reduction or lattice contraction is directly related to initial cell number. When equal numbers of fibroblasts derived from patients with epidermolysis bullosa dystrophica recessive, (EBdr), are used, there is delayed lattice contraction. The EBdr fibroblasts have an altered cellular shape, when compared to normal cells, in that the EBdr cells fail to flatten out and elongate, but do attach to collagen fibers like normal fibroblasts. EBdr fibroblasts maintain a rounded shape with numerous filopodia radiating from the cell periphery and such filopodia are attached to the collagen fibers of the lattice. In monolayer tissue culture on glass surfaces, EBdr fibroblasts are three times more likely to grow over neighboring fibroblasts. EBdr cell filopodia structures are attached to the cell surfaces lying beneath them, which demonstrates another condition of altered anchorage attachment of EBdr fibroblasts.

Cell Division↗