Roslyn W. Orkin

Undersulfated chondroitin sulfate in the cartilage matrix of brachymorphic mice.

Homozygous brachymorphic (bmbm) mice have a disproportionately short stature, similar to human achondroplasia. We previously showed that each zone of growth in young bmbm epiphyseal cartilages is smaller than normal and that the extracellular matrix appears to contain normal collagen fibrils, but smaller and reduced numbers of proteoglycan matrix granules. Our studies reported here indicate that mutant, like normal cartilage, synthesizes type II collagen and contains normal quantities of glycosaminoglycans as judged by uronic acid content. However, the glycosaminoglycans from the mutant differ from the normal in their chromatographic and electrophoretic properties. Further studies established that glycosaminoglycans from cartilages of brachymorphic animals were undersulfated. Whereas chondroitinase digests of glycosaminoglycans from cartilage of normal C57Bl6J 5-day-old mice contained predominantly disaccharides sulfated in the 4-position, that of the mutant contained appreciable unsulfated disaccharides as well.

Comparison of tissue factor pathway in human umbilical vein and adult saphenous vein endothelial cells: implications for newborn hemostasis and for laboratory models of endothelial cell function.

In this work we have undertaken a comparative study of human umbilical vein endothelial cells (HUVECs) and human saphenous vein endothelial cells (HSVECs) with respect to functional and antigenic tissue factor (TF), tissue factor pathway inhibitor (TFPI), and TF mRNA. Monolayers of each cell type (passage 2, except where specified) were grown to confluence and then activated for 4 h with either 50 U/mL IL-1-α or 10 μg/mL tumor necrosis factor-α. Activated factor X appearing in supernatant was measured using a chromogenic assay, and both Northern blots and quantitative RT-PCR were performed to assess concentrations of TF mRNA accompanying activation. The role of TFPI was separately determined by ELISA for supernatant TFPI antigen, and by measurements of production of activated factor X in the presence of 0, 5, 15, or 50 μg/mL of an antibody directed against TFPI. To address a non-TF pathway endothelial cell function, antigenic concentrations of tissue plasminogen activator for both cell types was also determined by ELISA. HUVECs were found to produce 2.4- to 3.5-fold more functional TF. No significant HUVEC-HSVEC differences were detected in TF antigen, supernatant TFPI, anti-TFPI affinity for endothelial cell-associated TFPI, TF mRNA or its amplification products, and tissue plasminogen activator. Immunostaining for TF antigen, however, may have failed to detect a modest HUVEC-HSVEC difference. Our finding with respect to functional TF indicates that HUVECs and HSVECs are not equivalent in terms of models for endothelial cell function in small children versus adults.

Ambient oxygen tension modulates endothelial fibrinolysis

PURPOSE Vascular procedures reoxygenate ischemic endothelial cells (EC) and arterialize saphenous vein (HSV) EC. The balance between the EC-derived fibrinolytic components, plasminogen activator (tPA), and plasminogen inhibitor (PAI-1) contributes to maintaining thromboresistance. This balance also affects proteolysis through plasmin generation, mediating matrix metabolism endothelial migration, angiogenesis, and theoretically affecting the development of intimal hyperplasia. METHODS To explore the impact of varying oxygen tensions on EC fibrinolysis, HSV and human umbilical vein (HUV) were subjected to Po2 of 40 mm Hg for 24 hours with restoration of Po2 to 150 mm Hg for 24 hours. The tPA and PAI-1 antigen and tPA/PAI-1 antigen ratio in conditioned media (CM), expressed as increases or decreases % change, normalized for cell count, versus controls, were analyzed by enzyme-linked immunosorbent assay. Cellular tPA and PAI-1 mRNAs were assessed by Northern analysis. RESULTS The tPA but not PAI-1 was significantly decreased after the first 24 hours in HSVEC and significantly decreased after 48 hours in both HUVEC and HSVEC when compared with controls. Messenger RNA for tPA was unchanged but PAI-1 mRNA increased significantly for HSVEC and HUVEC after 24 hours of Po2 of 40 mm Hg, returning to baseline within 24 hours of Po2 to 150 mm Hg restoration. CONCLUSIONS These data support the hypothesis of a fibrinolytic shift after altered ambient O2 tensions exposure in endothelium and demonstrate that HSVEC are more sensitive to altered O2 tension than HUVEC. Altered O2 tensions depress EC fibrinolysis in this model.

A compliant tubular device to study the influences of wall strain and fluid shear stress on cells of the vascular wall

PURPOSE Cellular constituents of the blood vessel wall are continuously subjected, in vivo, to both mechanical and hemodynamic forces, which elicit structural and biologic responses. We have developed a compliant tubular system, the vascular simulating device (VSD), that reproduces these forces, while supporting the attachment and the experimental manipulation of endothelial and smooth muscle cells. METHODS The VSD consists of a compliant silicone rubber tube coupled to a pump system, which permits the simultaneous application of known levels of pressure and flow, to vascular wall cells cultured on the inner surface of the tube. Seeded cells can be monitored visually under phase contrast or fluorescent optics, as well as harvested and analyzed for biologic responses. RESULTS The elastic modulus and compliance of the silicone rubber tube are similar to those of canine and human arteries. Endothelial and smooth muscle cells cultured on the lumenal surface of the tubes remain attached and viable after subjecting them to physiologic pulsatile flow and cyclic strain. CONCLUSION The VSD makes it possible to approximate, in vitro, those forces encountered by vascular wall cells, in vivo and therefore may make it possible to determine whether specific combinations of mechanical and hemodynamic forces are causally associated with specific vascular diseases.

Hyaluronidase activity and hyaluronate content of the developing chick embryo heart

Abstract Hyaluronidase activity and hyaluronate content were measured in the developing chick heart from embryonic day 3 through posthatching stages. High levels of both enzyme and substrate were found during the earliest stages examined. Hyaluronidase activity gradually declined to 63% of the initial (day 3) level by embryonic day 16. Enzyme activity decreased more sharply during the next 4 days to 30% of the initial level and remained constant through 2 weeks after hatching. Low levels of enzyme activity (about 10% initial levels) were still detectable in 10-week-old chicken hearts. The heart hyaluronidase is an endoglycosidase with an estimated molecular weight of 62,000, which degrades hyaluronate and, to a lesser extent, chondroitin sulfate at an acid pH optimum. Hyaluronate constituted approximately 50% of the total glycosaminoglycan content at embryonic day 5. Between embryonic days 5 and 12, the concentration of hyaluronate decreased to 25–30% of the initial level and remained constant thereafter. The level of other glycosaminoglycans decreased more gradually than hyaluronate and did not reach a constant level until hatching. This pattern of hyaluronidase activity and hyaluronate concentration presumably reflects the extensive tissue remodeling which transforms the developing heart from a thin-walled tube containing extensive regions of extracellular matrix to a compact, thick-walled myocardium having a limited extracellular compartment.

Current concepts of basement-membrane structure and function.

ConclusionIn this brief review we have attempted to describe the known components of basement membranes in relation to the morphology and function of these matrices. Further details of the molecular structures and biosynthesis of these components may be found in original papers and in various reviews (Kefalides, 1973; Spiro, 1976; Kefalides et al., 1979; Heathcote & Grant, 1981).Although basement membranes appear to contain essentially similar protein and carbohydrate moieties, the proportions and organization of these may differ and, in the opinion of the authors, the key to an understanding of basement membranes lies in the recognition of this heterogeneity. At present, structural models of basement membrane are far from satisfactory and should be regarded with reservation.

Shear Stress Decreases Endothelial Cell Tissue Factor Activity by Augmenting Secretion of Tissue Factor Pathway Inhibitor

Abstract—Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1&agr; (IL-1&agr;) for 3 hours and simultaneously conditioned with shear stresses of 0, 0.68, or 13.2 dyne/cm2 in a parallel-plate flow chamber. In the presence of an inflow buffer containing 100 nmol/L factor X and 10 nmol/L factor VII, production of factor Xa, a measure of functional tissue factor (TF), was determined as the product of outflow concentration of factor Xa (chromogenic assay performed under quasi-static flow conditions after the shear period) and flow rate. Similarly, production of TF pathway inhibitor (TFPI) was estimated as the product of antigenic TFPI (by enzyme-linked immunosorbent assay) in the supernatant and flow rate. In parallel experiments, total RNA was isolated for determination of amplification products of TF mRNA by reverse transcription–polymerase chain reaction. We found that shear stress reduced factor Xa production (mean±SE; n=number of experiments) from 13.33±1.14 (n=16) fmol/min×cm2 at 0 shear stress to 5.70±2.51 (n=5) and 0.54±0.54 (n=4) fmol/min×cm2 at shear stresses of 0.68 and 13.2 dyne/cm2, respectively. At the same time, immunogold labeling showed that TF antigen on the endothelial surface increased >5-fold with shear stress, whereas TFPI antigen on the surface increased 2-fold. The secretion of TFPI (appearance of new supernatant TFPI) rose from 7.4±2.4 (n=12) ×10−3 fmol/min×cm2 at 0 shear stress to 23.7±7.3 (n=9) and 50.2±14.3 (n=4) ×10−3 fmol/min×cm2 at 0.68 and 13.2 dyne/cm2, respectively. TF mRNA amplification products were not markedly changed by shear stress. We conclude that acute application of shear stress reduces functional, but not antigenic, expression of TF by intact, activated endothelial cell monolayers in a manner associated with shear stress–augmented endothelial cell secretion of TFPI.

Collagen in developing chick muscle in vivo and in vitro

Abstract Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.

Synthesis and distribution of collagen in the rat palate during shelf elevation

Abstract Previous studies have shown that drugs which block the crosslinking of collagen prevent formation of the secondary palate by inhibiting shelf elevation. In this paper, the authors establish that collagen synthesis increases significantly just prior to shelf elevation and that type I collagen is synthesized throughout this developmental period (gestational Days 14–16 in the rat embryo) by isolated shelves in vitro. The largest accumulation of collagen fibers, predominantly oriented in a rostral-caudal plane, was observed adjacent to the basement membrane of the oral palatal epithelium. The unique location and orientation of these collagen fibers suggest that they play a structural role in the elevation of the palate.

Hyaluronidase activity in embryonic chick heart muscle and cushion tissues and cells

Hyaluronidase activity was compared in embryonic chick cardiac cushion and noncushion segments, as well as in cultures of mesenchyme derived from cardiac cushion endocardium (cushion tissue-enriched cultures) and in cultures of myocardial cells at stages critical to heart valve and septum development. Enzyme levels were higher in both heart tissue regions at periods of active cushion tissue mesenchyme migration than after migration ceases, and higher in the cushion region than in the noncushion region at both periods. Hyaluronidase was measured in cells and medium in both types of cultures, with five times greater activity found in the myocardial cultures. The cardiac hyaluronidase from cells and medium of both culture types had an estimated molecular weight of 41,000 to 44,000 and degraded hyaluronate and, to a lesser degree, chondroitin sulfate, at an acidic pH optimum. Ion-exchange chromatography demonstrated that in both culture types, a proportion of the secreted enzyme was more acidic than that found in the cell layer. These studies indicate the potential for hyaluronate degradation by the major cell types present in the developing heart at early stages and that the enzyme responsible is probably a lysosomal enzyme. Therefore, hyaluronate internalization is a likely requirement for degradation, and thus, the turnover of hyaluronate in developing heart valves is more complex than the extracellular degradative process suggested by histochemical data.