chondroitin-sulfates and Hypoxia

Topics: Bone Marrow; Cell Differentiation; Cells, Cultured; Chondrocytes; Chondroitin Sulfates; Chromatography, Liquid; Disaccharides; Glycosaminoglycans; Humans; Hypoxia; Mesenchymal Stem Cells; Oxygen; Tandem Mass Spectrometry

Macrophages are prominent in hypoxic areas of atherosclerotic lesions. Their secreted proteoglycans (PG) can modulate the retention of lipoproteins as well as the activity of enzymes, cytokines, and growth factors involved in atherogenesis. Versican appears to be one of the main extracellular matrix components binding LDL in the arterial intima. We have recently shown that hypoxia increases versican and perlecan expression in macrophages, and that this increase was regulated by the hypoxia inducible factor (HIF). Here we report effects of hypoxia on human monocyte-derived macrophage (HMDM) secreted glycosaminoglycans (GAG), and its interaction with LDL. After 24 h exposure to 0.5% O2 (hypoxia), metabolically labeled GAG of secreted PG had higher affinity for LDL compared to 21% O2 (control cells). GAG secreted by HMDM in hypoxia were found to be more sulfated and longer which might be responsible for the increased affinity of LDL for these GAG chains. These results indicate that hypoxia induced changes in macrophage GAG biosynthesis have important consequences for the interaction with LDL. If present in vivo, an augmented association of GAG with LDL might contribute to the development of atherosclerosis in hypoxic intima.

Topics: Atherosclerosis; Chondroitin Sulfates; Chromatography, Gel; Glycosaminoglycans; Humans; Hypoxia; Lipoproteins, LDL; Macrophages; Proteoglycans; Sulfotransferases

Binding of basic fibroblast growth factor (FGF2) to its high affinity receptors requires the presence of specific heparan sulfate (HS) moieties on the cell surface that act as coreceptors. To determine the contribution of cell-surface HS to modulation of FGF2-dependent cell growth, we studied the changes in the cell mass and FGF2 binding of endothelial cell HS under normoxic and hypoxic conditions in vitro. Both large vein and cardiac microvascular endothelial cells cultured under hypoxic conditions demonstrated an increase in the ratio of cell-surface HS to chondroitin sulfate (CS), as well as an increase in the number of low affinity (HS-associated) binding sites for FGF2 with no change in the apparent K(d). This increase in the number of HS-FGF2 binding sites, in the absence of a significant change in FGF receptor expression, resulted in enhanced responsiveness of hypoxic, compared with normoxic, endothelial cells to FGF2 stimulation. Gene expression studies demonstrated increased expression of the key regulatory enzyme responsible for HS chain synthesis, 1,4 GlcNAc transferase (GlcNAcT-I), as well as increased expression of 2-O sulfotransferase (HS2ST), the enzyme responsible for sulfation of IdoA, a crucial part of the HS-FGF2 binding site. Transduction of cells with an adenovirus encoding a HIF-1alpha expression construct resulted in a similar increase in GlcNAcT-I and HS2ST expression. We conclude that hypoxia increases endothelial cell responsiveness to FGF2 by promoting preferential synthesis of HS rather than CS chains and increasing the number of FGF2-binding sites on HS chains. Both of these events are mediated by a HIF-1alpha-dependent increase in expression of the enzymes GlnNAcT-I and HS2ST. This shift in cell-surface HS composition results in enhanced cell sensitivity to FGF2-induced growth stimulation.

Topics: Animals; Animals, Newborn; Binding Sites; Cell Division; Cells, Cultured; Chondroitin Sulfates; Endothelium, Vascular; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Glycosaminoglycans; Heparitin Sulfate; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Neovascularization, Physiologic; Protein Binding; Rats; Receptors, Fibroblast Growth Factor; Signal Transduction; Transcription Factors

Pulmonary interstitial pressure was measured via micropuncture in anesthetized rabbits in normoxia and after breathing 12% O(2). In normoxia [arterial PO(2) = 88 +/- 2 (SD) mmHg], pulmonary arterial pressure and pulmonary interstitial pressure were 16 +/- 8 and -9.6 +/- 2 cmH(2)O, respectively. After 6 h of hypoxia (arterial PO(2) = 39 +/- 16 mm Hg), the corresponding values were 30+/-8 and 3.5+/-2.5 cm H(2)O (P<0.05). Pulmonary interstitial proteoglycan extractability, evaluated by hexuronate assay after 0.4 M guanidinium hydrochloride extraction, was 12.3, 32.4, and 60.6 microg/g wet tissue in normoxia and after 3 and 6 h of hypoxia, respectively, indicating a weakening of the noncovalent bonds linking proteoglycans to other extracellular matrix components. Gel filtration chromatography showed an increased fragmentation of chondroitin sulfate- and heparan sulfate-proteoglycans during hypoxic exposure, accounting for a loss of extracellular matrix native architecture and basement membrane structure. Gelatin zymography demonstrated increased amounts of the proteolytically activated form of gelatinase B (matrix metalloproteinase-9) after hypoxic exposure, providing evidence that the activation of proteinases may play a role in hypoxia-induced lung injury.

Topics: Animals; Blood Pressure; Chondroitin Sulfates; Chromatography, Gel; Extracellular Matrix; Extracellular Space; Heparitin Sulfate; Hexuronic Acids; Hydrostatic Pressure; Hypoxia; Intercostal Muscles; Lung; Matrix Metalloproteinase 9; Molecular Weight; Organ Size; Pulmonary Artery; Rabbits

Porcine aortic endothelium cultured at 20% oxygen concentration synthesizes collagen and three of its marker enzymes - proline and lysine hydroxylases and lysine oxidase, the cross-link enzyme. It also synthesizes and secretes hyaluronic acid, dermatan sulphate, possibly heparan sulphate, large amounts of chondroitin 4-sulphate and smaller amounts of chondroitin 6-sulphate. Growth of these cells for 24 h in 0%, or 2% oxygen results in little change in cell numbers or cell protein but a fall in collagen synthesis and in proline and lysine hydroxylases, but a rise in lysine oxidase. There is a considerable increase in synthesis and secretion of all the glycosaminoglycans found. The cell lipids appear qualitatively unchanged. Apart from increased lysosomes seen at 0% oxygen, no ultrastructural changes appear to occur. These findings illustrate the lability of the endothelial response to oxygen lack.

Topics: Animals; Aorta; Cells, Cultured; Chondroitin Sulfates; Collagen; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Endothelium; Glycosaminoglycans; Hypoxia; Lipids; Oxygen; Swine

The influence of 5% O2 hypoxia on the 35S-sulfate incorporation into different mucopolysaccharide fractions was studied in monolayer cultures of embryonic rat fibroblasts before reaching the stationary phase. Besides the O2 concentration also the pH value and lactate concentration were varied in the experiments. The results are valid for proliferating cell cultures.

Topics: Animals; Cells, Cultured; Chondroitin Sulfates; Dermatan Sulfate; Fibroblasts; Glycosaminoglycans; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Lactates; Oxygen Consumption; Rats; Sulfates; Sulfur Radioisotopes

Secondary cultures of embryonic rat fibroblasts [NEUPERT et al., Exp. Path. 7, 19-28 (1972)] were cultured in 21% and 5% O2 concentration at pH 6.6 and 7.4 for 6 or 8 days. The acid mucopolysaccharides were isolated and fractionated by alcohol precipitation, papain digestion, CPC-precipitation and fractionation after the microtechniques of SVEJCAR and ROBERTSON [see KITTLICK and NEUPERT, Exp. Path. 7, 7-18 (1972)]. Cells and medium were investigated together. The results were related to cell density and compared with the glucose and lactate values [see KITTLICK and NEUPERT, Exp. Path. 10, 109-114 (1975)]. Concerning the question of interrelations of MPS-synthesis and glycolysis a survey on literature is given. Our own test results were as follows: 1. Hypoxia (5% O2) did not influence MPS-total synthesis. 2. Depending on cell density the individual MPS fractions were different in their reaction to hypoxia. 3. Hyaluronic acid (and heparan sulphate) in the MPS-pattern showed other behaviour than chondroitin sulphate and dermatan sulphate, respectively. 4. At low cell density hypoxia effectuated increase in hyaluronic acid and decrease in chondroitin sulphate and dermatan sulphate, respectively. 5. At high cell density hypoxia effectuated decrease in hyaluronic acid and increase in chondroitin sulphate and dermatan sulphate, respectively. Possible relationship to processes in the tissue is discussed.

Topics: Animals; Cells, Cultured; Chondroitin Sulfates; Culture Media; Fibroblasts; Glycosaminoglycans; Hyaluronic Acid; Hydrogen-Ion Concentration; Hypoxia; Rats