Depletion of alveolar macrophages attenuates hypoxic pulmonary hypertension but not hypoxia-induced increase in serum concentration of MCP-1.

Exposure to hypoxia, leading to hypoxic pulmonary hypertension (HPH), is associated with activation of alveolar macrophages (AM). However, it remains unclear how AM participate in this process. There are studies which imply that the AM product monocyte chemoattractant protein-1 (MCP-1) plays an important role. Thus we tested: 1. if the selective elimination of AM attenuates HPH in rats, 2. the correlation of MCP-1 plasmatic concentrations with the presence and absence of AM during exposure to hypoxia, 3. the direct influence of hypoxia on MCP-1 production in isolated AM. We found that experimental depletion of AM attenuated the chronic hypoxia-induced increase in mean pulmonary arterial pressure, but did not affect the serum MCP-1 concentrations. Furthermore, the MCP-1 production by AM in vitro was unaffected by hypoxia. Thus we conclude that AM play a significant role in the mechanism of HPH, but MCP-1 release from these cells is most likely not involved in this process. The increase of MCP-1 accompanying the development of HPH probably originates from other sources than AM.

Comparison of the effects of tert-butyl hydroperoxide and peroxynitrite on the oxidative damage to isolated beef heart mitochondria.

Isolated beef heart mitochondria have been exposed to tert-butyl hydroperoxide (tBHP) and peroxynitrite (PeN) in order to model the effects of reactive oxygen and nitrogen species on mitochondria in vivo. The formation of malondialdehyde (MDA), protein carbonyls, lipofuscin-like pigments (LFP), and nitrotyrosine was studied during incubations with various concentrations of oxidants for up to 24 h. The oxidants differed in their ability to oxidize particular substrates. Fatty acids were more sensitive to the low concentrations of tBHP, whereas higher concentrations of PeN consumed MDA. Oxidation of proteins producing carbonyls had different kinetics and also a probable mechanism with tBHP or PeN. Diverse proteins were affected by tBHP or PeN. In both cases, prolonged incubation led to the appearance of proteins with molecular weights lower than 29 kDa bearing carbonyl groups that might have been caused by protein fragmentation. PeN induced nitration of protein tyrosines that was more intensive in the soluble proteins than in the insoluble ones. LFP, the end products of lipid peroxidation, were formed more readily by PeN. On the other hand, fluorometric and chromatographic techniques have confirmed destruction of LFP by higher PeN concentrations. This is a unique feature that has not been described so far for any oxidant.

Immunolocalization of protein-bound 3-nitrotyrosine in inflammatory myopathies.

3-nitrotyrosine (3NT) is regarded as a "footprint" of nitric oxide generation. The study aimed at documenting the presence and distribution of 3-nitrotyrosine (3NT) in muscle tissue samples from patients with idiopathic inflammatory myopathies (IIM) as well as from those with non-inflammatory myopathies to consider whether polymyositis (PM) and dermatomyositis (DM) could be distinguished based on 3NT immunohistochemistry in muscle biopsy. Cryosections prepared from muscle biopsies of 54 patients with either IIM, i.e., PM and DM, or various non-inflammatory myopathies were immunostained using monoclonal antibody against 3NT. The 3NT immunostaining was localized to endothelial cells and their close surroundings in muscle biopsies of DM and PM patients but only in those areas of tissue sections where inflammatory cell infiltrates were present. No 3NT positivity was found in tissue sections of IIM patients without inflammatory infiltrates in the studied sample as well as in muscle tissue sections of patients with non-inflammatory myopathies. However, the endothelial cells were also positive in cases of confirmed non-inflammatory myopathies with secondary lymphocytic infiltration (myodystrophies, myasthenia gravis). Despite the pathogenetic significance, the 3NT immunohistochemistry is of low diagnostic value for the differential diagnosis of IIM in muscle biopsy.

Isolation of rat lung mast cells for purposes of one-week cultivation using novel Percoll variant Percoll PLUS.

Prolonged cultivation of separated rat lung mast cells (LMC) in vitro is necessary to better investigate a possible role of LMC in different stages of tissue remodeling induced by hypoxia. Rat lung mast cells (LMC) were separated using a protocol including an improved proteolytic extraction and two subsequent density gradient separations on Ficoll-Paque PLUS and a new generation of Percoll, i.e. Percoll PLUS. Instead of usual isotonic stock Percoll solution, an alternative "asymptotically isotonic" stock solution was more successful in our density separation of LMC on Percoll PLUS. Separated cells were cultivated for six days in media including stem cell factor, interleukins IL-3 and IL-6, and one of two alternative mixtures of antibiotics. These cultivations were performed without any contamination and with only rare changes in cell size and morphology. Model co-cultivation of two allogenic fractions of LMC often caused considerable rapid changes in cell morphology and size. In contrast to these observations no or rare morphological changes were found after cultivation under hypoxic conditions. In conclusions, we modified separation on Percoll PLUS to be widely used, altered LMC separation with respect to purposes of long-lasting cultivation and observed some model morphological changes of LMC.

Effect of culture substrate and culture conditions on lens epithelial cell proliferation and alpha-smooth muscle actin expression.

The most common complication following cataract surgery is posterior capsule opacification. This results from migration, proliferation and transdifferentiation of residual lens epithelial cells (LECs). We studied the effect of several culture substrates and culture conditions on LEC proliferation and alpha-smooth muscle actin (alpha-SMA) expression. We used primary and secondary cultures of porcine LECs cultivated on collagen I, collagen IV, microscopic glass slides, and uncoated plastic dishes. We studied the cell proliferation and expression of alpha-SMA and alpha-, beta-, and gamma-crystallins. The effect of the medium exchange protocol was studied using the TOTL-86 rabbit epithelial lens cell line. There was no difference in growth characteristics of primary cultures on different substrates. In secondary cultures, LECs adhered better to collagen-coated surfaces. The culture substrate influenced LEC proliferation and alpha-SMA expression. The proliferation was greater when the medium was changed than when extra medium was added on the 4th day. The cells did not synthesize alpha-, beta- or gamma-crystallin. The culture substrate influences the adhesion ability, proliferation and alpha-SMA expression in lens epithelial cells. In addition, it is necessary to consider the effects of the medium exchange protocol, serum supplementation, cell density and other cell culture conditions in lens epithelial cell experiments.

In vitro hypoxia increases production of matrix metalloproteinases and tryptase in isolated rat lung mast cells.

Chronic hypoxia results in hypoxic pulmonary hypertension characterized by fibrotization and muscularization of the walls of peripheral pulmonary arteries. This vessel remodeling is accompanied by an increase in the amount of lung mast cells (LMC) and the presence of small collagen cleavage products in the vessel walls. We hypothesize that hypoxia activates LMC, which release matrix metalloproteinases (MMPs) cleaving collagen and starting increased turnover of connective tissue proteins. This study was designed to determine whether in vitro hypoxia stimulates production of MMPs in rat LMC and increases their collagenolytic activity. The LMC were separated on the Percoll gradient and then were divided into two groups and cultivated for 24 h in 21 % O(2) + 5 % CO(2) or in 10 % O(2) + 5 % CO(2). Presence of the rat interstitial tissue collagenase (MMP-13) in LMC was visualized by immunohistological staining and confirmed by Western blot analysis. Total MMPs activity and tryptase activity were measured in both cultivation media and cellular extracts. Exposure to hypoxia in vitro increased the amount of cells positively labeled by anti-MMP-13 antibody as well as activities of all measured enzymes. The results therefore support the concept that LMC are an important source of increased collagenolytic activity in chronic hypoxia.

Changes of extracellular matrix of rat cornea after exposure to hypoxia.

The purpose of the study was to check whether hypoxia of corneal tissue increases the collagenolytic activity due to release of reactive oxygen and nitrogen species. Rats were exposed to hypoxia 10% O(2) for 4, 14, and 21 days. The radical tissue injury was measured by the level of nitrotyrosine and changes in the lipoperoxide-related fluorophores. Collagen protein composition was analyzed by slab gel electrophoresis. The activity of gelatinolytic enzymes was studied using the zymography. The vascularization of the corneas was measured. We found no differences in the corneal tissue in the gel electrophoretic profile of collagenous proteins and gelatinolytic activity between normoxic and hypoxic rats. We did not find any sign of radical tissue injury. There were no changes in the vascularization of corneas after exposition to hypoxia. The environmental 10% hypoxia does not induce radical tissue injury and an increase of collagenolytic activity in the rat cornea.

Redox regulation of proliferation of lens epithelial cells in culture.

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.

The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves.

Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species.

Erythrocyte membranes inhibit respiratory burst and protein nitration during phagocytosis by macrophages.

Phagocytosis is associated with respiratory burst producing reactive oxygen and nitrogen species. Several studies imply that erythrocytes can inhibit the respiratory burst during erythrophagocytosis. In this work we studied the mechanisms of this effect using control and in vitro peroxidized erythrocyte membranes. We demonstrated that autofluorescence of peroxidation products can be used for visualization of phagocytozed membranes by fluorescence microscopy. We also found that respiratory burst induced by a phorbol ester was inhibited by control membranes (5 mg/ml) to 63 % (P < 0.001), and to 40 % by peroxidized membranes (P < 0.001). We proved that this effect is not caused by the direct interaction of membranes with free radicals or by the interference with luminol chemiluminescence used for the detection of respiratory burst. There are indications of the inhibitory effects of iron ions and free radical products. Macrophages containing ingested erythrocyte membranes do not contain protein-bound nitrotyrosine. These observations imply a specific mechanism of erythrocyte phagocytosis.

Hyperoxia attenuated nitrotyrosine concentration in the lung tissue of rats with experimental pneumonia.

Although nitrated proteins have been repeatedly used as markers of lung injury, little is known about their formation and metabolism under hyperoxia. We therefore measured 3-nitrotyrosine (3NTYR) concentrations in lung tissue and serum of rats with carrageenan-induced pneumonia exposed to hyperoxia. Twenty-nine Wistar male rats were assigned to one of 4 groups. Two experimental groups were treated by intratracheal application of carrageenan (0.5 ml of 0.7 % solution) and then one was exposed to hyperoxia for 7 days (FIO2 0.8), the other to air. Rats of two control groups breathed either hyperoxic gas mixture or air for 7 days. At the end of exposure the ventilation was determined in anesthetized, intubated animals in which 3NTYR concentrations were measured in the lung tissue and nitrites and nitrates (NOx) were estimated in the serum. Carrageenan instillation increased 3NTYR concentrations in lung tissue (carrageenan-normoxic group 147+/-7 pmol/g protein, control 90+/-10 pmol/g protein) and NOx concentration in the serum (carrageenan-normoxic group 126+/-13 ppb, control 78+/-9 ppb). Hyperoxia had no effect on lung tissue 3NTYR concentration in controls (control-hyperoxic 100+/-14 pmol/g protein) but blocked the increase of lung tissue 3NTYR in carrageenan-treated rats (carrageenan-hyperoxic 82+/-13 pmol/g protein), increased NOx in serum (control-hyperoxic 127+/-19 ppb) and decreased serum concentration of 3NTYR in both hyperoxic groups (carrageenan-hyperoxic 51+/-5 pmol/g protein, control-hyperoxic 67+/-7 pmol/g protein, carrageenan-normoxic 82+/-9 pmol/g protein, control 91+/-7 pmol/g protein). The results suggest that hyperoxia affects nitration of tyrosine residues, probably by increasing 3NTYR degradation.

Serum cartilage oligomeric matrix protein reflects the presence of clinically diagnosed synovitis in patients with knee osteoarthritis.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a component of articular cartilage whose serum levels show a strong correlation with radiographic osteoarthritis (OA) status. It has recently been found, however, that COMP is also produced in synovium. To assess the hypothesis that synovitis affects serum COMP levels in patients with knee OA, we collected sera for COMP simultaneous with a clinical examination for synovitis. DESIGN: Sera were collected from OA patients who fulfilled the American College of Rheumatology criteria for knee OA. Radiographs were classified according to the grading system of Kellgren and Lawrence. Synovitis was diagnosed clinically by joint tenderness plus swelling and/or increased warmth over the joint. COMP levels in sera were measured by inhibition ELISA with monoclonal antibody (mAb) 17-C10. RESULTS: Serum COMP levels were significantly correlated with age, synovitis and an interaction of synovitis and OA severity. Synovitis showed the strongest effect on COMP levels (R=0.1587, P< 0.01), in contrast to C-reactive protein, duration of OA and OA severity score which showed no significant effect on COMP levels. Individual signs of synovitis, namely, joint tenderness and warmth had a significant effect on serum COMP levels while swelling alone did not. CONCLUSION: Synovitis exerts a significant effect on serum COMP levels measured with mAb 17-C10 in OA patients. These findings underscore the importance of the clinical joint examination to assess for synovitis, when attempting to apply objective measures, such as COMP, to the clinical setting.

A possible role of the oxidant tissue injury in the development of hypoxic pulmonary hypertension.

Chronic sojourn in hypoxic environment results in the structural remodeling of peripheral pulmonary arteries and pulmonary hypertension. We hypothesize that the pathogenesis of changes in pulmonary vascular structure is related to the increase of radical production induced by lung tissue hypoxia. Hypoxia primes alveolar macrophages to produce more hydrogen peroxide. Furthermore, the increased release of oxygen radicals by other hypoxic lung cells cannot be excluded. Several recent reports demonstrate the oxidant damage of lungs exposed to chronic hypoxia. The production of nitric oxide is high in animals with hypoxic pulmonary hypertension and the serum concentration of nitrotyrosine (radical product of nitric oxide and superoxide interaction) is also increased in chronically hypoxic rats. Antioxidants were shown to be effective in the prevention of hypoxia induced pulmonary hypertension. We suppose that the mechanism by which the radicals stimulate of the vascular remodeling is due to their effect on the metabolism of vascular wall matrix proteins. Non-enzymatic protein alterations and/or activation of collagenolytic matrix metalloproteinases may also participate. The presence of low-molecular weight cleavage products of matrix proteins stimulates the mesenchymal proliferation in the wall of distal pulmonary arteries. Thickened and less compliant peripheral pulmonary vasculature is then more resistant to the blood flow and the hypoxic pulmonary hypertension is developed.

Characterization of monoclonal antibodies recognizing different fragments of cartilage oligomeric matrix protein in human body fluids.

Cartilage oligomeric matrix protein (COMP) is a high-molecular-weight glycoprotein found at a high concentration in articular cartilage. Recent studies have shown that the joint fluid and serum levels of antigenic COMP, measured by an enzyme-linked immunosorbent assay (ELISA) which uses a polyclonal antiserum raised against bovine COMP, provide important information about metabolic changes occurring in the cartilage matrix in joint disease. In this report, we describe the specificity of three monoclonal antibodies (mAbs) to human COMP and their usefulness in quantifying antigenic COMP fragments in body fluids. Two of the mAbs (16-F12 and 18-G3) recognized both oligomeric and monomeric forms of COMP, but the third (17-C10) reacted positively only with the former. Immunoblots of human COMP, predigested with trypsin for up to 6 h, showed that the three mAbs are directed against different epitopes identified on small tryptic fragments of 30 kDa (16-F12), 25 kDa (17-C10), and 40 kDa as well as 30 kDa (18-G3), respectively. The antibodies also recognized a different pattern of fragments in human pathological synovial fluids. This was particularly striking in the case of the medium size fragments (16-F12: 90 and 110 kDa; 17-C10: 70 and 90 kDa; 18-G3: up to five bands from 70 to 130 kDa). Competitive indirect inhibition ELISAs developed with mAbs 16-F12 and 17-C10 revealed further differences in the specificities of these antibodies. Thus, while mAb 16-F12 can be used only to quantify antigenic COMP in human synovial fluid and serum, mAb 17-C10 is useful in addition when analyzing canine and horse synovial fluid as well as canine serum. The results of analyses of synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis provided preliminary evidence in support of the contention that measurement of the different COMP epitopes recognized by these mAbs in body fluids could prove useful in the clinical assessment of patients with joint disease.

Age-dependent changes in proteoglycan biosynthesis in human intervertebral discs.

Proteoglycans from annulus fibrosus and nucleus pulposus of human intervertebral disc were investigated by electrophoresis in a composite agarose-polyacrylamide gel and immunohistochemically using various monoclonal antibodies against components of extracellular matrix. There were at least five different populations of proteoglycans in both annulus fibrosus and nucleus pulposus. Proteoglycans represented by individual electrophoretic bands differed from each other in hydrodynamic size but all of them contained epitopes present in keratan sulphate. Chondroitin sulphate could be detected in populations with molecular weight above 200,000 daltons. We could postulate that the cleavage of proteoglycan chains starts at the C-terminal end. This is supported by the finding that the globular region G2 on the protein core was detected in the same populations as keratan sulphate. We could confirm these results using tissue cultures of nucleus pulposus, inner and external part of annulus fibrosus. Of interest is the finding that there is de novo synthesis of globular domain G1 only in structures of intervertebral disc from a 16-year-old male and not in the tissue of a 69-year-old male. This might contribute to an explanation of decreased aggregation of proteoglycans during the aging process.

Electrostatic interaction influences cell adhesion?

The effect of electrostatic forces on the adhesion of LEP-19 diploid embryonal fibroblasts, Hep-2 laryngeal carcinoma cells, Raji lymphoblastoma cells and Sp 2/0 myeloma cells was examined in vitro. Adhesivity of all tested cell lines was higher on the cationized glass than on untreated or anionized glass. The negatively charged sialic acids on the cell surface play a role in cell adhesion. The participation of electrostatic interaction is independent of the energy metabolism in serum-free conditions.

A new cell line, GS, derived from a human renal cell carcinoma.

A new cell line, GS, of human renal cell carcinoma was established and characterized. It was derived from a metastasis of a human renal cell carcinoma, which appeared 6 years after nephrectomy. The GS cells exhibit basic characteristics of renal cell carcinoma: epithelial cell character, PAS and glycogen positivity, typical ultrastructural features. The cells have a pseudotriploid stemline with a modal number of 75 chromosomes and two marker chromosomes. GS cells formed neither colonies in soft agar nor transplantable tumours in nude mice but produced a factor(s) stimulating growth and colony forming activity of indicator cells.