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.

The contractile response of isolated small pulmonary arteries induced by activated macrophages.

To test whether macrophages can play any role in hypoxic pulmonary vasoconstriction, we tested the in vitro response of rings from small pulmonary arteries to the activation of macrophages by FMLP, a substance stimulating predominantly membrane-bound NADPH oxidase. A small vessel myograph was used to measure the responses of rings from small pulmonary arteries (300-400 microm) isolated from rat lungs. Rings from 5 rats were placed into both chambers of the myograph. The vessels were stabilized for 40 min and then normalized by automatic stretching to a wall tension equivalent to the intravascular pressure 30 mm Hg. At the start of each experiment, vessels were exposed to 80 mM K+ to obtain maximal contractile response, which was used to normalize subsequent contractile responses. 2x10(6) viable macrophages, obtained by peritoneal lavage, were added into one chamber, then 5 microM FMLP was administrated to both chambers and the tension measurement was started. The hydrogen peroxide concentration produced by stimulated macrophages was measured luminometrically. The concentrations of H2O2 in specimens from chambers containing activated macrophages rose from 3.5+/-1.5 nM to 110+/-28 nM within 25 min of stimulation, while FMLP itself didn't increase the H2O2 concentration from the baseline value (4.5+/-3 nM) in samples from control chambers. After FMLP administration, the tension of the vessel rings in the presence of macrophages reached 0.23+/-0.07 of maximal contractile response, it did not change in controls. The addition of ROS scavenger 4-hydroxy-TEMPO blocked the contractile response to the activation of macrophages. We conclude that the activation of macrophages stimulates the contraction of small pulmonary arteries and that this contraction is probably mediated by reactive oxygen species.

Hypercapnia attenuates the hypoxia-induced blunting of the reactivity in chronically hypoxic rats.

Chronic hypoxia causes oxidative injury of pulmonary vessels and attenuates their reactivity to different stimuli. When combined with hypercapnia, biochemical markers of this injury are reduced but the effect of concomitant hypoxia and hypercapnia on vascular reactivity is not fully understood. This study was therefore designed to test whether hypercapnia can prevent also the hypoxia-induced loss of reactivity of pulmonary vessels. The reactivity of vessels from rats exposed either to hypoxia or hypoxia combined with hypercapnia was tested using a small vessel myograph (M 500A, Linton, Norfolk, GB). The second and third intrapulmonary branches of pulmonary arteries were isolated under a dissecting microscope from lungs of 8 control rats (group N), 6 rats exposed to hypoxia for 5 days (isobaric, 10 % O(2), group H) and 7 rats exposed to hypoxia combined with hypercapnia for 5 days (10 % O(2), 5 % CO(2), group H+CO(2)). The transmural pressure was set by automatic normalization to 30 mm Hg. The vessel size did not vary among the groups. After stabilization we challenged the vessels twice with KCl (80 mM) and once with PGF(2alpha) (0.1 mM). There were no significant differences in KCl induced contractions among the groups. The responses to PGF(2alpha) were expressed as a ratio to the maximal tension obtained by the exposure to 80 mM KCl. Contractions induced by PGF(2alpha) were markedly reduced in group H (0.07+/-0.02) and in group H+CO(2) (0.26+/-0.03) in comparison with group N (0.83+/-0.07). The vessels of group H responded to PGF(2alpha) less than those of group H+CO(2). However we observed the attenuated reactivity also in group H+CO(2) in comparison with N. Hypercapnia therefore partially blunted the hypoxia-induced loss of reactivity in pulmonary arteries. This finding supports the hypothesis that hypercapnia significantly alters the nature of lung injury induced by chronic hypoxia.

Reactive oxygen species production in the early and later stage of chronic ventilatory hypoxia.

Pulmonary hypertension resulting from chronic hypoxia is at least partly caused by the increased production of reactive oxygen species (ROS). The goal of the presented study was to investigate the dynamics and the site of production of ROS during chronic hypoxia. In our study Wistar rats were kept for 1, 4 and 21 days in an isobaric hypoxic chamber (F(iO2)=0.1), while controls stayed in normoxia. We compared NO production in expired air, plasma and perfusate drained from isolated rat lungs and measured superoxide concentration in the perfusate. We also detected the presence of superoxide products (hydrogen peroxide and peroxynitrite) and the level of ROS-induced damage expressed as the concentration of lipid peroxydation end products. We found that the production and release of ROS and NO during early phase of chronic hypoxia has specific timing and differs in various compartments, suggesting the crucial role of ROS interaction for development of hypoxic pulmonary hypertension.

Lung mast cells and hypoxic pulmonary hypertension.

Hypoxic pulmonary hypertension (HPH) is a syndrome characterized by the increase of pulmonary vascular tone and the structural remodeling of peripheral pulmonary arteries. Mast cells have an important role in many inflammatory diseases and they are also involved in tissue remodeling. Tissue hypoxia is associated with mast cell activation and the release of proteolytic enzymes, angiogenic and growth factors which mediate tissue destruction and remodeling in a variety of physiological and pathological conditions. Here we focused on the role of mast cells in the pathogenesis of hypoxic pulmonary hypertension from the past to the present.

Disodium cromoglycate attenuates hypoxia induced enlargement of end-expiratory lung volume in rats.

Mechanism responsible for the enlargement of end-expiratory lung volume (EELV) induced by chronic hypoxia remains unclear. The fact that the increase in EELV persists after return to normoxia suggests involvement of morphological changes. Because hypoxia has been also shown to activate lung mast cells, we speculated that they could play in the mechanism increasing EELV similar role as in vessel remodeling in hypoxic pulmonary hypertension (HPH). We, therefore, tested an effect of mast cells degranulation blocker disodium cromoglycate (DSCG) on hypoxia induced EELV enlargement. Ventilatory parameters, EELV and right to left heart weight ratio (RV/LV+S) were measured in male Wistar rats. The experimental group (H+DSCG) was exposed to 3 weeks of normobaric hypoxia and treated with DSCG during the first four days of hypoxia, control group was exposed to hypoxia only (H), two others were kept in normoxia as non-treated (N) and treated (N+DSCG) groups. DSCG treatment significantly attenuated the EELV enlargement (H+DSCG = 6.1+/-0.8; H = 9.2+/-0.9; ml +/-SE) together with the increase in minute ventilation (H + DSCG = 190+/-8; H = 273 +/- 10; ml/min +/- SE) and RV/LV + S (H + DSCG = 0.39 +/- 0.03; H = 0.50 +/- 0.06).

Production of proteolytic enzymes in mast cells, fibroblasts, vascular smooth muscle and endothelial cells cultivated under normoxic or hypoxic conditions.

Matrix metalloproteinases (MMPs) is a family of proteolytic enzymes involved in remodeling of extracellular matrix. Although proteolytic enzymes are produced by many cell types, mast cells seem to be more important than other types in remodeling of pulmonary arteries during hypoxia. Therefore, we tested in vitro production of MMPs and serine proteases in four cell types (mast cells, fibroblasts, vascular smooth muscle cells and endothelial cells) cultivated for 48 h under normoxic or hypoxic (3% O2) conditions. MMP-13 was visualized by immunohistochemistry, MMP-2 and MMP-9 were detected by zymography in cell lysates. Enzymatic activities (MMPs, tryptase and chymase) were estimated in the cultivation media. Hypoxia had a minimal effect on total MMP activity in the cultivation media of all types of cells, but immunofluorescence revealed higher intensity of MMP-13 in the cells exposed to hypoxia except of fibroblasts. Tryptase activity was three times higher and chymase activity twice higher in mast cells cultivated in hypoxia than in those cultured in normoxia. Among all cell types studied here, mast cells are the most abundant source of proteolytic enzymes under normoxic and hypoxic conditions. Moreover, in these cells hypoxia increases the production of both specific serine proteases tryptase and chymase, which can act as MMPs activators.

[Disodium cromoglycate--mast cell degranulation blocker in the process of tissue remodelation]. / Kromoglykát sodný--blokátor degranulace zírných bunek v procesu tkánové remodelace.

Disodium cromoglycate (DSCG) is a compound commonly used in the treatment of allergic diseases. The effect of DSCG is due to its ability to stabilize the mast cell membrane and to prevent release of histamine and inflammatory mediators. Mast cells are also an abundant source of tissue metalloproteinases, serine proteases and growth factors, which play an important role in the processes of the tissue remodeling. In this view the DSCG is a substance which allows us to study the mechanisms of the pulmonary vascular bed remodeling in the experimental animals exposed to chronic hypoxia and in a phase of the recovery from hypoxia.

Hypercapnia attenuates hypoxic pulmonary hypertension by inhibiting lung radical injury.

Chronic lung hypoxia results in hypoxic pulmonary hypertension. Concomitant chronic hypercapnia partly inhibits the effect of hypoxia on pulmonary vasculature. Adult male rats exposed to 3 weeks hypoxia (Fi(02)=0.1) combined with hypercapnia (Fi(C02)=0.04-0.05) had lower pulmonary arterial blood pressure, increased weight of the right heart ventricle, and less pronounced structural remodeling of the peripheral pulmonary arteries compared with rats exposed only to chronic hypoxia (Fi(02)=0.1). According to our hypothesis, hypoxic pulmonary hypertension is triggered by hypoxic injury to the walls of the peripheral pulmonary arteries. Hypercapnia inhibits release of both oxygen radicals and nitric oxide at the beginning of exposure to the hypoxic environment. The plasma concentration of nitrotyrosine, the marker of peroxynitrite activity, is lower in hypoxic rats exposed to hypercapnia than in those exposed to hypoxia alone. Hypercapnia blunts hypoxia-induced collagenolysis in the walls of prealveolar pulmonary arteries. We conclude that hypercapnia inhibits the development of hypoxic pulmonary hypertension by the inhibition of radical injury to the walls of peripheral pulmonary arteries.

Hyperoxia blunts acute hypoxia- and PGF2alpha-induced pulmonary vasoconstriction in chronically hypoxic rats.

We investigated the influence of oxygenation of in vitro lung preparation on the pulmonary vascular reactivity. Small pulmonary vessels isolated from adult male Wistar rats exposed for 4 days to hypoxia (F(iO2) = 0.1, group CH) were compared with those of normoxic controls (group N). The bath in the chamber of small vessel myograph was saturated with gas mixture containing either 21% or 95% of O(2) with 5% CO(2) and we measured the reactions of vessels to acute hypoxic challenge with 0% O(2) or to PGF(2alpha). We did not observe any difference of the contractile responses between both groups when the normoxic conditions were set in the bath. When the bath oxygenation was increased to 95% O(2), the contractions induced by hypoxic challenge and PGF(2alpha) decreased in chronically hypoxic rats and did not change in normoxic controls. We hypothesize that reduced reactivity of vessels from hypoxic rats in hyperoxia results from the effect of chronic hypoxia on Ca(2+) signaling in the vascular smooth muscle, which is modulated by increased free radical production during the exposure to chronic hypoxia and further hyperoxia.

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.

Oxidative stress in the lung tissue--sources of reactive oxygen species and antioxidant defence.

Reactive oxygen species are oxygen-based molecules readily reacting with various compounds. It is already known that they play significant role in many physiological as well as pathological body processes. The aim of our review is to briefly summarize our knowledge of possible ROS sources in the lung tissue.

Mast cells, hypoxia and structure of the vascular bed.

Mast cells represent a heterogeneous and multifunctional cells population distributed throughout tissues. Their participation in the response to chronic hypoxia is discussed in consideration to their role in the angiogenesis and remodeling of pulmonary vasculature, including relevance of proangiogenic factors, mediators and proteolytic enzymes released by activated mast cells. Possible mechanism of mast cells activation by hypoxia is considered.

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.

Assessment of exhaled gases in ventilated preterm infants.

Hydrogen peroxide (H2O2) production in exhaled air was measured in ventilated preterm newborns at 5, 24 and 48 hours after delivery, using originally designed method of exhaled breath condensate (EBC) collection. H2O2 production in expired gas was 812+/-34 pmol/20 min during the first measurement and then declined to 389+/-21 at 24 hours and 259+/-26 pmol/20 min at 48 hours.

Hyperoxia prevents carrageenan-induced enlargement of functional residual lung capacity in rats.

Experimental pneumonia induced by intratracheal application of carrageenan or paraquat increases the functional residual lung capacity (FRC) in rats. The mechanism of this increase is not clear, but a decrease in PO(2) may be involved. To test this possibility, we attempted to eliminate the PO(2) decrease in carrageenan-treated rats by exposing them to hyperoxia. Animals of the first group were exposed to 7 days of hyperoxia (F(I)O(2) 0.78-0.84, group Car+O(2)) after intratracheal application of carrageenan (0.5 ml of 0.7 % carrageenan in saline), whereas animals of the second group were given the same dose of carrageenan but breathed air (group Car+A). The third group of rats was kept for seven days in hyperoxia (group O(2)) and the fourth group served as controls (C). The animals were then anesthetized and intubated and their ventilatory parameters and FRC were measured during air breathing. Carrageenan application induced a FRC increase (Car+A 2.0+/-0.2 ml, C 1.6+/-0.1 ml), which was not seen in carrageenan-treated rats exposed to hyperoxia (Car+O(2) 1.6+/-0.1 ml). Hyperoxia alone did not affect the value of FRC (O(2) 1.5+/-0.1 ml). These results support the hypothesis that a decrease in PO(2) plays an important role in the carrageenan-induced increase of FRC in rats.

Hypoxia and reoxygenation increase H2O2 production in rats.

To test the effect of transition from sustained hypoxia to normoxia on production of reactive oxygen species (ROS) in lungs, the authors measured hydrogen peroxide (H(2)O(2)) output in the expired air of rats breathing hypoxic, normoxic, and hyperoxic gas mixtures at the end of exposure to 72 hours of hypoxia. Twenty-one male Wistar rats (200 to 280 g) were randomly assigned to 1 of 3 groups. First two groups (experimental) were kept for 3 days in normobaric hypoxic chamber (F(1)O(2) 0.1), rats of the third group (controls) breathed air. The rats were then anesthetized, intubated, placed in the plethysmograph, and their ventilation measured. Two periods of exhaled breath condensate (EBC) collection, each lasting 1 hour, were then performed to assay H(2)O(2) output. The controls breathed during both samplings air, the first experimental group breathed during first sampling period hypoxic mixture (F(1)O(2) 0.1; SH-H measurement) and then, during second period, air (SH-H-A measurement), the second experimental group breathed first air (SH-A measurement) and then hyperoxic mixture (F(1)O(2) 1.0; SH-A-O(2) measurement). Concentration of H(2)O(2) in the EBC was assayed by chemiluminescence. H(2)O(2) production in the control group was low and similar in both measurements (20+/-10 and 13+/-5 pmol/h, mean+/-SEM). Exposure to 72 hours of hypoxia increased the H(2)O(2) production to 105+/-18 pmol/h (SH-H). Transition from hypoxia to normoxia resulted in an increase in the H(2)O(2) production (SH-A 421+/-24 pmol/h, and SH-H-A 366+/-19 pmol/h). Following transition from air breathing to hyperoxia did not affect the H(2)O(2) production (SH-A-O(2) 373+/-25 pmol/h). The results showed that sustained hypoxia and transition from sustained hypoxia to normoxia increased H(2)O(2) formation in the lungs.

Hypercapnia does not affect functional residual capacity enlargement induced by chronic hypoxia.

To determine whether changes in partial pressure of CO2 participate in mechanism enlarging the lung functional residual capacity (FRC) during chronic hypoxia, we measured FRC and ventilation in rats exposed either to poikilocapnic (group H, F(I)O2 0.1, F(I)CO2 <0.01) or hypercapnic (group H+CO2, F(I)O2 0.1, F(I)CO2 0.04-0.05) hypoxia for the three weeks and in the controls (group C) breathing air. At the end of exposure a body plethysmograph was used to measure ventilatory parameters (V'(E), f(R), V(T)) and FRC during air breathing and acute hypoxia (10 % O2 in N2). The exposure to hypoxia for three weeks increased FRC measured during air breathing in both experimental groups (H: 3.0+/-0.1 ml, H+CO2: 3.1+/-0.2 ml, C: 1.8+/-0.2 ml). During the following acute hypoxia, we observed a significant increase of FRC in the controls (3.2+/-0.2 ml) and in both experimental groups (H: 3.5+/-0.2 ml, H+CO2: 3.6+/-0.2 ml). Because chronic hypoxia combined with chronic hypercapnia and chronic poikilocapnic hypoxia induced the same increase of FRC, we conclude that hypercapnia did not participate in the FRC enlargement during chronic hypoxia.

Ventilatory response to sustained hypoxia in carotid body denervated rats.

Hypoxia stimulates ventilation, but when it is sustained, a decline in the ventilatory response is seen. The mechanism responsible for this decline lies within the CNS, but still remains unknown. In this study, we attempted to elucidate the possible role of hypoxia-induced depression of respiratory neurons by comparing the ventilatory response to hypoxia in intact rats and those with denervated carotid bodies. A whole-body plethysmograph was used to measure tidal volume, frequency of breathing and minute ventilation (VE) in awake and anesthetized intact rats and rats after carotid body denervation during exposure to hypoxia (FIO2 0.1). Fifteen-minute hypoxia induced an initial increase of VE in intact rats (to 248% of control ventilation in awake and to 227% in anesthetized rats) followed by a consistent decline (to 207% and 196% of control VE, respectively). Rats with denervated carotid bodies responded with a smaller increase in VE (to 134% in awake and 114% in anesthetized animals), but without a secondary decline (145% and 129% of control VE in the 15th min of hypoxia). These results suggest that afferentation from the carotid bodies and/or the substantial increase in ventilation are crucial for the biphasicity of the ventilatory response to sustained hypoxia and that a central hypoxic depression cannot fully explain the secondary decline in VE.