Matrix metalloproteinases-7, -8, -9 and TIMP-1 in the follow-up of diisocyanate-induced asthma.

BACKGROUND: Diisocyanate-induced asthma (DIA) is known to be associated with poor prognosis. We wished to clarify if matrix metalloproteinases (MMP)-7, -8 or -9 or tissue inhibitor of matrix metalloproteinases (TIMP-1) are associated with the functional or inflammatory outcome in DIA patients. METHODS: This is a longitudinal study where 17 patients with DIA diagnosed by a specific challenge test to diisocyanates were monitored. Exposure to diisocyanates was terminated seven (mean) months before the challenge test. The studies included spirometry, histamine challenge test and bronchoscopy. MMP-7, MMP-8, TIMP-1 [Enzyme-linked immunosorbent assay (ELISA)- and immunofluorometric assay-methods], MMP-9 (ELISA and zymography), interferon-gamma, tumour necrosis factor-alpha, interleukin-6, -8, -15, -17, CXCL-5/ENA-78, monocyte chemoattractant protein-1 and macrophage inhibitory factor (MIF) (ELISA) were assayed from bronchoalveolar lavage (BAL) fluid. Inhaled steroid therapy was initiated after the examinations, which were repeated at 6 months and at 3 years during the treatment. The results were compared with those of 15 healthy controls. RESULTS: Inhaled steroid medication increased BAL levels of MMP-9 and MMP-9/TIMP-1 and decreased MMP-7 and MMP-7/TIMP-1. The increase in MMP-9 levels was associated with a decline in the TH-2 type inflammation. CONCLUSIONS: Our data suggest that reduced TH-2 type inflammation in DIA after inhaled steroid medication is reflected as elevated MMP-9 and MMP-9/TIMP-1 levels in BAL. MIF may be the inducer of MMP-9. This might point to some protective role for MMP-9 in DIA.

Gremlin localization and expression levels partially differentiate idiopathic interstitial pneumonia severity and subtype.

Idiopathic pulmonary fibrosis (IPF) (histopathology of usual interstitial pneumonia, UIP) and non-specific interstitial pneumonia (NSIP) are diseases characterized by loss of normal lung architecture and function. The differential diagnosis between IPF/UIP and NSIP may be difficult. The levels of bone morphogenetic protein (BMP)-4 antagonist gremlin are up-regulated in IPF/UIP. The present study was performed to clarify whether the localization or the mRNA expression of gremlin or BMP-4 could be used in the differential diagnosis or assessment of severity of IPF/UIP and NSIP. Gremlin and BMP-4 immunoreactivities were quantitated from 24 UIP and 12 NSIP lung specimens. Quantitative real-time polymerase chain reaction analyses were performed to compare gremlin and BMP-4 expression between UIP (n = 8) and NSIP (n = 5) biopsies. Immunohistochemical positivity and mRNA levels were correlated to lung function parameters. In IPF/UIP biopsies, gremlin was detected mainly in the thickened lung parenchyma, whereas in NSIP it was observed in the alveolar epithelium. BMP-4-positive (BMP-4+) cells were detected solely in the alveolar wall. The percentage of gremlin-positive area was higher in IPF/UIP (5.1 +/- 0.6) than in NSIP (1.8 +/- 0.7) (n = 36, p < 0.0001). Gremlin mRNA levels were higher in advanced UIP (p = 0.008) and NSIP (p = 0.007) biopsies than in the normal control lung. A negative correlation was found between the specific diffusion capacity corrected for alveolar volume (DLCO/VA) and gremlin mRNA levels (r = - 0.69, p = 0.007). The highest numbers of BMP-4+ cells were found in NSIP biopsies. BMP-4 mRNA levels correlated positively with forced vital capacity (r = 0.801, p < 0.0001) and diffusion capacity. Parenchymal gremlin immunoreactivity is thus suggestive of a UIP-type interstitial pneumonia. Gremlin expression levels correlating negatively and BMP-4 levels positively with disease severity support recent observations of a fibroprotective role for the BMPs.

Thiol proteins, redox modulation and parenchymal lung disease.

The lung is a unique organ in terms of its direct exposure to high levels of oxygen and reactive compounds. Several parenchymal lung diseases (e.g. emphysema associated with smoking and a number of fibrotic lung disorders) have been proposed to be due to the exposure of the lung to exogenous irritants leading to local redox imbalance in the alveolar epithelium. The disease progression of emphysema/chronic obstructive pulmonary disease (COPD) and fibrosis share several common factors, such as the role of reactive oxygen species, disturbances of the pulmonary thiol status and activation of growth factors and tissue destructing proteases. Importantly in COPD or fibrosis, medication does not provide any significant therapeutic effect. This review concentrates on the key thiol (-SH)-regulated mechanisms leading to the development of COPD and/or pulmonary fibrosis and the major redox-regulated defense/oxidant repair mechanisms, thioredoxin/peroxiredoxin and glutaredoxin protein families in the lung. Redox-regulated proteins, both proteases and oxidant repair enzymes, undergo conformational changes during oxidative stress, a process that modulates their activation or inactivation. In addition, some of the redox-regulated proteins influence the metabolism of glutathione (GSH), a major small molecular antioxidant of human lung, and participate in the crosstalk between numbers of GSH associated enzymes functioning in the detoxification pathways of human lung. An understanding of the processes involved in oxidant-mediated lung damage may provide the key to devising interventional strategies that can actually prevent the progression of lung parenchymal disease.

Aminoterminal propeptide of the alpha1-homotrimer variant of human type I procollagen (hotPINP) in malignant pleural effusion.

The aminoterminal propeptide (hotPINP) of type I homotrimer, a putative malignancy-associated type I collagen variant, was purified for the first time and a method was established for its detection in pleural fluid. Samples of 58 patients, with malignant or benign disease, were studied with specific immunoassays for the two propeptides of type-I procollagen (PICP and PINP) and with HPLC-DEAE chromatography to separate the two PINP variants. HotPINP was present in 64% of both benign and malignant pleural effusion fluids, with the exception of malignant mesotheliomas, none of which showed the presence of hotPINP. Also the PICP to PINP ratios were lower than normal in both benign and malignant samples (altogether in 69% of samples), although this deviation was greater in malignancy. These two phenomena were independent of each other. As synthesis of the alpha1-homotrimer-variant of type-I collagen seems to be relatively common during the formation of pleural effusion, it may be generally related to a fibroproliferative reaction in the pleural wall.

Reversible ischemic inhibition of F(1)F(0)-ATPase in rat and human myocardium.

The physiological role of F(1)F(0)-ATPase inhibition in ischemia may be to retard ATP depletion although views of the significance of IF(1) are at variance. We corroborate here a method for measuring the ex vivo activity of F(1)F(0)-ATPase in perfused rat heart and show that observation of ischemic F(1)F(0)-ATPase inhibition in rat heart is critically dependent on the sample preparation and assay conditions, and that the methods can be applied to assay the ischemic and reperfused human heart during coronary by-pass surgery. A 5-min period of ischemia inhibited F(1)F(0)-ATPase by 20% in both rat and human myocardium. After a 15-min reperfusion a subsequent 5-min period of ischemia doubled the inhibition in the rat heart but this potentiation was lost after 120 min of reperfusion. Experiments with isolated rat heart mitochondria showed that ATP hydrolysis is required for effective inhibition by uncoupling. The concentration of oligomycin for 50% inhibition (I(50)) for oxygen consumption was five times higher than its I(50) for F(1)F(0)-ATPase. Because of the different control strengths of F(1)F(0)-ATPase in oxidative phosphorylation and ATP hydrolysis an inhibition of the F(1)F(0)-ATPase activity in ischemia with the resultant ATP-sparing has an advantage even in an ischemia/reperfusion situation.

Role of cellular energetics in ischemia-reperfusion and ischemic preconditioning of myocardium.

A short period of ischemia followed by reperfusion produces a state of affairs in which the cells' potential for surviving longer ischemia is enhanced. This is called ischemic preconditioning. The effects of preconditioning are also related to the reperfusion damage which ensues upon tissue oxygenation. The role of the cellular energy state in reperfusion damage remains an enigma, although ischemic preconditioning is known to trigger mechanisms which contribute to the prevention of unnecessary ATP waste. In some species up to 80% of ATP hydrolysis in ischemia can be attributed to mitochondrial F1-F0-ATPase (ATP synthase), and a role for its inhibitor protein (IF1) in ATP preservation has been proposed. Although originally regarded as limited to large animals with a slow heart beat, inhibition by IF1 is probably a universal phenomenon. Coincidentally with ATPase inhibition, the decline in cellular ATP slows down, but even so the difference in ATP concentration between preconditioned and non-conditioned hearts is still small at the final stages of a long ischemia, when the beneficial effect of preconditioning is observable, although the energy state during reperfusion remains low in hearts which do not recover.

Placental mitochondrial DNA and respiratory chain enzymes in the etiology of preeclampsia.

OBJECTIVE: To evaluate the occurrence of the most common mutations and deletions in mitochondrial DNA and deficiencies in the enzyme complexes of the mitochondrial respiratory chain in placentas from preeclamptic women. METHODS: Mitochondria were isolated from the placentas of 17 preeclamptic or 25 control women, and the activities of mitochondrial respiratory chain complexes were measured. Deletions and three common point mutations of mitochondrial DNA were searched for by the Southern blot and polymerase chain reaction (PCR) methods from the same placentas. RESULTS: Mean (+/- standard deviation) mitochondrial respiratory chain enzyme complex activities in placentas on protein basis (nmol/min/mg of protein) were similar in preeclamptics and controls (nicotinamide adenine dinucleotide, reduced form-ubiquinone oxidoreductase 25.84 +/- 9.29 versus 31.02 +/- 7.52; nicotinamide adenine dinucleotide, reduced form-cytochrome-c oxidoreductase 77.88 +/- 42.24 versus 104.06 +/- 56.73; succinate-cytochrome-c oxidoreductase 57.90 +/- 13.83 versus 64.44 +/- 20.16; cytochrome-c oxidase 106.43 +/- 35.46 versus 128.37 +/- 48.64, respectively) and they were similar also when referenced to the mitochondrial marker enzyme citrate synthase. The sample sizes in both patient and control groups were found to be large enough by post hoc test. Large-scale deletions or the common 5-kb and 7.4-kb deletions were not detected, even at the sensitivity level of PCR. The three most common point mutations were not found in either control or preeclamptic placental samples. CONCLUSION: Common mitochondrial DNA mutations seem to play no major role in the universal etiology of preeclampsia, as assessed by analysis of the mitochondrial genome and respiratory chain enzyme activities in vitro. This does not exclude possible alterations in the energy state of the preeclamptic placenta.

Aggressive breast cancer leads to discrepant serum levels of the type I procollagen propeptides PINP and PICP.

The propeptides PICP and PINP are derived from the synthesis of type I collagen, a major matrix protein of bone and soft tissues. The aim of this cross-sectional study was to investigate their value as indicators of the aggressivity of breast cancer. Serum PINP, PICP, and total alkaline phosphatase were determined from 89 breast cancer patients. Forty had major bone and/or soft tissue metastases with an aggressive disease course: the progressive disease (PD) group. Forty-nine had either none or minor bone and/or soft tissue metastases with a stable clinical course: the stable disease group (SD). The mean value of PINP in the PD group was 7.2 times higher than that in the SD group (276 +/- 79 microg/l versus 38 +/- 3 microg/l, respectively; P = 0.005), whereas PICP mean value was only 1.7 times higher in the PD group (174 +/- 20 microg/l versus 100 +/- 5 microg/l; P = 0.001). The ratio of PICP to PINP was 1.02 +/- 0.07 in the PD group and 3.07 +/- 0.18 in the SD group (P < 0.001). The correlation between PICP and PINP was linear in the SD group and nonlinear in the PD group. The results indicate that high serum PICP and PINP concentrations and a low PICP:PINP ratio are associated with a highly aggressive nature of breast cancer. Determination of PINP, in particular, may be valuable when evaluating the clinical status of a breast cancer patient.

Respiratory control in heart muscle during fatty acid oxidation. Energy state or substrate-level regulation by Ca2+?

Fatty acids are the main fuel for the myocardium in vivo. They increase oxygen consumption, but the regulation of their beta-oxidation is not well known. Since Ca2+ and matrix volume have been implicated in the regulation of fatty acid oxidation in liver mitochondria, we set out to investigate the effects of Ca2+ on cellular respiration and energetics in the isolated perfused rat heart when oxidizing a short-chain fatty acid. Infusion of hexanoate increased oxygen consumption, while stepwise changes in the perfusate Ca2+ concentration in the range 0.5-2.5 mM caused the mechanical work output and oxygen consumption to change in parallel. Hexanoate addition increased the cellular energy state as determined by 31P NMR and evaluated from the cytosolic [ATP]/[ADP][Pi] ratio. During fatty acid infusion the energy state decreased slightly upon Ca(2+)-induced inotropy, and after discontinuation of the hexanoate infusion the de-energization was more pronounced. The fatty acid caused an extensive partially reversible reduction of flavoproteins and NAD with a slight tendency for oxidation during Ca(2+)-induced inotropy. The data are in agreement with the notion that oxygen consumption during fatty acid oxidation is mainly determined by the energy expenditure, even in the presence of Ca(2+)-induced alterations in the inotropic state. The constancy of the redox states of mitochondrial flavins and NADH/NAD during large changes in oxygen consumption is interpreted as indicating stabilization of the mitochondrial redox states by Ca(2+)-linked regulation.

Mechanisms of ischemic preconditioning in rat myocardium. Roles of adenosine, cellular energy state, and mitochondrial F1F0-ATPase.

BACKGROUND: Adenosine has been proposed as one mediator for the preconditioning effect in the myocardium of some animals, but recent investigations have shown that this may not be the mechanism in the rat heart, although the effect itself is clearly demonstrable. The cellular energy state has been shown to be better in preconditioned hearts, and the role of ATP consumption has been discussed. The role of inhibition of mitochondrial F1F0-ATPase as a mechanism for the preservation of ATP in preconditioned hearts remains controversial. METHODS AND RESULTS: Three-minute global ischemia followed by 9 minutes of reperfusion was used to precondition Langendorff-perfused rat hearts, and control hearts were perfused under normoxic conditions for the same time. The duration of sustained ischemia in both groups of hearts was 21 minutes, after which the hearts were reperfused for 15 minutes to evaluate their mechanical and metabolic recovery. Separate experiments were performed for tissue metabolite determinations, mitochondrial ATPase activity measurements, and 31P nuclear magnetic resonance studies. The recovery of the rate-pressure product was better in the preconditioned group. Three-minute preconditioning ischemia caused inhibition of the mitochondrial ATPase that persisted throughout the 9-minute intervening reperfusion so that at the early stages of sustained ischemia the enzyme activity was still more inhibited in preconditioned hearts. ATP was better preserved in preconditioned hearts than in control hearts during sustained ischemia. The accumulation of adenosine and its degradation products during sustained ischemia was greater in the control group. More lactate and H+ ions accumulated in this group, indicating higher anaerobic glycolysis. Also, inhibition of mitochondrial ATPase by oligomycin slowed ATP depletion during ischemia. CONCLUSIONS: The results indicate that preconditioning causes inhibition of rat heart mitochondrial ATPase that persists during reperfusion so that the enzyme is inhibited from the very beginning of the sustained ischemia. This inhibition leads to sparing of high-energy phosphates and improves the time-averaged energy state during ischemia. Although a causal relationship is difficult to prove, this reversible inhibition may contribute to postischemic recovery of the heart.

Efflux of adenosine and total adenylate catabolites during alterations of the cellular energy state. An NMR study of continuous and discontinuous ischemia.

Reperfusion after continuous or discontinuous ischemia has a bearing on clinical interventions. An important question is the washout of metabolites after periods of diminished energy state of the myocardial cell. We therefore set out to determine the washout of adenosine and its metabolites after periods of ischemia in an experimental set-up which allowed non-destructive monitoring of the cellular energy state and cytosolic pH over consecutive time intervals. Isolated rat hearts were perfused with hemoglobin-free saline in a nuclear magnetic resonance spectrometer equipped for 31P NMR spectroscopy of phosphorus-containing metabolites, which could be measured over 3-min time blocks. The response of the heart when subjected to 18 min of continuous ischemia and subsequent reperfusion was compared with that when subjected to three 6-min periods of ischemia separated by 3-min periods of reperfusion. The mechanical performance of the hearts, oxygen consumption and efflux of adenosine and its metabolites were measured. The consecutive ischemic periods produced no evidence of preconditioning as judged from the cellular energy state, although the mechanical recovery was better than after continuous ischemia. During the repetitive ischemia/reperfusion protocol the efflux of adenosine was smaller, although the efflux of combined adenylate catabolites did not differ from that after continuous ischemia. The results do not support the view of adenosine being a major effector in the phenomenon of preconditioning.

Metabolic effects of propionate, hexanoate and propionylcarnitine in normoxia, ischaemia and reperfusion. Does an anaplerotic substrate protect the ischaemic myocardium?

It has been suggested that propionyl-L-carnitine administration to ischaemic hearts facilitates the restoration of cardiac function upon reperfusion, but it is still a matter of dispute whether its effect is conveyed via the metabolic effect of the propionyl moiety, the carnitine moiety or other mechanisms involving membrane receptor interactions. The metabolism of propionylcarnitine involves the formation of succinyl-CoA, which causes an increase in the total amount of tricarboxylic acid cycle intermediates. According to the current paradigm, anaplerosis ensures rapid restoration of tricarboxylic acid cycle activity during reperfusion. To evaluate the contribution of anaplerosis to the protective effect of propionylcarnitine during ischaemia and reperfusion, isolated rat hearts were perfused with Krebs-Henseleit bicarbonate buffer containing 5 mM glucose+insulin (12 IU per litre), to which 1 mM propionate, 0.8 mM hexanoate or 1 mM propionylcarnitine were added. Global 20 or 24 min no-flow ischaemia was followed by 10 min reperfusion. The flavoprotein redox state, myoglobin oxygenation, oxygen consumption and mechanical functioning of the heart were recorded and metabolites determined in freeze-trapped tissue. In parallel experiments, the cellular energy state was studied with phosphorus nuclear magnetic resonance spectrometry. The addition of 1 mM propionylcarnitine failed to cause an anaplerotic effect, but did bring about an oxidation of flavins, probably due to citrate synthase inhibition. Propionate showed similar but stronger effects and a marked anaplerosis, but still failed to improve the recovery of the heart upon reperfusion. The addition of hexanoate caused marked anaplerosis upon reperfusion and flavin reduction. The results failed to demonstrate that propionylcarnitine had any beneficial effect on the ischaemic myocardium.

Hypoxia stimulates release of ANP and BNP from perfused rat ventricular myocardium.

We determined the effect of hypoxia on cellular energy state and ventricular atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and endothelin-1 (ET-1) release in an isolated perfused heart preparation after removal of all atrial tissue in 21- to 24-mo-old Wistar-Kyoto rats. After a control period (14 min), the ventricles (n = 6) were exposed to 30 min of hypoxia by changing the gas mixture to N2-CO2 (95:5 vol/vol; hypoxic period) and back to O2-CO2 (95:5 vol/vol) for 30 min (reoxygenation period). Control hearts (n = 6) were perfused throughout the experiment (74 min) with oxygenated Krebs-Henseleit phosphate-free buffer. In parallel experiments, the metabolic state of oxygenated (n = 4) and hypoxic (n = 5) ventricles was assessed using 31P-nuclear magnetic resonance (31P-NMR). Hypoxia caused a rapid decrease in left ventricular peak systolic pressure associated with a 2.1-fold increase (27.6 +/- 2.2 to 58.0 +/- 13.1 fmol/ml; P < 0.05) in the concentration of immunoreactive (ir) ANP and a 1.6-fold increase (2.5 +/- 0.2 to 3.9 +/- 0.5 fmol/ml; P < 0.05) in the [irBNP] (where brackets signify concentration) in the perfusate. In contrast, perfusate [irET-1] (1.2 +/- 0.2 fmol/ml) did not change significantly during hypoxia. 31P-NMR showed that the [ATP]-to-[ADP].[Pi] ratio was reduced during hypoxia with a simultaneous increase in intracellular monophosphates and perfusate [irANP] and [irBNP]. The decrease in the cytosolic pH during hypoxia was small. High-performance liquid chromatography of the perfusates showed that the ANP-like immunoreactive material released corresponded to the processed, low-molecular weight peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Trace elements and antibacterial activity in amniotic fluid.

Antibacterial activity and trace element concentrations in amniotic fluid (AF) were determined in a population of 39 pregnant women in the second half of gestation. Antibacterial activity in each AF was measured by a spectrophotometric micromethod after 18 h incubation at 37 degrees C using Escherichia coli K 12 as a reference bacterium. Concentrations of zinc, iron, copper, calcium, potassium and bromine were measured by particle-induced X-ray emission method and the zinc concentration was also measured by atomic absorption spectrophotometry. Phosphate concentration was determined by direct albumin adding method. In AFs with good antibacterial activity significantly lower concentrations of potassium and bromine were found when compared to AFs with lower antibacterial activity. Concentrations of zinc, iron, copper, calcium or phosphate did not correlate with antibacterial activity in AF.

Copper and zinc metabolism in aspartylglycosaminuria and Salla disease.

Changes in the metabolism of copper and zinc are described in aspartylglycosaminuria (AGU) patients. AGU patients had significantly reduced serum zinc concentrations. However, hair zinc levels were normal, and hyperzincuria could not be demonstrated. The copper content in the hair of AGU patients was highly elevated. Serum copper and ceruloplasmin concentrations were within normal range. In AGU, small-molecular-weight glycoasparagine storage products accumulate in tissues and are excreted in urine in large amounts. They may interfere with the transport mechanisms of trace elements, and thus alter their distribution and availability for tissues. The changes in copper and zinc levels may contribute to the pathogenesis of some of the clinical signs of AGU and Salla disease.