Serum matrix metalloproteinase-2 as a predictor of level of hypoxemia and severity of obstructive sleep apnea.

PURPOSE: Intermittent hypoxia in obstructive sleep apnea (OSA) resembles ischemia/reperfusion. Oxidative stress during ischemia/reperfusion increases matrix metalloproteinase-2 (MMP-2) activity and leads to adverse cardiovascular consequences in animal models, but there is scarce information about MMP-2 in humans with OSA. The aim of this study was to determine if serum MMP-2 levels of patients with OSA differ from controls and if MMP-2 activity correlates with the severity of OSA and level of hypoxemia. METHODS: Patients with OSA (n = 124) were recruited from the Sleep Disorders Center (Saskatoon City Hospital, Canada) after in-lab polysomnography (PSG). Controls (n = 26) were subjects referred for PSG who did not have OSA. Severity of OSA was categorized according to American Academy of Sleep Medicine criteria. Level of hypoxemia was expressed as oxygen desaturation index (ODI; 3% desaturation). Gelatin zymography was performed to measure serum MMP-2 activity. RESULTS: Serum MMP-2 activity was significantly higher in patients with OSA than in controls (p = 0.029). MMP-2 activity in patients with severe OSA was significantly higher than in those with mild/moderate OSA and controls (p = 0.002). Linear regression showed positive associations with MMP-2 activity in serum for AHI (p < 0.001) and ODI (p = 0.003). The associations persisted after adjustment for multiple confounders, including age, sex, BMI, and cardiovascular disease. CONCLUSIONS: Serum MMP-2 activity was associated with OSA severity, and level of hypoxemia in patients with OSA, suggesting MMP-2 is worth considering as a potential biomarker to be included in future studies on sets of biomarkers for hypoxemic insult in OSA.

Protection of the transplant kidney during cold perfusion with doxycycline: proteomic analysis in a rat model.

BACKGROUND: It has been previously shown that doxycycline (Doxy) protects the kidney from preservation injury by inhibition of matrix metalloproteinase. However, the precise molecular mechanism involved in this protection from injury is not known. We used a pharmaco-proteomics approach to identify potential molecular targets associated with kidney preservation injury. METHODS: Rat kidneys were cold perfused with or without doxycycline (Doxy) for 22 h. Kidneys perfusates were analyzed for the presence of injury markers such as lactate dehydrogenase (LDH), and neutrophil-gelatinase associated lipocalin (NGAL). Proteins extracted from kidney tissue were analyzed by 2-dimensional gel electrophoresis. Proteins of interest were identified by mass spectrometry. RESULTS: Triosephosphate isomerase, PGM, dihydropteridine reductase-2, pyridine nucleotide-disulfide oxidoreductase, phosphotriesterase-related protein, and aminoacylase-1A were not affected by cold perfusion. Perfusion with Doxy increased their levels. N(G),N(G)-dimethylarginine dimethylaminohydrolase and phosphoglycerate kinase 1 were decreased after cold perfusion. Perfusion with Doxy led to an increase in their levels. CONCLUSIONS: This study revealed specific metabolic enzymes involved in preservation injury and in the mechanism whereby Doxy protects the kidney against injury during cold perfusion.

Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP-2/MLCK-mediated cardiomyocyte protection from hypoxic injury.

Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase-2 (MMP-2). It has been shown that simultaneous inhibition of MMP-2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML-7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co-administration of nitric oxide synthase (NOS) inhibitor (1400W or L-NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia-reoxygenation (H-R)-induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP-2 by Doxy (25-100 µM), MLCK by ML-7 (0.5-5 µM) and NOS by L-NAME (25-100 µM) or 1400W (25-100 µM) protected myocyte contractility after H-R in a concentration-dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H-R at the level of highest single-drug concentration. The combination of subthreshold concentrations of NOS, MMP-2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP-2. The observed protection with addition of L-NAME or 1400W was better than previously reported combination of ML-7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility.

The need for support among cancer patients - a preliminary study.

PURPOSE: To analyze the demand for support among cancer patients subjected to systemic treatment or radiotherapy. METHODS: The study included 321 cancer patients treated in three Polish oncology centers. More than 73% of the responders were diagnosed with cancer not earlier than a year prior to the study. Most of the patients received chemotherapy (74.8%); nearly a half of the subjects (46.7%) were subjected to radiotherapy and every tenth person received hormonal therapy. The subjects were examined with the Berlin Social Support Scales (BSSS). RESULTS: Emotional and informative support were the most frequently needed forms of support. Age and sex did not exert significant effect on the need for support and the level of received support. Individuals with higher education showed the lowest scores of support seeking. The availability of instrumental support displayed the lowest score, especially among the individuals treated at daily chemotherapy units. The type of oncological treatment did not change the patients' perception of support. CONCLUSIONS: Irrespective of their demographic and clinical characteristics, cancer patients should be provided with emotional, informative and instrumental support.

Patient satisfaction with oncological care during the virus pandemic - SARS-CoV-2 taking into account social and demographic factors.

INTRODUCTION AND OBJECTIVE: The aim of the study was to examine the perception of medical services by oncological patients during the pandemic, identifying the key factors influencing it. The assessment of patient satisfaction with the treatment and care provided by doctors and other hospital staff provides important information on the quality of health services. MATERIAL AND METHODS: The study involved 394 patients diagnosed with cancer treated as inpatients in five oncology departments. The diagnostic survey method was used with a proprietary questionnaire and the standardized EORTC IN-PATSAT32 questionnaire. Calculations were carried out using Statistica 10.0 with p≤0.05 s considered statistically significant. RESULTS: Overall patient satisfaction with cancer care was 80.77/100. Higher values were shown for the competences of nurses than for doctors, especially for their interpersonal skills (79.34 - nurses vs. 74.13 - doctors) and availability (80.11 - nurses vs. 75.6 - doctors). It was also shown that the level of satisfaction with cancer care increased with age; women rated cancer care lower than men (p = 0.031), particularly its aspect related to the competences of doctors. A lower degree of satisfaction was observed among rural residents (p=0.042). Other demographic data, such as marital status and education, determined satisfaction with cancer care on the selected scale although it did not affect the overall level of satisfaction. CONCLUSIONS: The analysed socio-demographic factors, primarily age, gender and place of residence, determined some of the scales concerning patient satisfaction with cancer care during the COVID-19 pandemic. The results of this and other studies of a similar profile should be used in the formation of health policy, particularly in implementing programmes to improve the quality of cancer care in Poland.

Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion.

Ischemia/reperfusion (I/R) injury is a major consequence of a cardiovascular intervention. The study of changes of the left and right ventricle proteomes from hearts subjected to I/R may be a key to revealing the pathological mechanisms underlying I/R-induced heart contractile dysfunction. Isolated rat hearts were perfused under aerobic conditions or subjected to 25 min global ischemia and 30 min reperfusion. At the end of perfusion, right and left ventricular homogenates were analyzed by 2DE. Contractile function and coronary flow were significantly reduced by I/R. 2DE followed by mass spectrometry identified ten protein spots whose levels were significantly different between aerobic left and right ventricles, eight protein spots whose levels were different between aerobic and I/R left ventricle, ten protein spots whose levels were different between aerobic and I/R right ventricle ten protein spots whose levels were different between the I/R groups. Among these protein spots were ATP synthase beta subunit, myosin light chain 2, myosin heavy chain fragments, peroxiredoxin-2, and heat shock proteins, previously associated with cardiovascular disease. These results reveal differences between proteomes of left and right ventricle both under aerobic conditions and in response to I/R that contribute to a better understanding of I/R injury.

Ischemia induced peroxynitrite dependent modifications of cardiomyocyte MLC1 increases its degradation by MMP-2 leading to contractile dysfunction.

Damage to cardiac contractile proteins during ischemia followed by reperfusion is mediated by reactive oxygen species such as peroxynitrite (ONOO(-)), resulting in impairment of cardiac systolic function. However, the pathophysiology of systolic dysfunction during ischemia only, before reperfusion, remains unclear. We suggest that increased ONOO(-) generation during ischemia leads to nitration/nitrosylation of myosin light chain 1 (MLC1) and its increased degradation by matrix metalloproteinase-2 (MMP-2), which leads to impairment of cardiomyocyte contractility. We also postulate that inhibition of ONOO(-) action by use of a ONOO(-) scavenger results in improved recovery from ischemic injury. Isolated rat cardiomyocytes were subjected to 15 and 60 min. of simulated ischemia. Intact MLC1 levels, measured by 2D gel electrophoresis and immunoblot, were shown to decrease with increasing duration of ischemia, which correlated with increasing levels of nitrotyrosine and nitrite/nitrate. In vitro degradation of human recombinant MLC1 by MMP-2 increased after ONOO(-) exposure of MLC1 in a concentration-dependent manner. Mass spectrometry analysis of ischemic rat cardiomyocyte MLC1 showed nitration of tyrosines 78 and 190, as well as of corresponding tyrosines 73 and 185 within recombinant human cardiac MLC1 treated with ONOO(-). Recombinant human cardiac MLC1 was additionally nitrosylated at cysteine 67 and 76 corresponding to cysteine 81 of rat MLC1. Here we show that increased ONOO(-) production during ischemia induces MLC1 nitration/nitrosylation leading to its increased degradation by MMP-2. Inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO(-) scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhibition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility.

Patient Satisfaction with Oncological Care during the SARS-CoV-2 Virus Pandemic.

Recently, the outbreak of the SARS-CoV-2 virus and the COVID-19 pandemic significantly affected the health situation of the entire society and necessitated reorganization of health care including oncology. The objective of this study was to examine the perception of medical services by cancer patients during the pandemic and to identify the key elements influencing the level of satisfaction with oncological care. Of note, 394 patients diagnosed with cancer treated in inpatient oncology wards participated in the study (Poland). The diagnostic survey method was used. A survey questionnaire developed by the authors was used and validated the EORTC IN-PATSAT32 questionnaire. The calculations were made in Statistica 10.0 (Statsoft; 2011, Dell Inc., Round Rock, TX, USA). The average general level of satisfaction with oncological care in the study group was 80.77 out of a total score of 100, representing the highest level of satisfaction. Levels of satisfaction varied according to time since diagnosis (longer time-greater satisfaction) and were lower where treatment was delayed or perceived as disorganised. Nearly half of the respondents felt the threat of the SARS-CoV-2 infection, despite the fact that most of them believed that the hospital was well prepared to diagnose and treat cancer patients during the COVID-19 pandemic. Convincing patients about the proper preparation of health care for diagnostics and therapy is an important element influencing patient satisfaction with oncological care.

Effect of the Rho kinase inhibitor Y-27632 on the proteome of hearts with ischemia-reperfusion injury.

Growing attention has been given to the role of the Rho kinase pathway in the development of heart disease and ischemia/reperfusion (I/R) injury. Y-27632 is a Rho kinase inhibitor demonstrated to protect against I/R injury, but the exact mechanism by which it does so remains to be elucidated. The goal of this project was to determine new targets by which Y-27632 can protect the heart against I/R injury. Isolated rat hearts were perfused under aerobic conditions or subjected to I/R in the presence or absence of Y-27632. Administration of Y-27632 (1 µM) before ischemia and during the first 10 min of reperfusion resulted in complete recovery of cardiac function. 2-D electrophoresis followed by MS identified four proteins whose levels were affected by Y-27632 treatment. Lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were significantly increased in the Y-27632 treated group, while creatine kinase was normalized to control levels. In addition, we found increased level of two different molecular fragments of ATP synthase, which were normalized by Y-27632. This increase suggests that during ischemia ATP synthase is subjected to degradation. The changes in metabolic enzymes' levels and their regulation by Y-27632 suggest that the cardioprotective effect of Y-27632 involves increased energy production.

Effects of MMP-9 inhibition by doxycycline on proteome of lungs in high tidal volume mechanical ventilation-induced acute lung injury.

BACKGROUND: Although mechanical ventilation (MV) is a major supportive therapy for patients with acute respiratory distress syndrome, it may result in side effects including lung injury. In this study we hypothesize that MMP-9 inhibition by doxycycline might reduce MV-related lung damage. Using a proteomic approach we identified the pulmonary proteins altered in high volume ventilation-induced lung injury (VILI). Forty Wistar rats were randomized to an orally pretreated with doxycycline group (n = 20) or to a placebo group (n = 20) each of which was followed by instrumentation prior to either low or high tidal volume mechanical ventilation. Afterwards, animals were euthanized and lungs were harvested for subsequent analyses. RESULTS: Mechanical function and gas exchange parameters improved following treatment with doxycycline in the high volume ventilated group as compared to the placebo group. Nine pulmonary proteins have shown significant changes between the two biochemically analysed (high volume ventilated) groups. Treatment with doxycycline resulted in a decrease of pulmonary MMP-9 activity as well as in an increase in the levels of soluble receptor for advanced glycation endproduct, apoliporotein A-I, peroxiredoxin II, four molecular forms of albumin and two unnamed proteins. Using the pharmacoproteomic approach we have shown that treatment with doxycycline leads to an increase in levels of several proteins, which could potentially be part of a defense mechanism. CONCLUSION: Administration of doxycycline might be a significant supportive therapeutic strategy in prevention of VILI.

Pre-arrest doxycycline protects donation after circulatory death kidneys.

Kidney injury during donation after circulatory determination of death (DCDD) includes warm ischemic (WI) injury from around the time of asystole, and cold ischemic (CI) injury during cold preservation. We have previously shown that Matrix Metalloproteinases (MMPs) are involved in CI injury and that Doxycycline (Doxy), an antibiotic and known MMP inhibitor, protects the transplant kidney during CI. The purpose of our study was to determine if Doxy given before asystole can also prevent injury during WI. A rat model of DCDD was used, including Control, Preemptive Doxy (45 mg/kg iv), and Preemptive and Perfusion (100 microM) Doxy groups. Thirty minutes after asystole, both kidneys were removed. The left kidney was perfused at 4 °C for 22 h, whereas the right was used to establish the degree of warm ischemic injury prior to cold preservation. MMP-2 in the perfusate was significantly reduced in both treatment groups [Control 43.7 ± 7.2 arbitrary units, versus Preemptive Doxy group 23.2 ± 5.5 (p = 0.03), and 'Preemptive and Perfusion' group 18.0 ± 5.6 (p = 0.02)]. Reductions in NGAL, LDH, and MMP-9 were also seen. Electron microscopy showed a marked reduction in mitochondrial injury scores in the treatment groups. Pre-arrest Doxy was associated with a reduction in injury markers and morphologic changes. Doxy may be a simple and safe means of protecting transplant kidneys from both WI and CI.

Cardiac dysfunction in an animal model of neonatal asphyxia is associated with increased degradation of MLC1 by MMP-2.

The purpose of this study was to determine if decreased heart function after hypoxia followed by reoxygenation (H-R) is associated with increased degradation of cardiac myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2), and to investigate the effects of the increased level of peroxynitrite in the hearts of H-R animals on MLC1 degradation by MMP-2. Total of 12 newborn piglets were acutely instrumented to monitor cardiac function as assessed by stroke volume. Anesthetized piglets were block randomized to the normoxic group (n = 6), which received ventilation with room air for 6 h, or to the H-R group (n = 6), which received ventilation with 10-15% oxygen for 2 h, followed by reoxygenation with 100% oxygen for 1 h and then with 21% oxygen for 3 h. Hearts were removed and snap frozen for subsequent biochemical analyses. At the end of the 2-h hypoxia period, cardiac output, mean arterial pressure and stroke volume were significantly decreased in the H-R group. After 1 h of 100% oxygen, these parameters had increased slightly, but remained significantly lower than the normoxic controls throughout the reoxygenation period. Compared to normoxic animals, cardiac MLC1 levels were decreased and MMP-2 activity was increased in H-R animals. MMP-2 was co-localized with MLC1, and the amount of MLC1 associated with MMP-2 was higher in the hearts of H-R animals. In normoxic animals, cardiac MLC1 level was negatively, and cardiac MMP-2 activity was positively, strongly correlated with stroke volume index. This relationship was not seen in the H-R group. However, in both the normoxic group and the H-R group, the activity of cardiac MMP-2 was negatively correlated with the level of cardiac MLC1. There was a more than twofold increase in the level of nitrates, a marker for peroxynitrite formation, in the hearts of H-R animals. Mass spectrometric analyses detected peroxynitrite-induced nitration and S-nitrosylation of MLC1 protein in the hearts of H-R animals. These peroxynitrite-induced modifications of MLC1 were localized directly adjacent to the site at which MMP-2 cleaves MLC1. Peroxynitrite, formed during cardiac reoxygenation following a period of hypoxia, modifies the structure of cardiac MLC1 by nitrating and nitrosylating amino acids adjacent to the site where MMP-2 cleaves MLC1. This facilitates the degradation of MLC1 by MMP-2 and may contribute to cardiac dysfunction induced by H-R and other forms of oxidative stress. The high correlation between MMP-2 activity and MLC1 level in control animals suggests that MMP-2 may play an important role in regulating MLC1 turnover under normal physiological conditions. Determining the optimal parameters for controlled reoxygenation after hypoxia, together with pharmacological treatment with MMP-2 inhibitors and/or inhibitors of nitration/nitrosylation of MLC1, could reduce heart injury during the resuscitation of asphyxiated newborns and improve their long-term prognosis by reducing MLC1 degradation. Since the degradation of MLC1 by MMP-2 appears to be a common feature of oxidative stress, these pharmacological interventions may be useful in reducing tissue damage in other oxidative stress-related disorders as well.

Effect of duration of ischemia on myocardial proteome in ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is a serious problem resulting from clinical setting of coronary revascularization. Despite extensive studies on I/R injury, the molecular bases of cardiac dysfunction caused by I/R are still unknown, but are likely to result from alterations in protein expression. Isolated rat hearts were subjected to 15-30 min of no-flow ischemia without (Ischemia protocol) or with 30 min of reperfusion (I/R protocol). 2-DE analysis of heart proteins from both experimental protocols showed wide-ranging changes in protein levels. In the Ischemia protocol, 39 protein spots were changed in ischemic groups and those changes correlated with duration of ischemia. Ninety percent of the affected proteins were increased. In contrast to increased protein levels, the total messenger RNA (mRNA) level decreased approximately two fold. Compared to the Ischemia protocol, changes in protein levels in the I/R protocol did not correlate with the duration of ischemia and the degree of recovery of mechanical function. The decrease of affected protein from I/R protocol was associated with the increase in total protein level in reperfusate. Our studies show that the protein increase is correlated with the mechanical function of the I/R hearts and the increase is not likely associated with an increase in protein synthesis.

Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.

The regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundamental roles these zinc-endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP-2 contains 29 potential phosphorylation sites. Mass spectrometry reveals that at least five of these sites are phosphorylated in hrMMP-2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP-2 immunoreactivity on 2D immunoblots consistent with phosphorylation, and purified MMP-2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP-2 from HT1080 cell-conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP-2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP-2 significantly affects its enzymatic properties. Consistent with this, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase significantly increases its activity. We conclude that MMP-2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.

L-NAME improves doxycycline and ML-7 cardioprotection from oxidative stress.

Matrix metalloproteinase-2 (MMP-2) mediated degradation of myosin light chain 1 (MLC1) and troponin I (TnI) contributes to myocardial ischemia/reperfusion (I/R) injury. Modifications of MLC1 triggered by oxidative stress are mediated by myosin light chain kinase (MLCK), nitric oxide synthase (NOS), and MMP-2. Previous studies have shown that inhibiting both MLCK and MMP-2 protects against I/R injury. Here, we hypothesized that the addition of NOS inhibitor (L-NAME) at subprotective concentration to the mixture of subprotective concentrations of ML-7 and doxycycline (Doxy), will increase a synergistic cardioprotection of Doxy and ML-7 during I/R. Isolated rat hearts were subjected to global ischemia without or with administration of the mixture of inhibitors. Markers of I/R injury were measured in hearts and coronary effluents. Addition of L-NAME to the mixture of Doxy and ML-7 led to full recovery of heart contractility in comparison to combination of Doxy and ML-7. Improved heart contractility was associated with reduced degradation of TnI and MLC1. The combined administration of NOS, MMP-2 and MLCK inhibitors provides a novel strategy to protect heart from I/R injury.

Proteomic Analysis of Perfusate from Machine Cold Perfusion of Transplant Kidneys: Insights Into Protection from Injury.

BACKGROUND Machine cold perfusion is beneficial to the preservation of kidneys for transplantation. At the end of preservation, the perfusion solution contains many proteins. Using a proteomics approach, we searched for useful biomarkers and potential therapeutic targets in the perfusate. Our program is unique in that all transplant kidneys (even living donor kidneys, LKD) are placed on machine cold perfusion prior to transplantation. MATERIAL AND METHODS Perfusates from donation after neurological and circulatory determination of death (DNDD and DCDD respectively) and LKD were collected (n=41) and analyzed for LDH, neutrophil gelatinase-associated lipocalin (NGAL), and matrix metalloproteinase-2 (MMP-2) as markers of injury. Perfusate from each kidney was subjected to 2-dimensional gel electrophoresis, then analyzed using software to identify those spots which are significantly different between the 3 groups. Mass spectrometry was used to identify the proteins and their identity was confirmed with Western blot. RESULTS The highest levels of MMP-2, LDH, and NGAL were seen for the DCDD kidneys, followed by the DNDD kidneys and then LDK. Peroxiredoxin-2, NGAL, and alpha-1-antitrypsin were identified as significantly different between the different types of donor kidneys, and their role and possible therapeutic strategies are discussed. Collagen fragments, albumin, and immunoglobulin were also identified as possible byproducts of the injury and may be useful is assessing the degree of injury. CONCLUSIONS Comparison of the perfusates from the different types of kidneys has allowed us to identify proteins that will be useful in future research into reducing injury in transplant kidneys.

Degradation of myosin light chain in isolated rat hearts subjected to ischemia-reperfusion injury: a new intracellular target for matrix metalloproteinase-2.

BACKGROUND: Matrix metalloproteinase-2 (MMP-2) contributes to cardiac dysfunction resulting from ischemia-reperfusion (I/R) injury. MMP-2 not only remodels the extracellular matrix but also acts intracellularly in I/R by degrading troponin I. Whether other intracellular targets exist for MMP-2 during I/R is unknown. METHODS AND RESULTS: Isolated rat hearts were subjected to 20 minutes of ischemia and 30 minutes of reperfusion. The impaired recovery of mechanical function of the heart was attenuated by the MMP inhibitors o-phenanthroline or doxycycline. Quantitative 2D electrophoresis of homogenates of aerobically perfused hearts (control) or those subjected to I/R injury (in the presence or absence of MMP inhibitors) showed 3 low-molecular-weight proteins with levels that were significantly increased upon I/R injury and normalized to control levels by MMP inhibitors. Mass spectrometry analysis identified all 3 proteins as fragments of myosin light chain 1, which possesses theoretical cleavage recognition sequences for MMP-2 and is rapidly degraded by it in vitro. The association of MMP-2 with the thick myofilament in fractions prepared from I/R hearts was observed with immunogold electron microscopy, gelatin zymography for MMP-2 activity, and immunoprecipitation. MMP-2 was found to cleave myosin light chain 1 between tyrosine 189 and glutamine 190 at the C terminus. CONCLUSIONS: Our results demonstrate that myosin light chain 1 is another novel substrate for MMP-2 in the cardiomyocyte and that its degradation may contribute to contractile dysfunction resulting from I/R injury to the heart.

Protection of the Transplant Kidney from Preservation Injury by Inhibition of Matrix Metalloproteinases.

BACKGROUND: Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, play an important role in ischemic injury to the heart, yet it is not known if these MMPs are involved in the injury that occurs to the transplant kidney. We therefore studied the pharmacologic protection of transplant kidneys during machine cold perfusion. METHODS: Human kidney perfusates were analyzed for the presence of injury markers such as cytochrome c oxidase, lactate dehydrogenase, and neutrophil-gelatinase associated lipocalin (NGAL), and MMP-2 and MMP-9 were measured. The effects of MMP inhibitors MMP-2 siRNA and doxycycline were studied in an animal model of donation after circulatory determination of death (DCDD). RESULTS: Markers of injury were present in all analyzed perfusates, with higher levels seen in perfusates from human kidneys donated after controlled DCDD compared to brain death and in perfusate from kidneys with delayed graft function. When rat kidneys were perfused at 4°C for 22 hours with the addition of MMP inhibitors, this resulted in markedly reduced levels of MMP-2, MMP-9 and analyzed injury markers. CONCLUSIONS: Based on our study, MMPs are involved in preservation injury and the supplementation of preservation solution with MMP inhibitors is a potential novel strategy in protecting the transplant kidney from preservation injury.

Imbalance between tissue inhibitor of metalloproteinase-4 and matrix metalloproteinases during acute myocardial [correction of myoctardial] ischemia-reperfusion injury.

BACKGROUND: We have previously reported that matrix metalloproteinase-2 (MMP-2) contributes to myocardial ischemia-reperfusion injury by degradation of troponin I, a regulatory element of the contractile proteins. MMP activities are also tightly regulated by tissue inhibitors of metalloproteinase (TIMPs). The change in TIMPs during acute myocardial ischemia-reperfusion injury is not clear. METHODS AND RESULTS: Isolated rat hearts were perfused either aerobically for 75 minutes or subjected to 15, 20, or 25 minutes of global, no-flow ischemia followed by 30 minutes of aerobic reperfusion. During reperfusion after ischemia, there was a rapid, enhanced release of TIMP-4, the most abundant TIMP in the heart, into the coronary effluent, as shown both by reverse zymography and Western blot. There was a negative correlation between the recovery of cardiac mechanical function and the release of TIMP-4 during reperfusion in hearts subjected to different durations of ischemia. Immunogold electron microscopy revealed a close association of TIMP-4 with the sarcomeres in aerobically perfused hearts. Moreover, TIMP-4 was present only in thin myofilaments prepared from aerobically perfused hearts but not in ischemic-reperfused hearts. An enhanced MMP activity was shown in ischemic-reperfused hearts by in situ zymography. CONCLUSIONS: Loss of TIMP-4 from the cardiac myocyte leads to an increase in net myocardial MMP activity that contributes to acute myocardial stunning injury.

Inhibition of endogenous nitric oxide in the heart enhances matrix metalloproteinase-2 release.

Matrix metalloproteinase (MMP) activity is upregulated in pathologies such as atherosclerosis during which endogenous nitric oxide (NO) biosynthesis is reduced. Diminished levels of NO, an antioxidant species, may result in higher oxidative stress. Oxidants are capable of activating MMPs from their zymogen forms. We examined whether basal biosynthesis of NO in the coronary circulation regulates MMP-2 activity. In isolated rat hearts perfused with Krebs-Henseleit buffer at a constant flow of 10 ml min(-1), we measured the release of MMP-2 into the coronary effluent by gelatin zymography. The main gelatinolytic activity of 72-kDa corresponds to MMP-2. Infusion of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) concentration dependently increased coronary perfusion pressure (CPP) (by 48+/-11 mmHg with 100 microM) and enhanced the release of the 72-kDa MMP-2 in the effluent. Coinfusion of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1 microM) with L-NAME abolished both the increase in CPP and the enhanced MMP-2 release. The thromboxane A2 mimetic U46619 increased CPP to the same extent as L-NAME without increasing 72-kDa activity in the effluent, suggesting that MMP-2 release is not caused simply by enhanced perfusion pressure. Infusion of either L-NAME or U46619 did not significantly enhance LDH release. L-NAME infusion concentration dependently increased the level of lipid hydroperoxides in homogenates prepared from the perfused hearts. Coinfusion of SNAP prevented this increase. These data reveal another cytoprotective mechanism of endogenous NO biosynthesis in the heart, the inhibition of MMP-2 release.