In Vivo Study on Doxycycline Protective Mechanisms during Myocardial Ischemia Injury in Rats.

BACKGROUND: The fact that during myocardial ischemia/reperfusion (I/R) injury, myosin light chain 1 (MLC1) and troponin I (TnI) are degraded by matrix metalloproteases activity has already been well established in both in vitro and ex vivo studies. However, I/R injury is a complex issue based on several overlapping mechanisms. Increased activity of myosin light chain kinase and nitric oxide synthase due to oxidative stress leads to post-translational modifications of MLC1, thus leading to the increased degradation of these proteins. METHODS: Wistar rats were subjected to left anterior descending coronary artery occlusion. To measure the pharmacological effect of doxycycline, transthoracic echocardiography as well as biochemical tests, concentrations of TnI, LDH, MLC1, MMP-2 and MMP-9 were performed. Gelatinize activity and cytotoxicity level were also assessed; Results: I.p., administration of doxycycline before LAD occlusion surgery increased TnI and LDH content in the heart and decreased cytotoxicity. A reduction of MMP-2 and MMP-9 concentration and MMP-2 activity after administration of Doxy was also observed, as well as improvement in echocardiographic parameters just 7 days after surgery. CONCLUSIONS: Inhibition of MMPs by doxycycline, in vivo, may serve as a protective agent in future therapy.

Mitoquinone Alleviates Donation after Cardiac Death Kidney Injury during Hypothermic Machine Perfusion in Rat Model.

Transplanted organs are subjected to harmful conditions through stopping blood flow, hypothermic storage of the graft, and subsequent reperfusion. In particular, kidneys donated from patients after cardiac arrest (DCD) are classified as more vulnerable to ischemia-reperfusion injury (IRI). Hypothermic machine perfusion is proposed as a solution for better kidney storage before transplantation, and it is a good platform for additional graft treatment. Antioxidants have gained interest in regenerative medicine due to their ability to scavenge reactive oxygen species (ROS), which play a key role in IRI. We evaluated the effect of Mitoquinone (MitoQ), a strong mitochondria-targeted antioxidant, administered directly to the perfusing buffer. Rat kidneys were isolated, randomly classified into one of the following groups, donation after brainstem death (DBD), DCD, and DCD with MitoQ, and perfused for 22 hours with a hypothermic machine perfusion system. Subsequently, we detected levels of kidney injury (KIM-1) and oxidative stress (ROS/RNS, cytochrome C oxidase, and mitochondrial integrity) markers. We compared the activation of the apoptosis pathway (caspase 3 and 9), the concentration of phosphorylated Akt (pAkt), and the pAkt/total Akt ratio. MitoQ reduces KIM-1 concentration, total ROS/RNS, and the level of caspases. We observed a decrease in pAkt and the pAkt/total Akt ratio after drug administration. The length of warm ischemia time negatively impacts the graft condition. However, MitoQ added to the perfusing system as an 'on pump' therapy mitigates injury to the kidney before transplantation by inhibiting apoptosis and reducing ROS/RNS levels. We propose MitoQ as a potential drug for DCD graft preconditioning.

Δ9-Tetrahydrocannabinol (Δ9-THC) Improves Ischemia/Reperfusion Heart Dysfunction and Might Serve as a Cardioprotective Agent in the Future Treatment.

BACKGROUND: Ischemia/reperfusion (I/R) is a pivotal mechanism of organ injury during clinical stetting for example for cardiopulmonary bypasses. The generation of reactive oxygen species (ROS) during I/R induces oxidative stress that promotes endothelial dysfunction, DNA dissociation and local inflammation. In turn, those processes induce cytokine release, resulting in damage to cellular structures and cell death. One of the major psychoactive compounds of Cannabis is delta-9-tetrahydrocannabinol (Δ9-THC), which is known as an anti-inflammatory mediator. Our research aimed to test if Δ9-THC may be protective in the treatment of cardiovascular system dysfunction arising from I/R heart injury. METHODS: Two experimental models were used: isolated rat hearts perfused with the Langendorff method and human cardiac myocytes (HCM) culture. Rat hearts and HCM underwent ex vivo/chemical in vitro I/R protocol with/without Δ9-THC treatment. The following parameters were measured: cell metabolic activity, morphology changes, cell damage as lactate dehydrogenase (LDH) activity, ceramide kinase (CERK) activity, ROS level, total antioxidant capacity (TAC) and heart hemodynamic parameters. RESULTS: Δ9-THC protected the heart, as evidenced by the improved recovery of cardiac function (p < 0.05, N = 3-6). Cells subjected to I/R showed lower cytoplasmic LDH activity, and 10 µM Δ9-THC treatment reduced cell injury and increased LDH content (p = 0.019, N = 6-9). Morphology changes of HCM-spherical shape, vacuolisation of cytoplasm and swollen mitochondria-were inhibited due to Δ9-THC treatment. I/R condition affected cell viability, but 10 µM Δ9-THC decreased the number of dead cells (p = 0.005, N = 6-9). The total level of CERK was lower in the I/R group, reflecting oxidative/nitrosative stress changes. The administration of Δ9-THC effectively increased the production of CERK to the level of aerobic control (p = 0.028, N = 6-9). ROS level was significantly decreased in I/R cells (p = 0.007, N = 6-8), confirming oxidative stress, while administration of 10 µM Δ9-THC enhanced TAC in cardiomyocytes subjected to I/R (p = 0.010, N = 6-8). CONCLUSIONS: Δ9-THC promotes the viability of cardiomyocytes, improves their metabolic activity, decreases cell damage and restores heart mechanical function, serving as a cardioprotective. We proposed the use of Δ9-THC as a cardioprotective drug to be, administered before onset of I/R protocol.

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.

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.

Mixture of MMP-2, MLC, and NOS Inhibitors Affects NO Metabolism and Protects Heart from Cardiac I/R Injury.

OBJECTIVES: Coronary reperfusion procedure leads to ischemia/reperfusion injury of the heart (IRI). IRI arises from increased degradation of myosin light chains and increased activity of matrix metalloproteinase 2 (MMP-2). Increased production of toxic peroxynitrite (ONOO-) during oxidative stress is a source of increased nitration/nitrosylation of contractile proteins, which enhance their degradation through MMP-2. Hence, an imbalance in nitric oxide (NO) metabolism along with oxidative stress is an important factor contributing to pathophysiology of cardiovascular disorders, including myocardial infarction. The aim of the current study was to provide an important insight into understanding the interaction of iNOS, eNOS, and ADMA during oxidative stress and to propose the beneficial therapy to modulate this interaction. Material and Methods. Pathogen-free Wistar rats were used in this study as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischemia with or without administration of DOXY (1 µM), ML-7 (0.5 µM), and L-NAME (2 µM) mixture. Haemodynamic parameters of heart function, markers of I/R injury, tissue expression of iNOS, eNOS, and phospho-eNOS, asymmetric dimethylarginine, and NO production as well as MMP-2 activity were measured. RESULTS: Mechanical heart function and coronary flow (CF) were decreased in the hearts subjected to I/R. Treatment of the hearts with the tested mixture resulted in a recovery of mechanical function due to decreased activity of MMP-2. An infusion of Doxy, ML-7, and L-NAME mixture into I/R hearts decreased the expression of iNOS, eNOS, and phospho-eNOS and in consequence reduced ADMA expression. Decreased ADMA production led to enhanced NO synthesis and improvement of cardiac function at 85% of aerobic control. CONCLUSIONS: Synergistic effect of the multidrug therapy with the subthreshold doses allows addressing a few pathways of I/R injury simultaneously to achieve protection of cardiac function during I/R.

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.

A drug cocktail for protecting against ischemia-reperfusion injury.

Ischemia followed by reperfusion (I/R) of cardiomyocytes causes release of a large amount of inducible nitric oxide (NO) synthase (iNOS) followed by an increase of asymmetric dimethylarginine (ADMA). ADMA disrupts NO signaling by switching of the NOS activity from NO to the production of reactive oxygen species (ROS). Previously, we have shown that pretreatment of the hearts by co-administration of sub-threshold concentrations of doxycycline, a matrix metalloproteinase (MMPs) inhibitor, ML-7 an inhibitor of myosin light-chain kinase (MLCK) and L-NAME a non-selective NOS inhibitor protects the heart against I/R injury. In this study, we replaced the L-NAME with 1400W (selective inhibitor of iNOS) in the drug cocktail that was Langendorff-perfused into the hearts of Wistar rats before (prevention) or after (treatment) the induction of I/R. This pre-treatment resulted in full protection of contractility, decreased production of iNOS and ADMA and normalized the bioavailability of NO in the I/R hearts. Thus, the formulated drug cocktail protects the heart from I/R injury.

Pharmacological Protection of Kidney Grafts from Cold Perfusion-Induced Injury.

One of the greatest challenges facing the field of organ transplantation is the shortage of donor organs for transplantation. Renal transplantation increases quality of life and survival of patients suffering from end-stage renal disease. Although kidney transplantation has evolved greatly over the past few decades, a not insignificant amount of injury occurs to the kidney during recovery, preservation, and implantation and leads to the loss of function and loss of years of dialysis-free living for many patients. The use of kidneys from expanded criteria donors (ECD) and donation after circulatory determination of death (DCDD) has been adopted partly in response to the shortage of donor kidneys; however these kidneys are even more susceptible to ischemic injury. It has been shown that matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) are involved in mechanisms of injury to the transplant kidney. There is also some evidence that inhibition of MMP activity and/or ROS production can protect the kidney from injury. We review possible pharmacological strategies for protection of kidney graft from injury during recovery, preservation, and implantation.

Matrix metalloproteinases as possible biomarkers of obstructive sleep apnea severity - A systematic review.

Obstructive sleep apnea is an underdiagnosed sleep-related breathing disorder affecting millions of people. Recurrent episodes of apnea/hypopnea result in intermittent hypoxia leading to oxidative stress. Obstructive sleep apnea is considered an independent risk factor for cardiovascular disease but the exact pathophysiology of adverse cardiovascular outcomes of obstructive sleep apnea has not been fully elucidated. Matrix metalloproteinases (MMPs) have been associated with both oxidative stress and cardiovascular diseases. Hypoxic conditions were shown to influence MMP expression, secretion and activity. Moreover, matrix metalloproteinases contribute to ischemia/reperfusion injury. Therefore, action of matrix metalloproteinases can provide a possible molecular mechanism linking obstructive sleep apnea with oxidative stress and cardiovascular disease. The aim of this paper was to review the current evidence of association between matrix metalloproteinases and obstructive sleep apnea with focus on hypoxemia and severity of obstructive sleep apnea.

Cardioprotective effect of MMP-2-inhibitor-NO-donor hybrid against ischaemia/reperfusion injury.

Hypoxic injury of cardiovascular system is one of the most frequent complications following ischaemia. Heart injury arises from increased degradation of contractile proteins, such as myosin light chains (MLCs) and troponin I by matrix metalloproteinase 2 (MMP-2). The aim of the current research was to study the effects of 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate (MMP-2-inhibitor-NO-donor hybrid) on hearts subjected to ischaemia/reperfusion (I/R) injury. Primary human cardiac myocytes and Wistar rat hearts perfused using Langendorff method have been used. Human cardiomyocytes or rat hearts were subjected to I/R in the presence or absence of tested hybrid. Haemodynamic parameters of heart function, markers of I/R injury, gene and protein expression of MMP-2, MMP-9, inducible form of NOS (iNOS), asymmetric dimethylarginine (ADMA), as well as MMP-2 activity were measured. Mechanical heart function, coronary flow (CF) and heart rate (HR) were decreased in hearts subjected to I/R Treatment of hearts with the hybrid (1-10 µmol/L) resulted in a concentration-dependent recovery of mechanical function, improved CF and HR. This improvement was associated with decreased tissue injury and reduction of synthesis and activity of MMP-2. Decreased activity of intracellular MMP-2 led to reduced degradation of MLC and improved myocyte contractility in a concentration-dependent manner. An infusion of a MMP-2-inhibitor-NO-donor hybrid into I/R hearts decreased the expression of iNOS and reduced the levels of ADMA. Thus, 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate protects heart from I/R injury.

Biocompatible Carbon-Based Coating as Potential Endovascular Material for Stent Surface.

Stainless steel 316L is a material commonly used in cardiovascular medicine. Despite the various methods applied in stent production, the rates of in-stent restenosis and thrombosis remain high. In this study graphene was used to coat the surface of 316L substrate for enhanced bio- and hemocompatibility of the substrate. The presence of graphene layers applied to the substrate was investigated using cutting-edge imaging technology: energy-filtered low-voltage FE-SEM approach, scanning electron microscopy (SEM), Raman spectroscopy, and atomic force microscopy (AFM). The potential of G-316L surface to influence endothelial cells phenotype and endothelial-to-mesenchymal transition (EndoMT) has been determined. Our results show that the bio- and hemocompatible properties of graphene coatings along with known radial force of 316L make G-316L a promising candidate for intracoronary implants.

Low frequency electromagnetic field decreases ischemia-reperfusion injury of human cardiomyocytes and supports their metabolic function.

Electromagnetic field at extremely low frequencies plays a significant role in the physiological function of human tissues and systems. It is shown that electromagnetic field inhibits production of reactive oxygen species which are involved in heart injury triggered by oxidative stress. We hypothesize that low frequency electromagnetic field protects function of cardiac cells from ischemia-reperfusion injury. Human cardiac myocytes, endothelial cells, and cardiac fibroblast underwent ischemia-reperfusion conditions in the presence or in the absence of low frequency electromagnetic field. LDH and MMP-2 activities (as markers of cell injury), and cell metabolic activity (by fluorescein diacetate staining) were measured to determine the protective role of low frequency electromagnetic field. Our data showed that short courses of low frequency electromagnetic field protect cardiac cells from cellular damage and preserve their metabolic activity during ischemia-reperfusion. This study demonstrates the possibility to use of low frequency electromagnetic field as strategy for the prevention or therapy of ischemia-reperfusion injury. Impact statement In our study, we showed that LF-EMF may be protective for heart during ischemia-reperfusion (I/R). Following is the short description of the main findings: (a) the response to the I/R injury was different for endothelial cells, fibroblasts, and cardiomyocytes; (b) I/R decreases MMP-2 activity in cardiac myocytes and fibroblasts;

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.

Multidrug prevention or therapy of ischemia-reperfusion injury of the heart-Mini-review.

Restoration of blood flow to myocardium previously subjected to ischemia leads to ischemia/reperfusion injury due to oxidative stress. An increased production of toxic peroxynitrite, an enhanced phosphorylation and nitration/nitrosylation of myocyte contractile proteins and overactivation of matrix metalloproteinases -are only one of the several causes of heart damage. Multifactorial basis of ischemia/reperfusion injury demands the use of multiple pharmacological agents, inhibiting several pathways of cardiac injury. Nevertheless, the use of these drugs in their therapeutic doses, apart from their role in the treatment of pathological events, may also disturb physiological processes leading to numerous side-effects. Therefore current preclinical studies focuses on multidrug therapies in their low concentration. Synergistic or additive effect of low multidrug therapy inhibit pathological processes while maintaining the proper cell function and avoid alteration of physiological role of important functional proteins. This study provides information about multidrug strategies for the prevention/treatment of cardiac injury induced by oxidative stress.

Early experience with hypothermic machine perfusion of living donor kidneys - a retrospective study.

Although hypothermic machine perfusion (HMP) has been shown to be beneficial to deceased donor kidneys, the effect of HMP on living donor kidneys (LDK) is unknown. LDK are subjected to minutes of normothermic ischemia at the time of recovery. Comparison of 16 LDK preserved by HMP with 16 LDK preserved by static cold storage (SCS). Outcomes of interest are resistive indices (RI), both while on HMP and postoperatively, and creatinine clearance (CrCl). Injury markers NGAL and LDH were seen in the perfusate of LDK in amounts similar to what is found for donation after neurological determination of death kidneys. Compared to SCS kidneys, CrCl was significantly higher in the HMP group from days 2 through 7 post-transplant [ie: day 7 (78.8 ± 5.4 vs. 54.0 ± 4.6 ml/min, P = 0.005)]. CrCl at 1 year was higher in the HMP group (81.2 ± 5.8 vs. 70.0 ± 5.3 ml/min, P = 0.03). Early post-transplant RI was significantly lower in the HMP group (0.61 ± 0.02 vs. 0.71 ± 0.02, P < 0.0001). Our data support the assertion that injury does occur during LDK procurement and suggest that some of this injury may be reversed with HMP, resulting in more favorable early RI and graft function compared to SCS kidneys.

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.

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.