Sarcoplasmic reticulum calcium ATPase (SERCA) proteolysis by matrix metalloproteinase-2 contributes to vascular dysfunction in early hypertension.

AIMS: Hypertension is associated with an increased activity of matrix metalloproteinase (MMP)-2 in the vasculature, which, in turn, proteolyzes extra- and intracellular proteins that lead to vascular dysfunction. The activity of sarcoplasmic reticulum calcium ATPase (SERCA) is decreased in the aortas of hypertensive rats. Increased activity of MMP-2 proteolyzed SERCA in rat heart during ischemia and reperfusion injury, thus impairing cardiac function. Therefore, we examined whether increased activity of MMP-2 in early hypertension contributes to proteolyze SERCA in the aortas, thus leading to maladaptive vascular remodeling and dysfunction. MAIN METHODS: Male Sprague-Dawley rats were submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with doxycycline. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. After 7 days, aortas were collected for zymography assays, Western blot to SERCA, ATPase activity assay, vascular reactivity, Ki-67 immunofluorescence and hematoxylin/eosin stain. KEY FINDINGS: SBP was increased in 2K-1C rats and doxycycline did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and doxycycline decreased Ki-67. In 2K-1C rats, arterial relaxation to acetylcholine was impaired and doxycycline ameliorated it. SIGNIFICANCE: doxycycline reduced MMP-2 activity in aortas of 2K-1C rats and prevented proteolysis of SERCA and its dysfunction, thus ameliorating hypertension-induced vascular dysfunction.

MMP inhibition attenuates hypertensive eccentric cardiac hypertrophy and dysfunction by preserving troponin I and dystrophin.

PURPOSE: Cardiac transition from concentric (C-LVH) to eccentric left ventricle hypertrophy (E-LVH) is a maladaptive response of hypertension. Matrix metalloproteinases (MMPs), in particular MMP-2, may contribute to tissue remodeling by proteolyzing extra- and intracellular proteins. Troponin I and dystrophin are two potential targets of MMP-2 examined in this study and their proteolysis would impair cardiac contractile function. We hypothesized that MMP-2 contributes to the decrease in troponin I and dystrophin in the hypertensive heart and thereby controls the transition from C-LVH to E-LVH and cardiac dysfunction. METHODS: Male Wistar rats were divided into sham or two kidney-1 clip (2K-1C) hypertensive groups and treated with water (vehicle) or doxycycline (MMP inhibitor, 15 mg/kg/day) by gavage from the tenth to the sixteenth week post-surgery. Tail-cuff plethysmography, echocardiography, gelatin zymography, confocal microscopy, western blot, mass spectrometry, in silico protein analysis and immunofluorescence were performed. RESULTS: 6 out of 23 2K-1C rats (26%) had E-LVH followed by reduced ejection fraction. The remaining had C-LVH with preserved cardiac function. Doxycycline prevented the transition from C-LVH to E-LVH. MMP activity is increased in C-LVH and E-LVH hearts which was inhibited by doxycycline. This effect was associated with an increase in troponin I cleavage products and a decline in dystrophin in the left ventricle of E-LVH rats, which was prevented by doxycycline. CONCLUSION: Hypertension causes increased cardiac MMP-2 activity which proteolyzes troponin I and dystrophin, contributing to the transition from C-LVH to E-LVH and cardiac dysfunction.

Predictive Value of Matrix Metalloproteinases and Their Inhibitors for Mortality in Septic Patients: A Cohort Study.

PURPOSE: Over 170 biomarkers are being investigated regarding their prognostic and diagnostic accuracy in sepsis in order to find new tools to reduce morbidity and mortality. Matrix metalloproteinases (MMPs) and their inhibitors have been recently studied as promising new prognostic biomarkers in patients with sepsis. This study is aimed at determining the utility of several cutoff points of these biomarkers to predict mortality in patients with sepsis. MATERIALS AND METHODS: A multicenter, prospective, analytic cohort study was performed in the metropolitan area of Bucaramanga, Colombia. A total of 289 patients with sepsis and septic shock were included. MMP-9, MMP-2, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2, TIMP-1/MMP-9 ratio, and TIMP-2/MMP-2 ratio were determined in blood samples. Value ranges were correlated with mortality to estimate sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiving operating characteristic curve. RESULTS: Sensitivity ranged from 33.3% (MMP-9/TIMP-1 ratio) to 60.6% (TIMP-1) and specificity varied from 38.8% (MMP-2/TIMP-2 ratio) to 58.5% (TIMP-1). As for predictive values, positive predictive value range was from 17.5% (MMP-9/TIMP-1 ratio) to 70.4% (MMP-2/TIMP-2 ratio), whereas negative predictive values were between 23.2% (MMP-2/TIMP-2 ratio) and 80.9% (TIMP-1). Finally, area under the curve scores ranged from 0.31 (MMP-9/TIMP-1 ratio) to 0.623 (TIMP-1). CONCLUSION: Although TIMP-1 showed higher sensitivity, specificity, and negative predictive value, with a representative population sample, we conclude that none of the evaluated biomarkers had significant predictive value for mortality.

Prognostic Value of MMP-9 -1562 C/T Gene Polymorphism in Patients With Sepsis.

INTRODUCTION: Matrix metalloproteinase-9 (MMP-9) plays an important role in the pathophysiology of sepsis. A single-nucleotide polymorphism (SNP) at position -1562 (C/T) in the MMP-9 gene has been associated with differential MMP-9 expression, being higher when the -1562 T allele is present. We evaluated the association of the SNP MMP9 -1562 C/T with severity and mortality in patients with sepsis to establish whether the prognosis of the disease is affected. MATERIALS AND METHODS: A case-control study exploratory was carried out in a cohort of infected patients. 540 individuals were selected in total, 270 patients with sepsis and 270 controls (infected but non-septic), classified according to the 2016 consensus (Sepsis-3). The presence of the single-nucleotide polymorphism (SNP; allele T and/or allele C) was determined through analyses of restriction fragment length polymorphism and plasma levels of MMP-9 were determined through enzyme-linked immunosorbent assay immunoassay. RESULTS: SNP MMP-9 -1562 has two known alleles (T and C), with predominance of the C over the T allele; in the group of patients with sepsis, T allele was found in 7.2% of cases, while C allele in the rest (92.8%); in comparison, in the group of infected but non-septic patients, frequencies were 9.4% for T allele and 90.6% for the C allele (P = .33). Also, the presence of the polymorphic T allele was not related to the levels of MMP-9 in patients with sepsis in comparison with infected but non-septic patients 780 (397-1375) ng/mL vs 646 (172-1249) ng/mL (P = .64). There was also no association between the SNP and sepsis mortality (P = .78). CONCLUSIONS: We concluded that there was no association between the SNP MMP9 -1562 C/T and sepsis or between the SNP MMP9 -1562 C/T and sepsis mortality in the Northeastern Colombian septic patient cohort. Further research is needed to clarify the correlation among sepsis, genetic factors with allele T and MMP-9 plasma concentration.

Matrix metalloproteinase (MMP)-2 activation by oxidative stress decreases aortic calponin-1 levels during hypertrophic remodeling in early hypertension.

Hypertension is characterized by maladaptive vascular remodeling and enhanced oxidative stress in the vascular wall. Peroxynitrite may directly activate latent matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMC) by its S-glutathiolation. MMP-2 may then proteolyze calponin-1 in aortas from hypertensive animals, which stimulates VSMC proliferation and medial hypertrophy. Calponin-1 is an intracellular protein which helps to maintain VSMC in their differentiated (contractile) phenotype. The present study therefore investigated whether aortic MMP-2 activity is increased by oxidative stress in early hypertension and then contributes to hypertrophic arterial remodeling by reducing the levels of calponin-1. Male Wistar rats were submitted to the two kidney, one clip (2 K-1C) model of hypertension or sham surgery and were treated daily with tempol (18 mg/kg/day) or its vehicle (water) by gavage from the third to seventh day post-surgery. Systolic blood pressure (SBP) was daily assessed by tail-cuff plethysmography. After one week, aortas were removed to perform morphological analysis with hematoxylin and eosin staining and to analyze reactive oxygen­nitrogen species levels by dihydroethidium and immunohistochemistry for nitrotyrosine. MMP-2 activity was analyzed by in situ and gelatin zymography and its S-glutathiolation was analyzed by Western blot for MMP-2 of anti-glutathione immunoprecipitates. Calponin-1 levels were identified in aortas by immunofluorescence. SBP increased by approximately 50 mmHg at the first week in 2 K-1C rats which was unaffected by tempol. However, tempol ameliorated the hypertension-induced increase in arterial media-to-lumen ratio and hypertrophic remodeling. Tempol also decreased hypertension-induced aortic oxidative stress and the enhanced MMP-2 activity. S-glutathiolation may be a potential mechanism by which oxidative stress activates MMP-2 in aortas of 2 K-1C rats. Furthermore, calponin-1 was decreased in aortas from 2 K-1C rats and tempol prevented this. In conclusion, oxidative stress may contribute to the increase in aortic MMP-2 activity, possibly by S-glutathiolation, and this may result in calponin-1 loss and maladaptive vascular remodeling in early hypertension.

High fat diet modulates inflammatory parameters in the heart and liver during acute Trypanosoma cruzi infection.

The high fat diet (HFD) can trigger metabolic and cardiovascular diseases. Trypanosoma cruzi infection induces progressive inflammatory manifestations capable to affect the structure and the function of important organs such as the heart and liver. Here we aimed to investigate the effects of a HFD on the immune response and matrix metalloproteinase (MMP) activities during acute infection with the T. cruzi strain VL-10. The VL-10 strain has cardiac tropism and causes myocarditis in mice. Male C57BL/6 mice were treated with either: (i) regular diet (Reg) or (ii) HFD for 8 weeks, after which mice in each group were infected with T. cruzi. Mice were euthanized on day 30 after infection, and the liver and heart were subjected to histology and zymography to determine MMP-2 activities and plasma levels of IL-10, TNF, CCL2, and CCL5. T. cruzi-infected HFD animals had higher parasitemia, LDL and total cholesterol levels. Regardless of diet, plasma levels of all inflammatory mediators and cardiac MMP-2 activity were elevated in infected mice in contrast with the low plasma levels of leptin. HFD animals presented micro- and macrovesicular hepatic steatosis, while cardiac leukocyte infiltration was mainly detected in T. cruzi-infected mice. Our findings suggested that a HFD promotes higher circulating T. cruzi load and cardiac and liver immunopathogenesis in an experimental model using the VL-10 strain of the T. cruzi.

TIMP1 and MMP9 are predictors of mortality in septic patients in the emergency department and intensive care unit unlike MMP9/TIMP1 ratio: Multivariate model.

INTRODUCTION: Matrix metalloproteinases and tissue inhibitors of metalloproteinases could be promising biomarkers for establishing prognosis during the development of sepsis. It is necessary to clarify the relationship between matrix metalloproteinases and their tissue inhibitors. We conducted a cohort study with 563 septic patients, in order to elucidate the biological role and significance of these inflammatory biomarkers and their relationship to the severity and mortality of patients with sepsis. MATERIALS AND METHODS: A multicentric prospective cohort was performed. The sample was composed of patients who had sepsis as defined by the International Conference 2001. Serum procalcitonin, creatinine, urea nitrogen, C-Reactive protein, TIMP1, TIMP2, MMP2 and MMP9 were quantified; each patient was followed until death or up to 30 days. A descriptive analysis was performed by calculating the mean and the 95% confidence interval for continuous variables and proportions for categorical variables. A multivariate logistic regression model was constructed by the method of intentional selection of covariates with mortality at 30 days as dependent variable and all the other variables as predictors. RESULTS: Of the 563 patients, 68 patients (12.1%) died within the first 30 days of hospitalization in the ICU. The mean values for TIMP1, TIMP2 and MMP2 were lower in survivors, MMP9 was higher in survivors. Multivariate logistic regression showed that age, SOFA and Charlson scores, along with TIMP1 concentration, were statistically associated with mortality at 30 days of septic patients; serum MMP9 was not statistically associated with mortality of patients, but was a confounder of the TIMP1 variable. CONCLUSION: It could be argued that plasma levels of TIMP1 should be considered as a promising prognostic biomarker in the setting of sepsis. Additionally, this study, like other studies with large numbers of septic patients does not support the predictive value of TIMP1 / MMP9.

Doxycycline and Benznidazole Reduce the Profile of Th1, Th2, and Th17 Chemokines and Chemokine Receptors in Cardiac Tissue from Chronic Trypanosoma cruzi-Infected Dogs.

Chemokines (CKs) and chemokine receptors (CKR) promote leukocyte recruitment into cardiac tissue infected by the Trypanosoma cruzi. This study investigated the long-term treatment with subantimicrobial doses of doxycycline (Dox) in association, or not, with benznidazole (Bz) on the expression of CK and CKR in cardiac tissue. Thirty mongrel dogs were infected, or not, with the Berenice-78 strain of T. cruzi and grouped according their treatments: (i) two months after infection, Dox (50 mg/kg) 2x/day for 12 months; (ii) nine months after infection, Bz (3,5 mg/kg) 2x/day for 60 days; (iii) Dox + Bz; and (iv) vehicle. After 14 months of infection, hearts were excised and processed for qPCR analysis of Th1 (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL11), Th2 (CCL1, CCL17, CCL24, and CCL26), Th17 (CCL20) CKs, Th1 (CCR5, CCR6, and CXCR3), and Th2/Th17 (CCR3, CCR4, and CCR8) CKR, as well as IL-17. T. cruzi infection increases CCL1, CCL2, CCL4, CCL5, CCL17, CXCL10, and CCR5 expression in the heart. Dox, Bz, or Dox + Bz treatments cause a reversal of CK and CKR and reduce the expression of CCL20, IL-17, CCR6, and CXCR3. Our data reveal an immune modulatory effect of Dox with Bz, during the chronic phase of infection suggesting a promising therapy for cardiac protection.

Matrix metalloproteinase inhibitors prevent sepsis-induced refractoriness to vasoconstrictors in the cecal ligation and puncture model in rats.

Previous studies have shown that the loss of contractility in aortas from lipopolysaccharide (LPS)-treated rats is related to intracellular activation of matrix metalloproteinase (MMPs). However, the role of MMPs in the vascular refractoriness to vasoconstrictors has not been investigated in a model of polymicrobial sepsis. We evaluated the effects of the oral administration of the MMP inhibitors doxycycline or ONO-4817 in the in vitro vascular reactivity of aortic rings from rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Both doxycycline and ONO-4817 did not change vascular responses in sham-operated rats, but fully prevented hyporeactivity to KCl, phenylephrine and angiotensin II in vessels from CLP rats. This protective effect was not associated with changes in hematological parameters or blood nitrate and nitrite. The refractoriness to contractile agents was accompanied by enhanced activity of MMP-2 in aorta from CLP rats, which was abrogated by MMP inhibitors. CLP-induced sepsis did not impair the levels of MMP-2 in aorta, but significantly reduced calponin-1, a regulatory protein of vascular contraction. In addition, augmented levels of TIMP-1 were found in vessels from CLP rats. All these differences were prevented by either doxycycline or ONO-4817. Our study shows, for the first time in the CLP rat model of sepsis, that the vascular refractoriness to different contractile agents induced by polymicrobial sepsis is associated with increased activity of MMP-2 and reduced amounts of calponin-1 in the aorta. These findings reinforce the importance of the enhanced activity of MMPs for vascular failure in septic shock.

Morus nigra leaf extract improves glycemic response and redox profile in the liver of diabetic rats.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia and alterations in the carbohydrate, lipid, and protein metabolism. DM is associated with increased oxidative stress and pancreatic beta cell damage, which impair the production of insulin and the maintenance of normoglycemia. Inhibiting oxidative damage and controlling hyperglycemia are two important strategies for the prevention of diabetes. The pulp and leaf extracts of mulberry (Morus nigra L.) have abundant total phenolics and flavonoids, and its antioxidant potential may be an important factor for modulating oxidative stress induced by diabetes. In this study, DM was induced by intraperitoneal injection of alloxan monohydrate (135 mg kg(-1)). Female Fischer rats were divided into four groups: control, diabetic, diabetic pulp, and diabetic leaf extract. Animals in the diabetic pulp and diabetic leaf extract groups were treated for 30 days with M. nigra L. pulp or leaf extracts, respectively. At the end of treatment, animals were euthanized and, liver and blood samples were collected for analysis of biochemical and metabolic parameters. Our study demonstrated that treatment of diabetic rats with leaf extracts decreased the superoxide dismutase (SOD)/catalase (CAT) ratio and carbonylated protein levels by reducing oxidative stress. Moreover, the leaf extract of M. nigra L. decreased the matrix metalloproteinase (MMP)-2 activity, increased insulinemia, and alleviated hyperglycemia-induced diabetes. In conclusion, our study found that the leaf extract of M. nigra L. improved oxidative stress and complications in diabetic rats, suggesting the utility of this herbal remedy in the prevention and treatment of DM.

Smoothelin-B is not a target of matrix metalloproteinase (MMP)-2 in the vasculature of endotoxemic rats.

Smoothelin-B (SMTL-B) and calponin-1 are important regulators of vascular contraction. SMTL-B contains a calponin-homology domain and is structurally similar to cardiac troponin T. As calponin-1 and troponin T are proteolyzed by intracellular matrix metalloproteinase (MMP)-2 in oxidative stress injury, we hypothesized that SMTL-B is also cleaved by MMP-2 and contributes to lipopolysaccharide (LPS)-induced vascular hypocontractility. Rats received ONO-4817 (an MMP inhibitor) or its vehicle, 2 h prior to being administered lipopolysaccharide (LPS). LPS-induced aorta hypocontractility to potassium chloride or phenylephrine, and reduction of calponin-1 levels, were abolished by ONO-4817 at 6 but not 3 h after LPS. However, the level of SMTL-B was unaltered in LPS aortas and further unaffected by ONO-4817. Despite the importance of SMTL-B in vascular tone, it is not a target of MMP-2 in LPS-induced hypocontractility.

Matrix metalloproteinase inhibition attenuates right ventricular dysfunction and improves responses to dobutamine during acute pulmonary thromboembolism.

Activated matrix metalloproteinases (MMPs) cause cardiomyocyte injury during acute pulmonary thromboembolism (APT). However, the functional consequences of this alteration are not known. We examined whether doxycycline (a MMP inhibitor) improves right ventricle function and the cardiac responses to dobutamine during APT. APT was induced with autologous blood clots (350 mg/kg) in anaesthetized male lambs pre-treated with doxycycline (Doxy, 10 mg/kg/day, intravenously) or saline. Non-embolized control lambs received doxycycline pre-treatment or saline. The responses to intravenous dobutamine (Dob, 1, 5, 10 µg/kg/min.) or saline infusions at 30 and 120 min. after APT induction were evaluated by echocardiography. APT increased mean pulmonary artery pressure and pulmonary vascular resistance index by ~185%. Doxycycline partially prevented APT-induced pulmonary hypertension (P < 0.05). RV diameter increased in the APT group (from 10.7 ± 0.8 to 18.3 ± 1.6 mm, P < 0.05), but not in the Doxy+APT group (from 13.3 ± 0.9 to 14.4 ± 1.0 mm, P > 0.05). RV dysfunction on stress echocardiography was observed in embolized lambs (APT+Dob group) but not in embolized animals pre-treated with doxycycline (Doxy+APT+Dob). APT increased MMP-9 activity, oxidative stress and gelatinolytic activity in the RV. Although doxycycline had no effects on RV MMP-9 activity, it prevented the increases in RV oxidative stress and gelatinolytic activity (P < 0.05). APT increased serum cardiac troponin I concentrations (P < 0.05), doxycycline partially prevented this alteration (P < 0.05). We found evidence to support that doxycycline prevents RV dysfunction and improves the cardiac responses to dobutamine during APT.

Increased activities of cardiac matrix metalloproteinases matrix metalloproteinase (MMP)-2 and MMP-9 are associated with mortality during the acute phase of experimental Trypanosoma cruzi infection.

The strong inflammatory reaction that occurs in the heart during the acute phase of Trypanosoma cruzi infection is modulated by cytokines and chemokines produced by leukocytes and cardiomyocytes. Matrix metalloproteinases (MMPs) have recently emerged as modulators of cardiovascular inflammation. In the present study we investigated the role of MMP-2 and MMP-9 in T. cruzi-induced myocarditis, by use of immunohistochemical analysis, gelatin zymography, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction to analyze the cardiac tissues of T. cruzi-infected C57BL/6 mice. Increased transcripts levels, immunoreactivity, and enzymatic activity for MMP-2 and MMP-9 were observed by day 14 after infection. Mice treated with an MMP inhibitor showed significantly decreased heart inflammation, delayed peak in parasitemia, and improved survival rates, compared with the control group. Reduced levels of cardiac tumor necrosis factor-alpha, interferon-gamma, serum nitrite, and serum nitrate were also observed in the treated group. These results suggest an important role for MMPs in the induction of T. cruzi-induced acute myocarditis.

Patients' recollections of stressful experiences while receiving prolonged mechanical ventilation in an intensive care unit.

OBJECTIVE: To describe stressful experiences of adult patients who received mechanical ventilation for > or =48 hrs in an intensive care unit. DESIGN: Prospective cohort study. SETTING: Four intensive care units within an East Coast tertiary-care university medical center. PATIENTS: Patients were 150 adult intensive care unit patients receiving mechanical ventilation for > or =48 hrs. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: As part of a study of the long-term outcomes of adult patients requiring prolonged mechanical ventilation, we used a 32-item questionnaire to collect data on patients' stressful experiences, both psychological (e.g., fearfulness, anxiety) and physical (e.g., pain, difficulty breathing), associated with the mechanical ventilation endotracheal tube and with being in an intensive care unit. Of 554 patients who met study criteria and survived prolonged mechanical ventilation, 150 consented and were oriented to person, place, and situation. Two thirds of these patients remembered the endotracheal tube and/or being in an intensive care unit. The median numbers of endotracheal tube and intensive care unit experiences remembered were 3 (of 7) and 9 (of 22), respectively. If a patient remembered an experience in the questionnaire, it was likely to be moderately to extremely bothersome. Some of the items that many patients found to be moderately to extremely bothersome were pain, fear, anxiety, lack of sleep, feeling tense, inability to speak/communicate, lack of control, nightmares, and loneliness. Stressful experiences associated with the endotracheal tube were strongly associated with subjects' experiencing spells of terror, feeling nervous when left alone, and poor sleeping patterns. CONCLUSIONS: Subjects were more likely to remember experiences that were moderately to extremely bothersome. This might be because the more bothersome experiences were easier to recall or because most of these experiences are common and significant stressors to many of these patients. In either case, these data indicate that these patients are subject to numerous stressful experiences, which many find quite bothersome. This suggests the potential for improved symptom management, which could contribute to a less stressful intensive care unit stay and improved patient outcomes.