The role of IL-10 in immune responses against Pseudomonas aeruginosa during acute lung infection.

Pseudomonas aeruginosa is considered an opportunistic pathogen of great clinical importance. The clearance of this bacterium occurs through recognition of the pathogen by innate immune system receptors, leading to a lung inflammatory response. However, this response must be controlled via immunoregulatory pathways. In this study, we evaluate the role of endogenous murine IL-10 after acute infection with the virulent strain P. aeruginosa PA14. To assess the role of IL-10, intratracheal infection with the PA14 strain was performed in C57BL/6 or IL-10 KO mice. The PA14 strain was recovered in both types of animals, although IL-10 KO mice presented a higher number of viable bacteria in the lung when compared to the C57BL/6 group. Histopathological and stereological analyses showed that IL-10 KO mice had higher tissue damage and inflammatory infiltrate when compared to control animals. The activity of MMP-9 but not MMP-2, as well as IL-6 and TNF-α expression, were augmented in the lungs of infected animals and was much more evident in IL-10 KO animals when compared to the other analyzed groups. This work indicates that endogenous IL-10 control P. aeruginosa infection, the expression of pro-inflammatory genes, MMP-9 activity and histopathological processes of the infectious process in question.

Inducible nitric oxide synthase (iNOS) mediates ethanol-induced redox imbalance and upregulation of inflammatory cytokines in the kidney.

Overexpression of the inducible isoform of the enzyme nitric oxide synthase (iNOS) has been associated to pathological processes in the kidney. Ethanol consumption induces the renal expression of iNOS; however, the contribution of this enzyme to the deleterious effects of ethanol in the kidney remains elusive. We examined whether iNOS plays a role in the renal dysfunction and oxidative stress induced by ethanol consumption. With this purpose, male C57BL/6 wild-type (WT) or iNOS-deficient (iNOS-/-) mice were treated with ethanol (20% v/v) for 10 weeks. Treatment with ethanol increased the expression of Nox4 as well as the concentration of thiobarbituric acid reactive substances and the levels of tumor necrosis factor α in the renal cortex of WT but not iNOS-/- mice. Augmented serum levels of creatinine and increased systolic blood pressure were found in WT and iNOS-/- mice treated with ethanol. WT mice treated with ethanol showed increased production of reactive oxygen species and myeloperoxidase activity, but these responses were attenuated in iNOS-/- mice. We concluded that iNOS played a role in ethanol-induced oxidative stress and pro-inflammatory cytokine production in the kidney. These are mechanisms that may contribute to the renal toxicity induced by ethanol.

An intracellular matrix metalloproteinase-2 isoform induces tubular regulated necrosis: implications for acute kidney injury.

Acute kidney injury (AKI) causes severe morbidity, mortality, and chronic kidney disease (CKD). Mortality is particularly marked in the elderly and with preexisting CKD. Oxidative stress is a common theme in models of AKI induced by ischemia-reperfusion (I-R) injury. We recently characterized an intracellular isoform of matrix metalloproteinase-2 (MMP-2) induced by oxidative stress-mediated activation of an alternate promoter in the first intron of the MMP-2 gene. This generates an NH2-terminal truncated MMP-2 (NTT-MMP-2) isoform that is intracellular and associated with mitochondria. The NTT-MMP-2 isoform is expressed in kidneys of 14-mo-old mice and in a mouse model of coronary atherosclerosis and heart failure with CKD. We recently determined that NTT-MMP-2 is induced in human renal transplants with delayed graft function and correlated with tubular cell necrosis. To determine mechanism(s) of action, we generated proximal tubule cell-specific NTT-MMP-2 transgenic mice. Although morphologically normal at the light microscopic level at 4 mo, ultrastructural studies revealed foci of tubular epithelial cell necrosis, the mitochondrial permeability transition, and mitophagy. To determine whether NTT-MMP-2 expression enhances sensitivity to I-R injury, we performed unilateral I-R to induce mild tubular injury in wild-type mice. In contrast, expression of the NTT-MMP-2 isoform resulted in a dramatic increase in tubular cell necrosis, inflammation, and fibrosis. NTT-MMP-2 mice had enhanced expression of innate immunity genes and release of danger-associated molecular pattern molecules. We conclude that NTT-MMP-2 "primes" the kidney to enhanced susceptibility to I-R injury via induction of mitochondrial dysfunction. NTT-MMP-2 may be a novel AKI treatment target.

Antioxidant effect of doxycycline decreases MMP activity and blood pressure in SHR.

Increased matrix metalloproteinase (MMP) levels are involved in vascular remodeling of hypertension. In this study, we hypothesized that doxycycline (a MMP inhibitor) could exert antioxidant effects, reverse establish vascular remodeling, and lower blood pressure in spontaneously hypertensive rats (SHR). SHR and Wistar-Kyoto rats received either doxycycline at 30 mg/kg/day by gavage or vehicle. Systolic blood pressure (SBP) was assessed weekly by tail cuff. After 5 weeks of treatment, morphologic changes in the aortic wall were studied in hematoxylin/eosin sections. MMP activity and expression were determined by in situ zymography using DQ gelatin and immunofluorescence for MMP-2. Dihydroethidium was used to evaluate aortic reactive oxygen species (ROS) production by fluorescence microscopy. Doxycycline reduced SBP by 25 mmHg. However, the antihypertensive effects were not associated with significant reversal of hypertension-induced vascular hypertrophy. SHR showed increased aortic MMP-2 levels which co-localized with higher aortic MMP activity and ROS levels, and all those biochemical alterations associated with hypertension were blunted by treatment with doxycycline. These results show that MMP inhibition with doxycycline in SHR with established hypertension resulted in antioxidant effects, lower gelatinolytic activity, and antihypertensive effects which were not associated with reversal of hypertension-induced vascular remodeling.

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.

Temporal changes in cardiac matrix metalloproteinase activity, oxidative stress, and TGF-ß in renovascular hypertension-induced cardiac hypertrophy.

Cardiovascular remodeling found in later phases of two-kidney, one-clip (2K1C) hypertension may involve key mechanisms particularly including MMP-2, oxidative stress, transforming growth factor-ß (TGF-ß), and inactivation of the endogenous MMP inhibitor, the tissue inhibitor of MMP (TIMP)-4. We examined whether temporal cardiac remodeling resulting from 2K1C hypertension occurs concomitantly with alterations in cardiac collagen, MMP activity, MMP-2, TIMP-4, TGF-ß, and reactive oxygen species (ROS) levels during the development of 2K1C hypertension. Sham-operated and 2K1C hypertensive rats were studied after 15, 30, and 75 days of hypertension. Systolic blood pressure was monitored weekly. Left ventricle (LV) morphometry and fibrosis were evaluated in hematoxylin/eosin and picrosirius red-stained sections, respectively. Cardiac MMP-2 levels/activity was determined by gelatin zymography, immunofluorescence, and in situ zymography. TIMP-4 levels were determined by western blotting. Cardiac TGF-ß levels were evaluated by immunofluorescence and ROS levels were evaluated with a dihydroethidium probe. 2K1C hypertension induced LV hypertrophy associated with augmented gelatinolytic activity at an early phase of hypertension and further increased after 75 days of hypertension. These alterations were associated with increased cardiac MMP-2, TGF-ß, and ROS in hypertensive rats. Higher TIMP-4 levels were found in hypertensive rats only after 75 days after surgery. Our findings show that increased MMP-2 activity is associated with concomitant development of LV hypertrophy and increased TGF-ß and ROS levels.

Three Generation ß-Blockers for Atrial Fibrillation Treatment.

The efficiency of blood flowing from the heart depends on its electrical properties. Myocardial electrical activity is associated with generating cardiac action potentials in isolated myocardial cells and their coordinated propagation, which are mediated by gap junctions. Atrial fibrillation (AF) is a common cardiac arrhythmia which causes an aggressive disturbance in cardiac electromechanical function. Moreover, AF increases the risk of stroke and mortality and is a major cause of death. The mechanisms underlying AF involve electrophysiological changes in ion channel expression and function. ß-blockers may be useful in patients with chronic AF or in preventing postoperative AF in subjects undergoing coronary artery bypass grafting (CABG) or other types of surgery. The reduction in heart rate induced by ß1-adrenergic receptor antagonists may be associated with the beneficial effect of this drug class. Second generation beta-blockers may be considered superior to the first generation due to their selectivity to the ß1 receptor as well as avoiding pulmonary or metabolic adverse effects. Third generation beta-blockers may be considered a great option for their vasodilation and antioxidant properties. There is also a new ß-blocker, named landilol that also results on reduced risk of post operative AF without adverse effects and its use has been increasing in clinical trials.

Doxycycline ameliorates 2K-1C hypertension-induced vascular dysfunction in rats by attenuating oxidative stress and improving nitric oxide bioavailability.

Vascular dysfunction associated with two-kidney, one-clip (2K-1C) hypertension may result from both altered matrix metalloproteinase (MMP) activity and higher concentrations of reactive oxygen species (ROS). Doxycycline is considering the most potent MMP inhibitor of tetracyclines and attenuates 2K-1C hypertension-induced high blood pressure and chronic vascular remodeling. Doxycycline might also act as a ROS scavenger and this may contribute to the amelioration of some cardiovascular diseases associated with increased concentrations of ROS. We hypothesized that in addition to its MMP inhibitory effect, doxycycline attenuates oxidative stress and improves nitric oxide (NO) bioavailability in 2K-1C hypertension, thus improving hypertension-induced arterial endothelial dysfunction. Sham operated or 2K-1C hypertensive rats were treated with doxycycline 30 mg/kg/day (or vehicle). After 8 weeks of treatment, aortic rings were isolated to assess endothelium dependent vasorelaxation to A23187. Arterial and systemic levels of ROS were respectively measured using dihydroethidine (DHE) and thiobarbituric acid reactive substances (TBARS). Neutrophils-derived ROS were tested in vitro using the fluoroprobe Carboxy-H(2)DCFDA and human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). NO levels were assessed in rat aortic endothelial cells by confocal microscopy. Aortic MMP activity was determined by in situ zymography. Doxycycline attenuated 2K-1C hypertension (169 ± 17.3 versus 209 ± 10.9mm Hg in hypertensive controls, p<0.05) and protected against hypertension-induced reduction in endothelium-dependent vasorelaxation to A23187 (p<0.05). Doxycycline also decreased hypertension-induced oxidative stress (p<0.05), higher MMP activity (p<0.01) and improved NO levels in aortic endothelial cells (p<0.01). Therefore, doxycycline ameliorates 2K-1C hypertension-induced endothelial dysfunction in aortas by inhibiting oxidative stress generation and improving NO bioavailability, in addition to its inhibitory effects on MMP activity.

Tamoxifen and its metabolites cause acute vasorelaxation of aortic rings by inducing vasodilator prostanoid synthesis.

The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.

Epigenetic Regulation of the N-Terminal Truncated Isoform of Matrix Metalloproteinase-2 (NTT-MMP-2) and Its Presence in Renal and Cardiac Diseases.

Several clinical and experimental studies have documented a compelling and critical role for the full-length matrix metalloproteinase-2 (FL-MMP-2) in ischemic renal injury, progressive renal fibrosis, and diabetic nephropathy. A novel N-terminal truncated isoform of MMP-2 (NTT-MMP-2) was recently discovered, which is induced by hypoxia and oxidative stress by the activation of a latent promoter located in the first intron of the MMP2 gene. This NTT-MMP-2 isoform is enzymatically active but remains intracellular in or near the mitochondria. In this perspective article, we first present the findings about the discovery of the NTT-MMP-2 isoform, and its functional and structural differences as compared with the FL-MMP-2 isoform. Based on publicly available epigenomics data from the Encyclopedia of DNA Elements (ENCODE) project, we provide insights into the epigenetic regulation of the latent promoter located in the first intron of the MMP2 gene, which support the activation of the NTT-MMP-2 isoform. We then focus on its functional assessment by covering the alterations found in the kidney of transgenic mice expressing the NTT-MMP-2 isoform. Next, we highlight recent findings regarding the presence of the NTT-MMP-2 isoform in renal dysfunction, in kidney and cardiac diseases, including damage observed in aging, acute ischemia-reperfusion injury (IRI), chronic kidney disease, diabetic nephropathy, and human renal transplants with delayed graft function. Finally, we briefly discuss how our insights may guide further experimental and clinical studies that are needed to elucidate the underlying mechanisms and the role of the NTT-MMP-2 isoform in renal dysfunction, which may help to establish it as a potential therapeutic target in kidney diseases.

Three Generations of ß-blockers: History, Class Differences and Clinical Applicability.

BACKGROUND: Beta-adrenergic receptors are expressed in cardiomyocytes and activated by either noradrenaline released from sympathetic synapses or circulating catecholamines. Their corresponding receptors have three subtypes, namely, ß1, ß2 and ß3, which are members of the G protein-coupled receptors (GPCRs) family. Activation of ß1-adrenergic receptors causes various physiological reactions including cardiac contraction and renin secretion from juxtaglomerular cells of the kidney. Antagonists of ß-adrenergic receptors, known as ß-blockers, have been used effectively for over four decades and have beneficial effects in the treatment of cardiovascular diseases. There are three generations of ß-blockers according to their pharmacological properties. Firstgeneration ß-blockers are non-selective, blocking both ß1- and ß2-receptors; second-generation ß- blockers are more cardioselective in that they are more selective for ß1-receptors; and thirdgeneration ß-blockers are highly selective drugs for ß1-receptors. The latter also display vasodilator actions by blocking α1-adrenoreceptors and activating ß3-adrenergic receptors. In addition, thirdgeneration ß-blockers exhibit angiogenic, antioxidant, anti-proliferative, anti-hypertrophic and antiapoptotic activities among other effects that are still under investigation. CONCLUSION: The objective of this review is to describe the evolution observed during the development of the three distinctive generations, thereby highlighting the advantages of third-generation ß- blockers over the other two drug classes.

Chronic ethanol consumption increases vascular oxidative stress and the mortality induced by sub-lethal sepsis: Potential role of iNOS.

We hypothesized that long-term ethanol consumption would increase the mortality and aggravate the deleterious effects of sub-lethal cecal ligation and puncture (SL-CLP) in the vasculature by inducing the expression of inducible nitric oxide (NO) synthase (iNOS). Male C57BL/6J wild-type (WT) or iNOS-deficient mice (iNOS-/-) were treated with ethanol (20% v/v) for 12 weeks and then subjected to SL-CLP. Mice were killed 24 h post-operatively or followed six days for survival. Septic ethanol-treated mice showed a higher mortality than septic WT mice. However, septic iNOS-deficient mice treated with ethanol showed a decreased mortality rate when compared to ethanol-treated WT mice. Ethanol and SL-CLP augmented superoxide anion (O2-) generation in the mesenteric arterial bed (MAB) of both WT and iNOS-deficient mice. Treatment with ethanol and SL-CLP enhanced lipoperoxidation in the MAB of WT, but not iNOS-deficient mice. SL-CLP enhanced nitrate/nitrite (NOx) concentrations in the MAB of WT, but not iNOS-deficient mice. Both, ethanol and SL-CLP increased TNF-α and IL-6 levels in the MAB. Treatment with ethanol as well as SL-CLP up-regulated the expression of iNOS in the MAB of WT mice. The major finding of our study is that chronic ethanol consumption increases the mortality induced by SL-CLP and that iNOS plays a role in such response. Although ethanol led to vascular alterations, it did not aggravate the vascular injury induced by SL-CLP. Finally, iNOS mediated the increase in oxidative stress and pro-inflammatory cytokines induced by SL-CLP in the vasculature.

Quercetin decreases the activity of matrix metalloproteinase-2 and ameliorates vascular remodeling in renovascular hypertension.

BACKGROUND AND AIMS: Increased activity of matrix metalloproteinase (MMP)-2 is observed in aortas of different models of hypertension, and its activation is directly mediated by oxidative stress. As quercetin is an important flavonoid with significant antioxidant effects, the hypothesis here is that quercetin will reduce increased MMP-2 activity by decreasing oxidative stress in aortas of hypertensive rats and then ameliorate hypertension-induced vascular remodeling. METHODS: Male two-kidney one-clip (2K1C) hypertensive Wistar rats and controls were treated with quercetin (10 mg/kg/day) or its vehicle for three weeks by gavage. Rats were then analyzed at five weeks of hypertension. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography. Aortas were used to determine MMP activity by in situ zymography and reactive oxygen species (ROS) levels by dihydroethidium. Western blot was performed to detect focal adhesion kinase (FAK) and phosphorylated-FAK levels. RESULTS: SBP was increased in 2K1C rats and only a borderline reduction in SBP was observed after treating 2K1C rats with quercetin. Cross-sectional area and the number of vascular smooth muscle cells were significantly increased in aortas of hypertensive rats, and quercetin reduced them. Quercetin reduced ROS levels in aortas of 2K1C rats and the increased activity of gelatinases in situ. However, quercetin did not affect the levels of tissue inhibitor of MMP (TIMP)-2 and did not interfere with FAK and p-FAK levels in aortas of hypertensive rats. Furthermore, different concentrations of quercetin did not directly reduce the activity of human recombinant MMP-2 in vitro. CONCLUSIONS: Quercetin reduces hypertension-induced vascular remodeling, oxidative stress and MMP-2 activity in aortas.

Direct renin inhibition is not enough to prevent reactive oxygen species generation and vascular dysfunction in renovascular hypertension.

Renin-angiotensin system activation promotes oxidative stress and endothelial dysfunction. However, no previous study has examined the effects of the renin inhibitor aliskiren, either alone or combined with angiotensin II type 1 antagonists on alterations induced by two-kidney, one-clip (2K1C) hypertension. We compared the vascular effects of aliskiren (50mg/kg/day), losartan (10mg/kg/day), or both by gavage for 4 weeks in 2K1C and control rats. Treatment with losartan, aliskiren, or both exerted similar antihypertensive effects. Aliskiren lowered plasma Ang I concentrations in sham rats and in hypertensive rats treated with aliskiren or with both drugs. Aliskiren alone or combined with losartan decreased plasma angiotensin II concentrations measured by high performance liquid chromatography, whereas losartan alone had no effects. In contrast, losartan alone or combined with aliskiren abolished hypertension-induced increases in aortic angiotensin II concentrations, whereas aliskiren alone exerted no such effects. While hypertension enhanced aortic oxidative stress assessed by dihydroethidium fluorescence and by lucigenin chemiluminescence, losartan alone or combined with aliskiren, but not aliskiren alone, abolished this alteration. Hypertension impaired aortic relaxation induced by acetylcholine, and losartan alone or combined with aliskiren, but not aliskiren alone, reversed this alteration. Losartan alone or combined with aliskiren, but not aliskiren alone, increased plasma nitrite concentrations in 2K1C rats. These findings show that antihypertensive effects of aliskiren do not prevent hypertension-induced vascular oxidative stress and endothelial dysfunction. These findings contrast those found with losartan and suggest that renin inhibition is not enough to prevent hypertension-induced impaired redox biology and vascular dysfunction.

Data on the effects of losartan on protein expression, vascular reactivity and antioxidant capacity in the aorta of ethanol-treated rats.

We describe the effects of losartan, a selective AT1 receptor antagonist on the alterations induced by treatment with ethanol in the rat aorta. The data shown here are related to the article entitled "Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress" (P. Passaglia, C.S. Ceron, A.S. Mecawi, J. Antunes-Rodrigues, E.B. Coelho, C.R. Tirapelli, 2015) [1]. Here we include new data on the protective effect of losartan against ethanol-induced oxidative stress. Male Wistar rats treated for 2 weeks with ethanol (20%, vol./vol.) exhibited increased aortic production of reactive oxygen species (ROS) and losartan (10 mg/kg/day; p.o. gavage) prevented this response. Ethanol did not alter the expression of eNOS in the rat aorta. Losartan prevented ethanol-induced increase in the aortic expression of nNOS. Neither ethanol nor losartan affected superoxide dismutase (SOD) or catalase (CAT) activities in the rat aorta. Treatment with ethanol increased the contraction induced by phenylephrine in both endothelium-intact and endothelium-denuded aortas and these responses were prevented by losartan. Conversely, neither ethanol nor losartan affected the endothelium-dependent relaxation induced by acetylcholine.