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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

Delayed graft function (DGF) is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2), as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2) that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC), in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are associated with tubular injury in DGF and offer novel therapeutic targets for the prevention of this disorder.

Atorvastatin and sildenafil decrease vascular TGF-ß levels and MMP-2 activity and ameliorate arterial remodeling in a model of renovascular hypertension.

Imbalanced matrix metalloproteinase (MMP)-2 activity and transforming growth factor expression (TGF-ß) are involved in vascular remodeling of hypertension. Atorvastatin and sildenafil exert antioxidant and pleiotropic effects that may result in cardiovascular protection. We hypothesized that atorvastatin and sildenafil alone or in association exert antiproliferative effects by down-regulating MMP-2 and TGF-ß, thus reducing the vascular hypertrophy induced by two kidney, one clip (2K1C) hypertension. Sham and 2K1C rats were treated with oral atorvastatin 50 mg/kg, sildenafil 45 mg/kg, or both, daily for 8 weeks. Blood pressure was monitored weekly. Morphologic changes in the aortas were studied. TGF-ß levels were determined by immunofluorescence. MMP-2 activity and expression were determined by in situ zymography, gel zymography, Western blotting, and immunofluorescence. The effects of both drugs on proliferative responses of aortic smooth muscle cells to PDGF and on on MMP-2 activity in vitro were determined. Atorvastatin, sildenafil, or both drugs exerted antiproliferative effects in vitro. All treatments attenuated 2K1C-induced hypertension and prevented the increases in the aortic cross-sectional area and media/lumen ratio in 2K1C rats. Aortas from 2K1C rats showed higher collagen deposition, TGF-ß levels and MMP-2 activity and expression when compared with Sham-operated animals. Treatment with atorvastatin and/or sildenafil was associated with attenuation of 2K1C hypertension-induced increases in these pro-fibrotic factors. However, these drugs had no in vitro effects on hr-MMP-2 activity. Atorvastatin and sildenafil was associated with decreased vascular TGF-ß levels and MMP-2 activity in renovascular hypertensive rats, thus ameliorating the vascular remodeling. These novel pleiotropic effects of both drugs may translate into protective effects in patients.

Gastric S-nitrosothiol formation drives the antihypertensive effects of oral sodium nitrite and nitrate in a rat model of renovascular hypertension.

Many effects of nitrite and nitrate are attributed to increased circulating concentrations of nitrite, ultimately converted into nitric oxide (NO(•)) in the circulation or in tissues by mechanisms associated with nitrite reductase activity. However, nitrite generates NO(•) , nitrous anhydride, and other nitrosating species at low pH, and these reactions promote S-nitrosothiol formation when nitrites are in the stomach. We hypothesized that the antihypertensive effects of orally administered nitrite or nitrate involve the formation of S-nitrosothiols, and that those effects depend on gastric pH. The chronic effects of oral nitrite or nitrate were studied in two-kidney, one-clip (2K1C) hypertensive rats treated with omeprazole (or vehicle). Oral nitrite lowered blood pressure and increased plasma S-nitrosothiol concentrations independently of circulating nitrite levels. Increasing gastric pH with omeprazole did not affect the increases in plasma nitrite and nitrate levels found after treatment with nitrite. However, treatment with omeprazole severely attenuated the increases in plasma S-nitrosothiol concentrations and completely blunted the antihypertensive effects of nitrite. Confirming these findings, very similar results were found with oral nitrate. To further confirm the role of gastric S-nitrosothiol formation, we studied the effects of oral nitrite in hypertensive rats treated with the glutathione synthase inhibitor buthionine sulfoximine (BSO) to induce partial thiol depletion. BSO treatment attenuated the increases in S-nitrosothiol concentrations and antihypertensive effects of oral nitrite. These data show that gastric S-nitrosothiol formation drives the antihypertensive effects of oral nitrite or nitrate and has major implications, particularly to patients taking proton pump inhibitors.

Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress.

OBJECTIVES: We hypothesized that chronic ethanol intake enhances vascular oxidative stress and induces hypertension through renin-angiotensin system (RAS) activation. METHODS AND RESULTS: Male Wistar rats were treated with ethanol (20% v/v). The increase in blood pressure induced by ethanol was prevented by losartan (10mg/kg/day; p.o. gavage), a selective AT1 receptor antagonist. Chronic ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels and serum aldosterone levels. No differences on plasma osmolality and sodium or potassium levels were detected after treatment with ethanol. Ethanol consumption did not alter ACE activity, as well as the levels of ANG I and ANG II in the rat aorta or mesenteric arterial bed (MAB). Ethanol induced systemic and vascular oxidative stress (aorta and MAB) and these effects were prevented by losartan. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was prevented by losartan. Ethanol intake did not alter protein expression of ACE, AT1 or AT2 receptors in both aorta and MAB. Aortas from ethanol-treated rats displayed decreased ERK1/2 phosphorylation and increased protein expression of SAPK/JNK. These responses were prevented by losartan. MAB from ethanol-treated rats displayed reduced phosphorylation of p38MAPK and ERK1/2 and losartan did not prevent these responses. CONCLUSIONS: Our study provides novel evidence that chronic ethanol intake increases blood pressure, induces vascular oxidative stress and decreases nitric oxide (NO) bioavailability through AT1-dependent mechanisms.

The Nuclear Factor kappaB Inhibitor Pyrrolidine Dithiocarbamate Prevents Cardiac Remodelling and Matrix Metalloproteinase-2 Up-Regulation in Renovascular Hypertension.

Imbalanced matrix metalloproteinase (MMP) activity is involved in hypertensive cardiac hypertrophy. Pharmacological inhibition of nuclear factor kappaB (NF-кB) with pyrrolidine dithiocarbamate (PDTC) can prevent MMP up-regulation. We suggested that treatment with PDTC could prevent 2-kidney, 1-clip (2K1C) hypertension-induced left ventricular remodelling. Sham-operated controls or 2K1C rats with hypertension received either vehicle or PDTC (100 mg/kg/day) by gavage for 8 weeks. Systolic blood pressure was monitored every week. Histological assessment of left ventricles was carried out with haematoxylin/eosin sections, and fibrosis was quantified in picrosirius red-stained sections. Oxidative stress was evaluated in heart samples with the dihydroethidium probe. Cardiac MMP activity was determined by in situ zymography, and cardiac MMP-2 was assessed by immunofluorescence. 2K1C surgery significantly increased systolic blood pressure in the 2K1C vehicle. PDTC exerted antihypertensive effects after 2 weeks of treatment. Histology revealed increased left ventricular and septum wall thickness associated with augmented myocyte diameter in hypertensive rats, which were reversed by treatment with PDTC. Hypertensive rats developed pronounced cardiac fibrosis with increased interstitial collagen area, increased cardiac reactive oxygen species levels, gelatinase activity and MMP-2 expression. PDTC treatment decreased these alterations. These findings show that PDTC modulates myocardial MMP-2 expression and ameliorates cardiac remodelling in renovascular hypertension. These results suggest that interfering with MMP expression at transcriptional level may be an interesting strategy in the therapy of organ damage associated with hypertension.