p-Aminobenzamidine attenuates cardiovascular dysfunctions in spontaneously hypertensive rats.

AIMS: Diminazene aceturate, a putative ACE2 activator, is susceptible to cleavage resulting in the formation of p-aminobenzamidine (PAB). This study aimed to investigate the effects of PAB in addressing cardiovascular dysfunctions in spontaneously hypertensive rats (SHR). MAIN METHODS: Acute effects of PAB on mean arterial pressure (MAP), heart rate (HR), and aortic (AVC) and mesenteric vascular conductance (MVC) were evaluated in anesthetized SHR. Isolated aortic rings and the Langendorff technique were used to investigate the acute and chronic effects of PAB in the artery and heart. Chronic treatment with PAB (1 mg/kg, gavage) was carried out for 60 days. During this period, systolic blood pressure (SBP) and HR were measured by tail-cuff plethysmography. After the treatment, the left ventricle was collected for histology analyses, western blotting, and ACE2 activity. KEY FINDINGS: Bolus infusion of PAB acutely reduced MAP and increased both AVC and MVC in SHR. Additionally, PAB induced coronary and aorta vasodilation in isolated organs from Wistar and SHR in an endothelial-dependent manner. The chronic PAB treatment in SHR significantly attenuated the increase of SBP and improved the aorta vasorelaxation induced by acetylcholine and bradykinin-induced coronary vasodilation. In addition, chronic treatment with PAB attenuated the cardiomyocyte hypertrophy and extracellular matrix deposition in hearts from SHR. PAB did not alter the protein expression of the AT1, AT2, Mas, ACE, ACE2, or ACE2 activity. SIGNIFICANCE: PAB induced beneficial effects on cardiovascular dysfunctions induced by hypertension, suggesting that this molecule could be used in the development of new drugs for the treatment of cardiovascular diseases.

Diminazene Aceturate, an angiotensin converting enzyme 2 (ACE2) activator, promotes cardioprotection in ischemia/reperfusion-induced cardiac injury.

This study aimed to investigate whether the Diminazene Aceturate (DIZE), an angiotensin-converting enzyme 2 (ACE2) activator, can revert cardiac dysfunction in ischemia reperfusion-induced (I/R) injury in animals and examine the mechanism underlying this effect. Wistar rats systemically received DIZE (1 mg/kg) for thirty days. Cardiac function in isolated rat hearts was evaluated using the Langendorff technique. After I/R, ventricular non-I/R and I/R samples were used to evaluate ATP levels. Mitochondrial function was assessed using cardiac permeabilized fibers and isolated cardiac mitochondria. Cardiac cellular electrophysiology was evaluated using the patch clamp technique. DIZE protected the heart after I/R from arrhythmia and cardiac dysfunction by preserving ATP levels, independently of any change in coronary flow and heart rate. DIZE improved mitochondrial function, increasing the capacity for generating ATP and reducing proton leak without changing the specific citrate synthase activity. The activation of the ACE2 remodeled cardiac electrical profiles, shortening the cardiac action potential duration at 90 % repolarization. Additionally, cardiomyocytes from DIZE-treated animals exhibited reduced sensibility to diazoxide (KATP agonist) and a higher KATP current compared to the controls. DIZE was able to improve mitochondrial function and modulate cardiac electrical variables with a cardio-protective profile, resulting in direct myocardial cell protection from I/R injury.

Hemodynamic phenotyping of transgenic rats with ubiquitous expression of an angiotensin-(1-7)-producing fusion protein.

Activation of the angiotensin (Ang)-converting enzyme (ACE) 2/Ang-(1-7)/MAS receptor pathway of the renin-angiotensin system (RAS) induces protective mechanisms in different diseases. Herein, we describe the cardiovascular phenotype of a new transgenic rat line (TG7371) that expresses an Ang-(1-7)-producing fusion protein. The transgene-specific mRNA and the corresponding protein were shown to be present in all evaluated tissues of TG7371 with the highest expression in aorta and brain. Plasma Ang-(1-7) levels, measured by radioimmunoassay (RIA) were similar to control Sprague-Dawley (SD) rats, however high Ang-(1-7) levels were found in the hypothalamus. TG7371 showed lower baseline mean arterial pressure (MAP), assessed in conscious or anesthetized rats by telemetry or short-term recordings, associated with increased plasma atrial natriuretic peptide (ANP) and higher urinary sodium concentration. Moreover, evaluation of regional blood flow and hemodynamic parameters with fluorescent microspheres showed a significant increase in blood flow in different tissues (kidneys, mesentery, muscle, spleen, brown fat, heart and skin), with a resulting decrease in total peripheral resistance (TPR). TG7371 rats, on the other hand, also presented increased cardiac and global sympathetic tone, increased plasma vasopressin (AVP) levels and decreased free water clearance. Altogether, our data show that expression of an Ang-(1-7)-producing fusion protein induced a hypotensive phenotype due to widespread vasodilation and consequent fall in peripheral resistance. This phenotype was associated with an increase in ANP together with an increase in AVP and sympathetic drive, which did not fully compensate the lower blood pressure (BP). Here we present the hemodynamic impact of long-term increase in tissue expression of an Ang-(1-7)-fusion protein and provide a new tool to investigate this peptide in different pathophysiological conditions.

Oral methylmercury intoxication aggravates cardiovascular risk factors and accelerates atherosclerosis lesion development in ApoE knockout and C57BL/6 mice.

Methylmercury (MeHg) intoxication is associated with hypertension, hypercholesterolemia, and atherosclerosis by mechanisms that are not yet fully understood. We investigated the effects of MeHg intoxication in atherosclerosis-prone (ApoE-KO) and resistant C57BL/6 mice. Mice were submitted to carotid stenosis surgery (to induce atherosclerosis faster) and received water or MeHg solution (20 mg/L) for 15 days. Tail plethysmography was performed before and after MeHg exposure. Food and MeHg solution intakes were monitored weekly. On the 15th day, mice were submitted to intravital fluorescence microscopy of mesenteric vasculature to observe in vivo leukocyte rolling and adhesion. Results showed that despite the high hair and liver Hg concentrations in the MeHg group, food and water (or MeHg solution) consumption and liver function marker levels were similar to those in controls. MeHg exposure increased total cholesterol, the atherogenic (non-HDL) fraction and systolic and diastolic blood pressure. MeHg exposure also induced inflammation, as seen by the increased rolling and adhered leukocytes in the mesenteric vasculature. Atherosclerosis lesions were more extensive in the aorta and carotid sites of MeHg-ApoE knockout mice. Surprisingly, MeHg exposure also induced atherosclerosis lesions in C57BL/6 mice, which are resistant to atherosclerosis formation. We concluded that MeHg intoxication might represent a risk for cardiovascular diseases since it accelerates atherogenesis by exacerbating several independent risk factors.

Angiotensin II triggers knee joint lesions in experimental osteoarthritis.

OBJECTIVES: This study aimed to evaluate the involvement of Angiotensin II (Ang II) in joint lesions associated with osteoarthritis (OA) in vitro and in vivo. METHODS: Chondrocyte cultures were obtained from knee joints of neonatal rats and stimulated with Ang II/MIA/ACE inhibitors. In vivo, rats treated or not with the ACE inhibitor captopril, received daily injections of Ang II or sodium monoiodoacetate (MIA) in knee joints for evaluation of cartilage, bone, and synovial lesions. RESULTS: Cultured chondrocytes expressed the mRNA for Ace, Agtr1, Agtr2, and Mas1. Stimulating cells with Ang II reduced chondrocyte viability and metabolism. Accordingly, in vivo Ang II injection into the knees of rats triggered hyperalgesia, joint edema, increased the number of leukocytes in the joint cavity, and induced cartilage lesions associated with OA alterations. In further experiments, Ang II synthesis was prevented with the ACE inhibitor Captopril in the context of MIA-induced OA. Ang II inhibition with captopril improved the OARSI score, induced chondroprotection, and reduced the leukocyte recruitment from synovium after MIA. Additionally, captopril prevented MIA-induced bone resorption, by decreasing the number of osteoclasts and increasing the expression of IL-10 in the bone. In vitro, inhibiting Ang II synthesis decreased MIA-induced chondrocyte death and increased Col2a1 transcription. CONCLUSION: Ang II induces chondrocyte death and joint tissue damages associated with OA and its modulation can be a therapeutic strategy in osteoarthritis.

New antiglaucomatous agent for the treatment of open angle glaucoma: Polymeric inserts for drug release and in vitro and in vivo study.

A benzamidine derivative from diminazene was tested for a novel activity: treatment of primary open-angle glaucoma. This drug was incorporated into mucoadhesive polymeric inserts prepared using chitosan (Chs) and chondroitin sulfate (CS). Of current interest is the mucoadhesion, which increases the contact time with the ocular surface, resulting in improved bioavailability; also, the inserts are made to act as a prolonged release system. In the present work the inserts were prepared by the solvent casting method using different polymeric proportions (30:70, 50:50, 75:25% w/w Chs:CS and 100% Chs). Thermal analysis and infrared spectroscopy both demonstrated physical dispersion of the active drug. The most promising was the 50:50% Chs:CS which demonstrated that it was not fragile and has an in vitro release profile of up to 180 minutes. In addition, it presented greater adhesion strength in relation to the other formulations. These physicochemical results corroborate the in vivo tests performed. In this sense, we also demonstrated that the treatment with the 50:50% insert can control the intraocular pressure (IOP) for at least 3 weeks and prevents damage to the retinal ganglion cells (RGCs) compared to the placebo insert. Thus, this indicates thus that the new drug is quite viable and promising in glaucoma treatment.

Use of chitosan as pharmaceutical excipient in ocular drug delivery systems: Sterilization and pharmacokinetics.

The use of chitosan as a pharmaceutical excipient in the ocular field is already established. Nevertheless, some aspects related to its ocular administration, such as sterilization and excipient's pharmacokinetics, remain unclear. So, in this study, we evaluated those two relevant aspects, related to chitosan administration in eye. We used chitosan-based ocular inserts (CI) as formulation model. CI were produced by solvent/casting method and sterilized by saturated steam. Sterilization was confirmed by direct inoculation of inserts in suitable microbiological growth media. Physicochemical characterization of inserts before and after sterilization was performed. Results suggested that, although steam sterilization changed the arrangement of the matrix, the heat and the humidity did not modify the structure of the main polymeric chain. Pharmacokinetics of CI radiolabeled with technetium-99m (99m Tc) was assessed by scintigraphic images and ex vivo biodistribution study, after ocular administration in male Wistar rats. Scintigraphic and images analysis and ex vivo biodistribution study showed that the insert remained mainly in the eye until 6 hr after administration and its degradation products began to migrate to the abdominal cavity after 18 hr. Together, these data represent an important step forward the manufacturing and the clinical application of CI in the ophthalmic field.

Diminazene aceturate (DIZE) has cellular and in vivo antiarrhythmic effects.

Diminazene aceturate (DIZE) is an anti-protozoan compound that has been previously reported to increase the activity of the angiotensin-converting enzyme 2 (ACE2) and thus increase Angiotensin-(1-7) production, leading to cardioprotection against post-myocardial infarction dysfunction and structural remodelling. Moreover, DIZE is able to ameliorate morpho-functional changes after myocardial infarction by enhancing ACE2 activity, thus increasing Angiotensin-(1-7) production (a benefic peptide of the renin-angiotensin system). However, despite the improvement in cardiac function/structure, little is known about DIZE effects on arrhythmia suppression, contraction/excitable aspects of the heart and importantly its mechanisms of action. Thus, our aim was to test the acute effect of DIZE cardioprotection at the specific level of potential antiarrhythmic effects and modulation in excitation-contraction coupling. For this, we performed in vitro and in vivo techniques for arrhythmia induction followed by an acute administration of DIZE. For the first time, we described that DIZE can reduce arrhythmias which is explained by modulation of cardiomyocyte contraction and excitability. Such effects were independent of Mas receptor and nitric oxide release. Development of a new DIZE-based approach to ameliorate myocardial contractile and electrophysiological dysfunction requires further investigation; however, DIZE may provide the basis for a future beneficial therapy to post-myocardial infarction patients.

Chitosan/hydroxyethyl cellulose inserts for sustained-release of dorzolamide for glaucoma treatment: In vitro and in vivo evaluation.

Eye drops containing hydrophilic drugs are commonly used to reduce intraocular pressure (IOP) in glaucoma patients, but compliance to the treatement is commonly reduced by frequent dosing and eventual systemic side effects. Sustained-release drug delivery systems, such as ocular inserts, can reduce dosing, limit systemic exposure, reduce side effects, and, then, improve patient adherence to therapy. Here, we developed and evaluated chitosan/hydroxyethyl cellulose-based ocular inserts for sustained release of dorzolamide, a hydrophilic drug. Dorzolamide inserts (DI) were produced by solvent/casting method and characterized by various physicochemical techniques. Pharmacokinetics studies were performed using scintigraphic images and ex vivo biodistribution. The effectiveness of inserts was tested in glaucomatous rats. Characterization studies showed that the drug strongly interacted with the polymeric matrix, but in vitro results showed that DI took only 3 h to release 75% of dorzolamide entraped. However, scintigraphic images and ex vivo biodistribution studies revealed that more than 50% of 99mTc-dorzolamide remained in the eye after 18 h of DI administration, while only about 30% of the drug remained in the eye after drops instilation. DI exerted significant hypotensive effect for two weeks, after single administration, while IOP values remained high in placebo and untreated groups. Eye drops were effective only during the treatment period. Only DI treatment prevented retinal ganglion cells death. Altogether, these findings evidenced the potential application of polymeric-based inserts for sustained release of dorzolamide in glaucoma management.

Efeitos anti-hipertensivos do aceturato de diminazeno: um ativador da enzima conversora de angiotensina 2 em ratos / Antihypertensive effects of diminazene aceturate: an activator of angiotensin-2 converting enzyme in rats

INTRODUÇÃO: O sistema renina-angiotensina-aldosterona desempenha importante papel cardiovascular. A enzima conversora de angiotensina 2 (ECA2) degrada a Angiotensina II em Angiotensina-(1-7), que se liga ao receptor Mas e há liberação de óxido nítrico (NO). Recentemente, foi descrito o aceturato de diminazeno (DIZE), ativador da ECA2. OBJETIVOS: Avaliar a ação do DIZE na pressão arterial (PA) e frequência cardíaca (FC) de ratos normotensos e hipertensos, além de explorar seu mecanismo de ação em vasos isolados. Métodos: Estudo de intervenção em ratos submetidos a implante de cânulas na artéria femoral para registrar PA e FC e na veia femoral para injeção de DIZE e salina (controle). O efeito no fluxo sanguíneo de arteríolas mesentéricas foi avaliado por microscopia intravital. Para induzir hipertensão arterial foi usado o modelo de hipertensão renovascular (2-kidney1clip; 2K1C). Após o procedimento, os animais foram tratados com DIZE, captopril ou salina e posteriormente feita análise histológica dos corações. No estudo in vitro de vasos isolados, o mecanismo de ação do DIZE foi avaliado usando o A-779, bloqueador do Mas, e o L-NAME, inibidor da NO sintase. RESULTADOS e CONCLUSÕES: O DIZE causou redução na PA com aumento compensatório da FC de maneira dose-dependente em ratos normotensos (Fig.1). O efeito hipotensor do DIZE foi semelhante ao do captopril em ratos hipertensos e não houve alteração na FC (Fig.1). Houve diminuição na velocidade do fluxo nas arteríolas no leito vascular mesentérico em ratos normotensos. A vasodilatação foi dependente do Mas e da liberação de NO. O DIZE preveniu o desenvolvimento de hipertrofia cardíaca em ratos hipertensos. Conclui-se que o DIZE tem efeito hipotensor em ratos normotensos e hipertensos devido a liberação de NO após ativação do Mas. (AU)

Physical Exercise-Induced Cardiovascular and Thermoregulatory Adjustments Are Impaired in Rats Subjected to Cutaneous Artery Denervation.

This study aimed to investigate the chronic effects of caudal artery denervation on morphometric parameters of the tail vascular smooth muscle and on physical exercise-induced thermoregulatory and cardiovascular adjustments in rats. Male Wistar rats were subjected to caudal artery denervation or the sham procedure. Approximately 26-28 days after these procedures, their thermoregulatory and cardiovascular parameters were evaluated at rest and during or following a fatiguing treadmill run. At the end of the experiments, the rats were euthanized, and samples of their tails were removed to evaluate morphometric parameters of the vascular smooth muscle surrounding the caudal artery. Denervated rats showed morphological adaptations, including increased arterial wall thickness and wall-to-lumen ratios. In resting rats and following the fatiguing exercise, caudal artery denervation barely affected the thermoregulatory and cardiovascular parameters evaluated. By contrast, caudal artery denervation attenuated the increase in tail skin temperature, decreased the spontaneous baroreflex sensitivity, and exacerbated the increases in mean arterial pressure in exercising rats. The increased wall-to-lumen ratio of denervated rats correlated negatively with the maximum tail skin temperature attained or cutaneous heat loss sensitivity but correlated positively with the maximum diastolic blood pressure attained during exercise. In conclusion, cutaneous denervation induces vascular remodeling characterized by morphological adaptations of the tail vascular smooth muscle. This vascular remodeling likely underlies the impaired tail heat loss and blood pressure adjustments in denervated rats subjected to physical exercise. Therefore, we have highlighted the importance of cutaneous vascular innervation integrity in thermal and cardiovascular control in stress-challenged rats. In this sense, our findings advance the understanding of thermoregulatory and cardiovascular system reactions after a sustained cutaneous vascular innervation injury, which is essential for the treatment of some diseases, such as Parkinson's disease and type 1 and type 2 diabetes mellitus.

Cardiovascular effects of small peptides of the renin angiotensin system.

The renin-angiotensin system (RAS) is a unique hormonal cascade which is composed by multiple enzymes and effector peptides. Recently, new peptides presenting biological activity have been discovered, increasing the complexity of the RAS Here, we evaluated the effects of small peptides of the RAS in coronary bed of rats. Firstly, we examined the direct effect of small angiotensinergic peptides [Angiotensin (Ang) -(1-5), Ang-(1-4) Ang-(1-3), and Ang-(1-2)] in coronary vessels. Noteworthy, it was observed that Ang-(1-4), Ang-(1-3), and Ang-(1-2) caused a significant reduction in pressure perfusion. Because Ang-(1-2) was the smallest peptide tested and presented the major effect, we decided to investigate its mechanisms of action. The effect of Ang-(1-2) was partially dependent on the Mas receptor, nitric oxide release and angiotensin-converting enzyme. Importantly, Ang-(1-2) reduced the blood pressure of Wistar rats and SHR Interestingly, SHR presented a more pronounced decrease in blood pressure levels than Wistar rats. Altogether, these data showed that angiotensinergic small peptides hold biological activities in coronary bed of rats.

Enhancement of Bone Healing by Local Administration of Carbon Nanotubes Functionalized with Sodium Hyaluronate in Rat Tibiae.

It has been reported that carbon nanotubes (CNTs) serve as nucleation sites for the deposition of bone matrix and cell proliferation. Here, we evaluated the effects of multi-walled CNTs (MWCNTs) on bone repair of rat tibiae. Furthermore, because sodium hyaluronate (HY) accelerates bone restoration, we associated CNTs with HY (HY-MWCNTs) in an attempt to boost bone repair. The bone defect was created by a 1.6-mm-diameter drill. After 7 and 14 days, tibiae were processed for histological and morphometric analyses. Immunohistochemistry was used to evaluate the expression of vascular endothelial growth factor (VEGF) in bone defects. Expression of osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (Col I) was assessed by real-time PCR. Histomorphometric analysis showed a similar increase in the percentage of bone trabeculae in tibia bone defects treated with HY and HY-MWCNTs, and both groups presented more organized and thicker bone trabeculae than nontreated defects. Tibiae treated with MWCNTs or HY- MWCNTs showed a higher expression of VEGF. Treatment with MWCNTs or HY-MWCNTs increased the expression of molecules involved in the bone repair process, such as OCN and BMP-2. Also, HY- and MWCNT-treated tibiae had an increased expression of Col I. Thus, it is tempting to conclude that CNTs associated or not with other materials such as HY emerged as a promising biomaterial for bone tissue engineering.

Vasodilator Effect of Angiotensin-(1-7) on Vascular Coronary Bed of Rats: Role of Mas, ACE and ACE2.

BACKGROUND: Angiotensin(Ang)-(1-7) is a biologically active member of the reninangiotensin system that participates of the regulation of blood pressure. Although Ang-(1-7) is able to potentiate the vasodilator effect of bradykinin in coronary bed of rats, a direct vasodilator effect of Ang-(1-7) in this vascular bed has not been characterized. OBJECTIVES: The aim of this study was to evaluate the mechanisms involved in the vasodilator effect of Ang-(1-7) in the vasculature of isolated rat hearts perfused according to the Langendorff technique at constant flow. METHODS: Isolated hearts, after approximately 30 minutes of stabilization, were perfused with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(1-7). The participation of the Ang-(1-7) receptor Mas, AT1 receptor, angiotensin-converting enzyme (ACE) and ACE2 was evaluated perfusing hearts with a combination of Ang-(1-7) plus A779, Ang-(1-7) plus losartan, Ang-(1-7) plus captopril/enalapril and Ang-(1-7) plus DX-600, respectively. RESULTS: Ang-(1-7) induced a significant decrease in the perfusion pressure, indicating a direct vasodilatation action of this peptide in the coronary bed. This effect was abolished by A779, captopril, enalapril and DX-600 an ACE2-specific inhibitor. However, AT1 blockade did not blunt the Ang-(1-7) effect. No significant changes were observed in heart rate, as well as in contractile tension and ±dT/dt. Moreover, immunohistochemical analysis showed the presence of Ang-(1-7) and Mas in coronary vessels. CONCLUSION: The Ang-(1-7) concentration used in this study was unable to induce changes in the cardiac function since no consistent alterations in contraction force and HR were viewed after Ang- (1-7) perfusion. In summary, this study showed that Ang-(1-7) induces vasodilation in the coronary bed of rats and this effect involves coupling to Mas receptor and interaction with ACE and ACE2.

N-Methyl-d-Aspartate (NMDA) Receptor Blockade Prevents Neuronal Death Induced by Zika Virus Infection.

Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration.IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.

Vascular Reactivity of Isolated Aorta to Study the Angiotensin-(1-7) Actions.

Since the early 1950s, vascular reactivity using isolated vessel rings has been a useful and efficient model for physiological and pharmacological studies. This experimental model was utilized in the milestone study of Dr. Robert Furchgott to discover the endothelium-derived relaxation factor (EDRF) nitric oxide (NO), opening new avenues and scientific perspectives in the vascular pathophysiology. Moreover, the isolated vessel ring preparation had an important contribution to understand many vasoactive systems. Here, we described the isolated aorta technique and pitfalls about the use of angiotensin-(1-7) peptide in this preparation.

Original Research: ACE2 activator associated with physical exercise potentiates the reduction of pulmonary fibrosis.

The interstitial lung diseases are poorly understood and there are currently no studies evaluating the association of physical exercise with an ACE2 activator (DIZE) as a possible treatment for this group of diseases. We evaluate the effects of pharmacological treatment with an angiotensin-converting enzyme 2 activator drug, associated with exercise, on the pulmonary lesions induced by bleomycin. From the 96 male Balb/c mice used in the experiment, only 49 received 8 U/kg of bleomycin (BLM, intratracheally). The mice were divided into control (C) and bleomycin (BLM) groups, sedentary and trained (C-SED, C-EXE, BLM-SED, BLM-EXE), control and bleomycin and also sedentary and trained treated with diminazene (C-SED/E, C-EXE/E, BLM-SED/E, BLM-EXE/E). The animals were trained five days/week, 1 h/day with 60% of the maximum load obtained in a functional capacity test, for four weeks. Diminazene groups were treated (1 mg/kg, by gavage) daily until the end of the experiment. The lungs were collected 48 h after the training program, set in buffered formalin and investigated by Gomori's trichrome, immunohistochemistry of collagen type I, TGF-ß1, beta-prolyl-4-hydroxylase, MMP-1 and -2. The BLM-EXE/E group obtained a significant increase in functional capacity, reduced amount of fibrosis and type I collagen, decreased expression of TGF-ß1 and beta-prolyl-4-hydroxylase and an increase of metalloproteinase -1, -2 when compared with the other groups. The present research shows, for the first time, that exercise training associated with the activation of ACE2 potentially reduces pulmonary fibrosis.

Severe Calorie Restriction Reduces Cardiometabolic Risk Factors and Protects Rat Hearts from Ischemia/Reperfusion Injury.

BACKGROUND AND AIMS: Recent studies have proposed that if a severe caloric restriction (SCR) is initiated at the earliest period of postnatal life, it can lead to beneficial cardiac adaptations later on. We investigated the effects of SCR in Wistar rats from birth to adult age on risk factors for cardiac diseases (CD), as well as cardiac function, redox status, and HSP72 content in response to ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: From birth to the age of 3 months, CR50 rats were fed 50% of the food that the ad libitum group (AL) was fed. Food intake was assessed daily and body weight were assessed weekly. In the last week of the SCR protocol, systolic blood pressure and heart rate were measured and the double product index was calculated. Also, oral glucose and intraperitoneal insulin tolerance tests were performed. Thereafter, rats were decapitated, visceral fat was weighed, and blood and hearts were harvested for biochemical, functional, tissue redox status, and western blot analyzes. Compared to AL, CR50 rats had reduced the main risk factors for CD. Moreover, the FR50 rats showed increased cardiac function both at baseline conditions (45% > AL rats) and during the post-ischemic period (60% > AL rats) which may be explained by a decreased cardiac oxidative stress and increased HSP72 content. CONCLUSION: SCR from birth to adult age reduced risk factors for CD, increased basal cardiac function and protected hearts from the I/R, possibly by a mechanism involving ROS.

Mefenamic acid decreases inflammation but not joint lesions in experimental osteoarthritis.

Mefenamic acid is a non-steroidal anti-inflammatory drug able to control the symptoms of osteoarthritis (OA), but its effects on protection of cartilage and bone are still unclear. This study aimed to investigate whether the control of inflammation by mefenamic acid translates into decreased joint lesions in experimental OA in rats. OA was induced by injecting 1 mg of monosodium iodoacetate (MIA) into the joints of rats. The animals were treated with mefenamic acid (50 mg/kg, daily, oral gavage) either pre-MIA injection (preventive) or post-MIA injection (therapeutic). Joint swelling and hyperalgesia were evaluated at baseline and 1, 3, 14 and 28 days after induction of OA. Intra-articular lavage and kinetics of cell migration into the synovium were measured 3 and 28 days after OA induction. Histopathological analysis, Osteoarthritis Research Society International (OARSI) score, total synovium cells count, cartilage area and levels of proteoglycans in joints were also evaluated. Mefenamic acid prevented joint oedema and hyperalgesia induced by MIA in the acute phase (3 days) of the disease. In the chronic phase (28 days), preventive and therapeutic regimens decreased the number of mononuclear cells in the joint cavity. In contrast, thickening of the synovium, bone resorption, loss of cartilage and levels of proteoglycans were unaffected by mefenamic acid when it was administered either preventively or therapeutically. Thus, mefenamic acid had anti-inflammatory effects but did not reduce the progression of OA lesions, thereby indicating that it is only effective for symptomatic control of OA.

Anti-hypertensive Effects of Diminazene Aceturate: An Angiotensin- Converting Enzyme 2 Activator in Rats.

Previous studies have shown that activation of endogenous angiotensin-converting enzyme 2 (ACE2) results in various beneficial effects in the cardiovascular system. Recently, a new ACE2 activator, named diminazene aceturate (DIZE), was described. Here, we evaluated the actions of this compound in blood pressure (BP) and heart rate (HR) of conscious normotensive and hypertensive rats, as well as explored its mechanism of actions using isolated vessels. The renovascular model of hypertension was utilized. The participation of the Angiotensin-(1-7) receptor Mas and nitric oxide (NO) in the effects of DIZE was evaluated using A-779 and L-NAME, respectively. It was observed that DIZE caused a marked decrease in BP with a compensatory increase in HR in nornotensive rats. Accordingly, a significant reduction in the blood flow of the mesenteric bed was evidenced using intravital microscopy. Moreover, in rats with renovascular hypertension, DIZE caused a decrease in BP similar to the hypotensive effect induced by captopril. Importantly, this compound also prevented the development of cardiac hypertrophy induced by hypertension. The isolated vessels technique revealed that the vasodilator effects of DIZE were dependent on Mas activation and NO release. Thus, our findings demonstrated that DIZE reduces the BP of normotensinve and hypertensive rats possibly by a mechanism involving Mas and NO.