Leandro E. Souza

An Oral Formulation of Angiotensin-(1-7) Produces Cardioprotective Effects in Infarcted and Isoproterenol-Treated Rats

In this study we evaluated the cardiac effects of a pharmaceutical formulation developed by including angiotensin (Ang)-(1-7) in hydroxypropyl &bgr;-cyclodextrin (HP&bgr;CD), in normal, infarcted, and isoproterenol-treated rats. Myocardial infarction was produced by left coronary artery occlusion. Isoproterenol (2 mg/kg, IP) was administered daily for 7 days. Oral administration of HP&bgr;CD/Ang-(1-7) started immediately before infarction or associated with the first dose of isoproterenol. After 7 days of treatment, the rats were euthanized, and the Langendorff technique was used to analyze cardiac function. In addition, heart function was chronically (15, 30, 50 days) analyzed by echocardiography. Cardiac sections were stained with hematoxylin/eosin and Masson trichrome to evaluate cardiac hypertrophy and damage, respectively. Pharmacokinetic studies showed that oral HP&bgr;CD/Ang-(1-7) administration significantly increased Ang-(1-7) on plasma whereas with the free peptide it was without effect. Oral administration of HP&bgr;CD/Ang-(1-7) (30 &mgr;g/kg) significantly reduced the deleterious effects induced by myocardial infarction on systolic and diastolic tension, ±dT/dt, perfusion pressure, and heart rate. Strikingly, a 50% reduction of the infarcted area was observed in HP&bgr;CD/Ang-(1-7)–treated rats. Furthermore, HP&bgr;CD/Ang-(1-7) attenuated the heart function impairment and cardiac remodeling induced by isoproterenol. In infarcted rats chronically treated with HP&bgr;CD/Ang-(1-7), the reduction of ejection fraction and fractional shorting and the increase in systolic and diastolic left ventricular volumes observed in infarcted rats were attenuated. Altogether, these findings further confirm the cardioprotective effects of Ang-(1-7). More importantly, our data indicate that the HP&bgr;CD/Ang-(1-7) is a feasible formulation for oral administration of Ang-(1-7), which can be used as a cardioprotective drug.

Role of Exercise Training in Cardiovascular Autonomic Dysfunction and Mortality in Diabetic Ovariectomized Rats

Diabetes and menopause markedly increase the risk of cardiovascular disease in women. The objective of the present study was to investigate the effects of exercise training on cardiovascular autonomic dysfunction and on total mortality in diabetic female rats undergoing ovarian hormone deprivation. Female Wistar rats were divided into ovariectomized groups: sedentary and trained controls and sedentary and trained diabetic rats (streptozotocin, 50 mg/kg IV). Trained groups were submitted to an exercise training protocol on a treadmill (8 weeks). The baroreflex sensitivity was evaluated by heart rate responses to arterial pressure changes. Heart rate variability was determined using the SD of the basal heart rate. Vagal and sympathetic tonus were evaluated by pharmacological blockade. Diabetes impaired baroreflex sensitivity (≈55%), vagal tonus (≈68%), and heart rate variability (≈38%). Exercise training improved baroreflex sensitivity and heart rate variability in control and diabetic groups in relation to their sedentary groups. Trained control rats presented increased vagal tonus compared with that of sedentary ones. The sympathetic tonus was reduced in the trained diabetic group as compared with that of other studied groups. Significant correlations were obtained between heart rate variability and vagal tonus with baroreflex sensitivity. Mortality, assessed during the training period, was reduced in trained diabetic (25%) rats compared with mortality in sedentary diabetic rats (60%). Together, these findings suggest that decreases in baroreflex sensitivity and heart rate variability may be related to increased mortality in female diabetic subjects and that improved autonomic regulation induced by exercise training may contribute to decreased mortality in this population.

Beneficial Effects of Long-Term Administration of an Oral Formulation of Angiotensin-(1–7) in Infarcted Rats

In this study was evaluated the chronic cardiac effects of a formulation developed by including angiotensin(Ang)-(1–7) in hydroxypropyl β-cyclodextrin (HPβCD), in infarcted rats. Myocardial infarction (MI) was induced by left coronary artery occlusion. HPβCD/Ang-(1–7) was administered for 60 days (76 μg/Kg/once a day/gavage) starting immediately before infarction. Echocardiography was utilized to evaluate usual cardiac parameters, and radial strain method was used to analyze the velocity and displacement of myocardial fibers at initial time and 15, 30, and 50 days after surgery. Real-time PCR was utilized to evaluate the fibrotic signaling involved in the remodeling process. Once-a-day oral HPβCD/Ang-(1–7) administration improved the cardiac function and reduced the deleterious effects induced by MI on TGF-β and collagen type I expression, as well as on the velocity and displacement of myocardial fibers. These findings confirm cardioprotective effects of Ang-(1–7) and indicate HPβCD/Ang-(1–7) as a feasible formulation for long-term oral administration of this heptapeptide.

Cholinergic stimulation with pyridostigmine improves autonomic function in infarcted rats

In the present study we evaluated the effects of short‐term pyridostigmine bromide (0.14 mg/mL) treatment started early after myocardial infarction (MI) on left ventricular (LV) and autonomic functions in rats. Male Wistar rats were divided into control, pyridostigmine, infarcted and infarcted + pyridostigmine‐treated groups. Pyridostigmine was administered in the drinking water, starting immediately after MI or sham operation, for 11 days. Left ventricular function was evaluated indirectly by echocardiography and directly by LV catheterization. Cardiovascular autonomic control was evaluated by baroreflex sensitivity (BRS), heart rate variability (HRV) and pharmacological blockade. All evaluations started after 7 days pyridostigmine treatment and were finalized after 11 days treatment. Pyridostigmine prevented the impairment of +dP/dT and reduced the MI area in infarcted + pyridostigmine compared with infarcted rats (7 ± 3% vs 17 ± 4%, respectively). Mean blood pressure was restored in infarcted + pyridostigmine compared with infarcted rats (103 ± 3 vs 94 ± 3 mmHg, respectively). In addition, compared with the infarcted group, pyridostigmine improved BRS, as evaluated by tachycardic (1.6 ± 0.2 vs 2.5 ± 0.2 b.p.m./mmHg, respectively) and bradycardic (−0.42 ± 0.01 vs −1.9 ± 0.1 b.p.m./mmHg) responses, and reduced the low frequency/high frequency ratio of HRV (0.81 ± 0.11 vs 0.24 ± 0.14, respectively). These improvements are probably associated with increased vagal tone and reduced sympathetic tone in infarcted + pyridostigmine compared with infarcted rats. In conclusion, the data suggest that short‐term pyridostigmine treatment started early after MI can improve BRS, HRV and parasympathetic and sympathetic tone in experimental rats. These data may have potential clinical implications because autonomic markers have prognostic significance after MI.

Renal cyst growth is the main determinant for hypertension and concentrating deficit in Pkd1-deficient mice

We have bred a Pkd1 floxed allele with a nestin-Cre expressing line to generate cystic mice with preserved GFR to address the pathogenesis of complex ADPKD phenotypes. Hypertension affects about 60% of these patients before loss of renal function, leading to significant morbimortality. Cystic mice were hypertensive at 5 and 13 weeks of age, a phenotype not seen in non-cystic controls and Pkd1-haploinsufficient animals, which do not develop renal cysts. Fractional sodium excretion was reduced in cystic mice at these ages. Angiotensinogen gene expression was higher in cystic than non-cystic kidneys at 18 weeks, while ACE and the AT1 receptor were expressed in renal cyst epithelia. Cystic animals displayed increased renal cAMP, cell proliferation and apoptosis. At 24 weeks mean arterial pressure and fractional sodium excretion did not significantly differ between the cystic and non-cystic groups, whereas cardiac mass increased in cystic mice. Renal concentrating deficit is also an early finding in ADPKD. Maximum urine osmolality and urine nitrite excretion were reduced in 10–13 and 24-week-old cystic mice, deficits not found in haploinsufficient and non-cystic controls. A trend of higher plasma vasopressin was observed in cystic mice. Thus, cyst growth most probably plays a central role in early-stage ADPKD-associated hypertension, with activation of the intrarenal renin-angiotensin system as a key mechanism. Cyst expansion is also likely essential for the development of the concentrating deficit in this disease. Our findings are consistent with areas of reduced perfusion in the kidneys of patients with ADPKD.

Cardiac Impairment Evaluated by Transesophageal Echocardiography and Invasive Measurements in Rats Undergoing Sinoaortic Denervation

Background Sympathetic hyperactivity may be related to left ventricular (LV) dysfunction and baro- and chemoreflex impairment in hypertension. However, cardiac function, regarding the association of hypertension and baroreflex dysfunction, has not been previously evaluated by transesophageal echocardiography (TEE) using intracardiac echocardiographic catheter. Methods and Results We evaluated exercise tests, baroreflex sensitivity and cardiovascular autonomic control, cardiac function, and biventricular invasive pressures in rats 10 weeks after sinoaortic denervation (SAD). The rats (n = 32) were divided into 4 groups: 16 Wistar (W) with (n = 8) or without SAD (n = 8) and 16 spontaneously hypertensive rats (SHR) with (n = 8) or without SAD (SHRSAD) (n = 8). Blood pressure (BP) and heart rate (HR) did not change between the groups with or without SAD; however, compared to W, SHR groups had higher BP levels and BP variability was increased. Exercise testing showed that SHR had better functional capacity compared to SAD and SHRSAD. Echocardiography showed left ventricular (LV) concentric hypertrophy; segmental systolic and diastolic biventricular dysfunction; indirect signals of pulmonary arterial hypertension, mostly evident in SHRSAD. The end-diastolic right ventricular (RV) pressure increased in all groups compared to W, and the end-diastolic LV pressure increased in SHR and SHRSAD groups compared to W, and in SHRSAD compared to SAD. Conclusions Our results suggest that baroreflex dysfunction impairs cardiac function, and increases pulmonary artery pressure, supporting a role for baroreflex dysfunction in the pathogenesis of hypertensive cardiac disease. Moreover, TEE is a useful and feasible noninvasive technique that allows the assessment of cardiac function, particularly RV indices in this model of cardiac disease.

Cardiovascular autonomic dysfunction in non-obese diabetic mice.

It is known that diabetes is associated with autonomic dysfunction; however, data about autonomic function in non-obese diabetic mice (NOD) remain scarce. We evaluated the autonomic profile of NOD mice. Female mice, 24-28 week old, were divided in two groups: NOD (n = 6) and control (n = 6, Swiss mice). NOD mice with glycemia ≥ 300 mg/dl were used. Heart rate variability (HRV) and arterial pressure variability (APV) in time and frequency domains, symbolic analysis of heart rate (HR) and baroreflex sensitivity were evaluated. HR and arterial pressure (AP) were similar between the groups; however, HRV (total variance of RR interval: NOD=21.07 ± 3.75 vs. C = 42.02 ± 6.54 ms(2)) and the vagal modulation index RMSSD were lower in NOD group (4.01 ± 0.32 vs. 8.28 ± 0.97 ms). Moreover, the absolute and normalized low-frequency (LF) components were also enhanced in NOD (normalized = 61.0 ± 4.0%) as compared to control mice (normalized = 20.0 ± 4.0%). Both the absolute and normalized high-frequency (HF) components were lower in NOD (normalized = 39.0 ± 4.0%) when compared to the control group (normalized = 80.0 ± 4.0). In the symbolic analysis the 0V pattern, an indication of sympathetic activity, was higher in NOD and 2 LV pattern, an indication of parasympathetic activity, was lower in the NOD than in the control group. Both bradycardic and tachycardic responses were decreased in NOD (3.01 ± 0.72 vs. 4.54 ± 0.36 bpm/mmHg and 2.49 ± 0.31 vs. C = 3.43 ± 0.33 bpm/mmHg) when compared to the control group. Correlation analysis showed negative correlations between vagal indexes (RMSSD, %HF and 2LV) and glycemic levels. In conclusion, NOD mice develop severe diabetes correlated with autonomic dysfunction.

Sevoflurane preconditioning during myocardial ischemia-reperfusion reduces infarct size and preserves autonomic control of circulation in rats

PURPOSE To investigate the myocardial ischemia-reperfusion with sevoflurane anesthetic preconditioning (APC) would present beneficial effects on autonomic and cardiac function indexes after the acute phase of a myocardial ischemia-reperfusion. METHODS Twenty Wistar rats were allocated in three groups: control (CON, n=10), myocardial infarction with sevoflurane (SEV, n=5) and infarcted without sevoflurane (INF, n=5). Myocardial ischemia (60 min) and reperfusion were performed by temporary coronary occlusion. Twenty-one days later, the systolic and diastolic function were evaluated by echocardiography; spectral analysis of the systolic arterial pressure (SAPV) and heart rate variability (HRV) were assessed. After the recording period, the infarct size (IS) was evaluated. RESULTS The INF group presented greater cardiac dysfunction and increased sympathetic modulation of the SAPV, as well as decreased alpha index and worse vagal modulation of the HRV. The SEV group exhibited attenuation of the systolic and diastolic dysfunction and preserved vagal modulation (square root of the mean squared differences of successive R-R intervals and high frequency) of HRV, as well as a smaller IS. CONCLUSION Sevoflurane preconditioning better preserved the cardiac function and autonomic modulation of the heart in post-acute myocardial infarction period.

Characterization of the renal renin-angiotensin system in transgenic mice that express rat tonin

Introduction: Tonin is an enzyme that is able to generate angiotensin II (Ang II) from angiotensin I (Ang I) or directly from angiotensinogen. Our goal was to characterize the renal renin-angiotensin system in transgenic mice that express rat tonin (TGM`(rTon)). Materials and methods: Mice were euthanized and the kidneys removed for analysis. Tonin activity was evaluated by radioimmunoassay and angiotensin I-converting enzyme (ACE) activity by HPLC. Tonin, ACE and angiotensin II-converting enzyme (ACE2) expression was analyzed by Western blotting. Results: Tonin activity was significantly increased in TGM`(rTon) compared to their respective wild-type (WT) littermates (1.7 ± 0.21 vs 0.11 ± 0.02 nmol of Ang II/min/mg of protein). Tonin activity had a strong positive correlation with tonin expression in both TGM`(rTon) and their respective wild-type littermates. The ACE activity and expression levels of 65-kDa N-domain angiotensin I-converting enzyme isoform were significantly increased in the TGM`(rTon) when compared with WT. ACE2 expression levels were statistically significantly higher in the TGM`(rTon) when compared with WT. Angiotensin 1–7 (Ang(1–7)) and Ang I levels were significantly lower in the TGM`(rTon). Conclusions: We suggest that the environment of tonin abundance may increase N-domain ACE activity liberated by a secretase able to cleave somatic ACE.

Increase in Vascular Injury of Sodium Overloaded Mice May be Related to Vascular Angiotensin Modulation.

This study aimed to analyzing the effect of chronic sodium overload upon carotid and femoral injury, and its relation to vascular angiotensin modulation. Male C57Bl6 mice were divided in: control (cont), receiving 1% NaCl solution for 2 weeks (salt-2) or 12 weeks (salt-12). Two-weeks before the end of the study, a 2mm catheter was implanted around the left femoral and carotid arteries to induce injury. Blood pressure (BP) and heart rate (HR) were measured at the end of the study by tail plethysmography. Arteries were collected and prepared for histological analysis to determine arterial thickening and perivascular collagen deposition. Angiotensin II and Ang(1-7) were quantified in fresh arteries using the HPLC method. There were no differences in body weight, BP and HR. Intima/media ratio had a similar increase in both injured arteries of cont and salt-2 mice, but a more pronounced increase was observed in salt-12 mice (31.1±6%). On the other hand, sodium overload modified perivascular collagen deposition, increasing thick fibers (cont: 0.5%; salt-2: 3.4%; salt-12: 0.6%) and decreasing thin fibers (cont: 7.4%; salt-2: 0.5%; salt-12: 6.8%) in non-injured arteries. Injured arteries presented similar collagen fiber distribution. Angiotensin quantification showed increased Ang(1-7) in salt treated mice (salt-2: +72%; salt-12: +45%) with a concomitant decrease in Ang II (salt-2: -54%; salt-12: -60%). Vascular injury increased significantly Ang(1-7) in salt-12 mice (+80%), maintaining Ang II reduction similar to that of a non-injured artery. The lack of changes in BP and HR suggests that the structural changes observed may be due to non-hemodynamic mechanisms such as local renin-angiotensin system. Collagen evaluation suggests that sodium overload induces time-related changes in vascular remodeling. The increase of artery injury with concomitant increase in Ang(1-7) in 12-week treated mice shows a direct association between the duration of salt treatment and the magnitude of vascular injury.