Somatic gene therapy for hypertension

Gene therapy for hypertension is needed for the next generation of antihypertensive drugs. Current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). Gene therapy offers a way to produce long-lasting antihypertensive effects (weeks, months or years). We are currently using two strategies: a) antisense oligodeoxynucleotides (AS-ODN) and b) antisense DNA delivered in viral vectors to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODN are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODN, targeted to angiotensin type 1 receptors (AT1-R), angiotensinogen (AGT), angiotensin converting enzyme, and ß1-adrenergic receptors effectively reduce hypertension in rat models (SHR, 2K-1C) and cold-induced hypertension. A single dose is effective up to one month when delivered with liposomes. No side effects or toxic effects have been detected, and repeated injections can be given. For the vector, adeno-associated virus (AAV) is used with a construct to include a CMV promoter, antisense DNA to AGT or AT1-R and a reporter gene. Results in SHR demonstrate reduction and slowing of development of hypertension, with a single dose administration. Left ventricular hypertrophy is also reduced by AAV-AGT-AS treatment. Double transgenic mice (human renin plus human AGT) with high angiotensin II causing high blood pressure, treated with AAV-AT1-R-AS, show a normalization of blood pressure for over six months with a single injection of vector. We conclude that ODNs will probably be developed first because they can be treated like drugs for the treatment of hypertension with long-term effects. Viral vector delivery needs more engineering to be certain of its safety, but one day may be used for a very prolonged control of blood pressure.

Experimental myocardial hypertrophy induced by a minimally invasive ascending aorta coarctation

Ascending aorta coarctation was produced by a minimally invasive technique in rabbits. Animal mortality was 5%. Morphometric and hemodynamic parameters were evaluated. A parabiotically isolated heart model was used to assess the hemodynamic parameters. Left ventricular weight/body weight ratio and muscle area showed clear evidence of hypertrophy when compared to control. The hemodynamic changes in the isolated heart model suggested decreased diastolic and systolic function in the coarcted group. The present model produced hypertrophy with low mortality rates as a result of its less invasive nature.

Head-to-head comparison of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia in an animal model of coronary artery stenosis

To compare the sensitivity of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia we produced a physiologically significant stenosis in the left circumflex artery of 14 open-chest dogs (range: 50 to 89% reduction in luminal diameter). In each study, dobutamine (5 to 40 µg kg-1 min-1 in 3-min stages) and pacing (20 bpm increments, each 2 min, up to 260 bpm) were performed randomly, and then followed by dipyridamole (up to 0.84 mg/kg over 10 min). The positivity of stress echocardiography tests was quantitatively determined by a significant (P<0.05) reduction of or failure to increase absolute and percent systolic wall thickening in the stenotic artery supplied wall, as compared to the opposite wall (areas related to the left anterior descending artery). Systolic and diastolic frozen images were analyzed off-line by two blinded observers in the control and stress conditions. The results showed that 1) the sensitivity of dobutamine, dipyridamole and pacing stress tests was 57, 57 and 36%, respectively; 2) in animals with positive tests, the mean percent change of wall thickening in left ventricular ischemic segments was larger in the pacing (-19 ± 11%) and dipyridamole (-18 ± 16%) tests as compared to dobutamine (-9 ± 6%) (P = 0.05), but a similar mean reduction of wall thickening was observed when this variable was normalized to a control left ventricular segment (area related to the left anterior descending artery) (pacing: -16 ± 7%; dipyridamole: -25 ± 16%; dobutamine: -26 ± 10%; not significant), and 3) a significant correlation was observed between magnitude of coronary stenosis and left ventricular segmental dysfunction induced by ischemia in dogs submitted to positive stress tests. We conclude that the dobutamine and dipyridamole stress tests showed identical sensitivities for the detection of myocardial ischemia in this one-vessel disease animal model with a wide range of left circumflex artery stenosis. The pacing stress test was less sensitive, but the difference was not statistically significant. The magnitude of segmental left ventricular dysfunction induced by ischemia was similar in all stress tests evaluated.

Ambulatory blood pressure and Doppler echocardiographic indexes of borderline hypertensive men presenting an exaggerated blood pressure response during dynamic exercise

Borderline hypertension (BH) has been associated with an exaggerated blood pressure (BP) response during laboratory stressors. However, the incidence of target organ damage in this condition and its relation to BP hyperreactivity is an unsettled issue. Thus, we assessed the Doppler echocardiographic profile of a group of BH men (N = 36) according to office BP measurements with exaggerated BP in the cycloergometric test. A group of normotensive men (NT, N = 36) with a normal BP response during the cycloergometric test was used as control. To assess vascular function and reactivity, all subjects were submitted to the cold pressor test. Before Doppler echocardiography, the BP profile of all subjects was evaluated by 24-h ambulatory BP monitoring. All subjects from the NT group presented normal monitored levels of BP. In contrast, 19 subjects from the original BH group presented normal monitored BP levels and 17 presented elevated monitored BP levels. In the NT group all Doppler echocardiographic indexes were normal. All subjects from the original BH group presented normal left ventricular mass and geometrical pattern. However, in the subjects with elevated monitored BP levels, fractional shortening was greater, isovolumetric relaxation time longer, and early to late flow velocity ratio was reduced in relation to subjects from the original BH group with normal monitored BP levels (P<0.05). These subjects also presented an exaggerated BP response during the cold pressor test. These results support the notion of an integrated pattern of cardiac and vascular adaptation during the development of hypertension.

Relationship between microalbuminuria and cardiac structural changes in mild hypertensive patients

The aim of this study was to determine the relationship between urinary albumin excretion (UAE), cardiac structural changes upon echocardiography and 24-h ambulatory blood pressure (ABPM) levels. Twenty mild hypertensive patients (mean age 56.8 ± 9.6 years) were evaluated. After 2 weeks of a washout period of all antihypertensive drugs, all patients underwent an echocardiographic evaluation, a 24-h ABPM and an overnight urine collection. Systolic and diastolic blood pressure during 24-h ABPM was 145 ± 14/91 ± 10 mmHg (daytime) and 130 ± 14/76 ± 8 mmHg (nighttime), respectively. Seven (35%) patients presented UAE > or = 15 µg/min, and for the whole group, the geometric mean value for UAE was 10.2 x/÷ 3.86 µg/min. Cardiac measurements showed mean values of interventricular septum thickness (IVS) of 11 ± 2.3 mm, left ventricular posterior wall thickness (PWT) of 10 ± 2.0 mm, left ventricular mass (LVM) of 165 ± 52 g, and left ventricular mass index (LVMI) of 99 ± 31 g/m². A forward stepwise regression model indicated that blood pressure levels did not influence UAE. Significant correlations were observed between UAE and cardiac structural parameters such as IVS (r = 0.71, P<0.001), PWT (r = 0.64, P<0.005), LVM (r = 0.65, P<0.005) and LVMI (r = 0.57, P<0.01). Compared with normoalbuminuric patients, those who had microalbuminuria presented higher values of all cardiac parameters measured. The predictive positive and negative values of UAE > or = 15 µg/min for the presence of geometric cardiac abnormalities were 75 and 91.6%. These data indicate that microalbuminuria in essential hypertension represents an early marker of cardiac structural damage.

Slaughterhouse blood as a perfusate for studying myocardial function under ischemic conditions

Metabolic studies using the in vitro non-recirculating blood-perfused isolated heart model require large volumes of blood. The present study was designed to determine whether heterologous pig blood collected from a slaughterhouse can be used as perfusate for isolated pig hearts perfused under aerobic and constant reduced flow conditions. Eight isolated working pig hearts perfused for 90 min at a constant flow of 1.5 ml g-1 min-1 with non-recirculated blood diluted with Krebs-Henseleit bicarbonate buffer at a hematocrit of 23% were compared to eight hearts subjected to the same protocol but perfused only with Krebs-Henseleit bicarbonate buffer solution. Hearts were paced at 100 bpm and subjected to aerobic perfusion at 38ºC. Hearts were weighed before perfusion and at the end of the experiment and the results are reported as percent weight gain (mean ± SD). Comparisons between groups were performed by the Student t-test (P<0.05). After 90 min of perfusion with modified Krebs-Henseleit, perfused hearts presented a larger weight gain than blood-perfused hearts (39.34 ± 9.27 vs 23.13 ± 5.42%, P = 0.003). Left ventricular end-diastolic pressure was higher in the modified Krebs-Henseleit-perfused group than in the blood group (2.8 ± 0.4 vs 2.3 ± 0.3 mmHg, respectively, P = 0.01). We conclude that heterologous blood perfusion, by preserving a more physiological myocardial water content, is a better perfusion fluid than modified Krebs-Henseleit solution for quantitative studies of myocardial metabolism and heart function under ischemic conditions.

Effects of losartan combined with exercise training in spontaneously hypertensive rats

We investigate whether combined treatment with losartan, an angiotensin II receptor blocker, and exercise training (ET) in spontaneously hypertensive rats (SHR) would have an additive effect in reducing hypertension and improving baroreflex sensitivity when compared with losartan alone. Male SHR (8 weeks old) were assigned to 3 groups: sedentary placebo (SP, N = 16), sedentary under losartan treatment (SL, N = 11; 10 mg kg-1 day-1, by gavage), and ET under losartan treatment (TL, N = 10). ET was performed on a treadmill 5 days/week for 60 min at 50% of peak VO2, for 18 weeks. Blood pressure (BP) was measured with a catheter inserted into the carotid artery, and cardiac output with a microprobe placed around the ascending aorta. The baroreflex control of heart rate was assessed by administering increasing doses of phenylephrine and sodium nitroprusside (iv). Losartan significantly reduced mean BP (178 ± 16 vs 132 ± 12 mmHg) and left ventricular hypertrophy (2.9 ± 0.4 vs 2.5 ± 0.2 mg/g), and significantly increased baroreflex bradycardia and tachycardia sensitivity (1.0 ± 0.3 vs 1.7 ± 0.5 and 2.0 ± 0.7 vs 3.2 ± 1.7 bpm/mmHg, respectively) in SL compared with SP. However, losartan combined with ET had no additional effect on BP, baroreflex sensitivity or left ventricular hypertrophy when compared with losartan alone. In conclusion, losartan attenuates hypertension and improves baroreflex sensitivity in SHR. However, ET has no synergistic effect on BP in established hypertension when combined with losartan, at least at the dosage used in this investigation.

Alternative parameters for echocardiographic assessment of fetal diastolic function

Alternative methods to assess ventricular diastolic function in the fetus are proposed. Fetal myocardial hypertrophy in maternal diabetes was used as a model of decreased left ventricular compliance (LVC), and fetal respiratory movements as a model of increased LVC. Comparison of three groups of fetuses showed that, in 10 fetuses of diabetic mothers (FDM) with septal hypertrophy (SH), the mean excursion index of the septum primum (EISP) (ratio between the linear excursion of the flap valve and the left atrial diameter) was 0.36 ± 0.09, in 8 FDM without SH it was 0.51 ± 0.09 (P = 0.001), and in the 8 normal control fetuses (NCF) it was 0.49 ± 0.12 (P = 0.003). In another study, 28 fetuses in apnea had a mean EISP of 0.39 ± 0.05 which increased to 0.57 ± 0.07 during respiration (P < 0.001). These two studies showed that the mobility of the septum primum was reduced when LVC was decreased and was increased when LVC was enhanced. Mean pulmonary vein pulsatility was higher in 14 FDM (1.83 ± 1.21) than in 26 NCF (1.02 ± 0.31; P = 0.02). In the same fetuses, mean left atrial shortening was decreased (0.40 ± 0.11) in relation to NCF (0.51 ± 0.09; P = 0.011). These results suggest that FDM may have a higher preload than normal controls, probably as a result of increased myocardial mass and LV hypertrophy. Prenatal assessment of LV diastolic function by fetal echocardiography should include analysis of septum primum mobility, pulmonary vein pulsatility, and left atrial shortening.

Imidazoline receptors in the heart: a novel target and a novel mechanism of action that involves atrial natriuretic peptides

Chronic stimulation of sympathetic nervous activity contributes to the development and maintenance of hypertension, leading to left ventricular hypertrophy (LVH), arrhythmias and cardiac death. Moxonidine, an imidazoline antihypertensive compound that preferentially activates imidazoline receptors in brainstem rostroventrolateral medulla, suppresses sympathetic activation and reverses LVH. We have identified imidazoline receptors in the heart atria and ventricles, and shown that atrial I1-receptors are up-regulated in spontaneously hypertensive rats (SHR), and ventricular I1-receptors are up-regulated in hamster and human heart failure. Furthermore, cardiac I1-receptor binding decreased after chronic in vivo exposure to moxonidine. These studies implied that cardiac I1-receptors are involved in cardiovascular regulation. The presence of I1-receptors in the heart, the primary site of production of natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), cardiac hormones implicated in blood pressure control and cardioprotection, led us to propose that ANP may be involved in the actions of moxonidine. In fact, acute iv administration of moxonidine (50 to 150 µg/rat) dose-dependently decreased blood pressure, stimulated diuresis and natriuresis and increased plasma ANP and its second messenger, cGMP. Chronic SHR treatment with moxonidine (0, 60 and 120 µg kg-1 h-1, sc for 4 weeks) dose-dependently decreased blood pressure, resulted in reversal of LVH and decreased ventricular interleukin 1ß concentration after 4 weeks of treatment. These effects were associated with a further increase in already elevated ANP and BNP synthesis and release (after 1 week), and normalization by 4 weeks. In conclusion, cardiac imidazoline receptors and natriuretic peptides may be involved in the acute and chronic effects of moxonidine.

Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease

The objective of the present study was to determine the relationship between nitric oxide synthases (NOS) and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35% (N = 10), and from patients with CAD and LVEF >60% (N = 10) during cardiac surgery. NOS activity was measured by the conversion of L-[H³]-arginine to L-[H³]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS) activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 ± 0.18 vs 1.51 ± 0.31 pmol mg-1 min-1 (P < 0.0001) and 0.37 ± 0.08 vs 0.78 ± 0.09 relative cDNA absorbance at 320 nm (P < 0.0001), respectively. In contrast, inducible NOS (iNOS) activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 ± 0.90 vs 1.54 ± 0.65 pmol mg-1 min-1 (P < 0.0001) and 2.19 ± 0.27 vs 1.43 ± 0.13 cDNA absorbance at 320 nm (P < 0.0001), respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35%. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

Pressure and time dependence of the cardiopulmonary reflex response in patients with hypertensive cardiomyopathy

The first minutes of the time course of cardiopulmonary reflex control evoked by lower body negative pressure (LBNP) in patients with hypertensive cardiomyopathy have not been investigated in detail. We studied 15 hypertensive patients with left ventricular dysfunction (LVD) and 15 matched normal controls to observe the time course response of the forearm vascular resistance (FVR) during 3 min of LBNP at -10, -15, and -40 mmHg in unloading the cardiopulmonary receptors. Analysis of the average of 3-min intervals of FVR showed a blunted response of the LVD patients at -10 mmHg (P = 0.03), but a similar response in both groups at -15 and -40 mmHg. However, using a minute-to-minute analysis of the FVR at -15 and -40 mmHg, we observed a similar response in both groups at the 1st min, but a marked decrease of FVR in the LVD group at the 3rd min of LBNP at -15 mmHg (P = 0.017), and -40 mmHg (P = 0.004). Plasma norepinephrine levels were analyzed as another neurohumoral measurement of cardiopulmonary receptor response to LBNP, and showed a blunted response in the LVD group at -10 (P = 0.013), -15 (P = 0.032) and -40 mmHg (P = 0.004). We concluded that the cardiopulmonary reflex response in patients with hypertensive cardiomyopathy is blunted at lower levels of LBNP. However, at higher levels, the cardiopulmonary reflex has a normal initial response that decreases progressively with time. As a consequence of the time-dependent response, the cardiopulmonary reflex response should be measured over small intervals of time in clinical studies.

Eucalyptol, an essential oil, reduces contractile activity in rat cardiac muscle

Eucalyptol is an essential oil that relaxes bronchial and vascular smooth muscle although its direct actions on isolated myocardium have not been reported. We investigated a putative negative inotropic effect of the oil on left ventricular papillary muscles from male Wistar rats weighing 250 to 300 g, as well as its effects on isometric force, rate of force development, time parameters, post-rest potentiation, positive inotropic interventions produced by Ca2+ and isoproterenol, and on tetanic tension. The effects of 0.3 mM eucalyptol on myosin ATPase activity were also investigated. Eucalyptol (0.003 to 0.3 mM) reduced isometric tension, the rate of force development and time parameters. The oil reduced the force developed by steady-state contractions (50% at 0.3 mM) but did not alter sarcoplasmic reticulum function or post-rest contractions and produced a progressive increase in relative potentiation. Increased extracellular Ca2+ concentration (0.62 to 5 mM) and isoproterenol (20 nM) administration counteracted the negative inotropic effects of the oil. The activity of the contractile machinery evaluated by tetanic force development was reduced by 30 to 50% but myosin ATPase activity was not affected by eucalyptol (0.3 mM), supporting the idea of a reduction of sarcolemmal Ca2+ influx. The present results suggest that eucalyptol depresses force development, probably acting as a calcium channel blocker.

The decreased oxygen uptake during progressive exercise in ischemia-induced heart failure is due to reduced cardiac output rate

We tested the hypothesis that the inability to increase cardiac output during exercise would explain the decreased rate of oxygen uptake (VO2) in recent onset, ischemia-induced heart failure rats. Nine normal control rats and 6 rats with ischemic heart failure were studied. Myocardial infarction was induced by coronary ligation. VO2 was measured during a ramp protocol test on a treadmill using a metabolic mask. Cardiac output was measured with a flow probe placed around the ascending aorta. Left ventricular end-diastolic pressure was higher in ischemic heart failure rats compared with normal control rats (17 ± 0.4 vs 8 ± 0.8 mmHg, P = 0.0001). Resting cardiac index (CI) tended to be lower in ischemic heart failure rats (P = 0.07). Resting heart rate (HR) and stroke volume index (SVI) did not differ significantly between ischemic heart failure rats and normal control rats. Peak VO2 was lower in ischemic heart failure rats (73.72 ± 7.37 vs 109.02 ± 27.87 mL min-1 kg-1, P = 0.005). The VO2 and CI responses during exercise were significantly lower in ischemic heart failure rats than in normal control rats. The temporal response of SVI, but not of HR, was significantly lower in ischemic heart failure rats than in normal control rats. Peak CI, HR, and SVI were lower in ischemic heart failure rats. The reduction in VO2 response during incremental exercise in an ischemic model of heart failure is due to the decreased cardiac output response, largely caused by depressed stroke volume kinetics.

Minor segmental wall motion abnormalities detected in patients with Chagas' disease have adverse prognostic implications

Recent data from our laboratory have shown that patients with the indeterminate form of Chagas' disease can have impairment of left ventricular contractility, as evaluated by the slope of the left ventricle end-systolic pressure-dimension relationship. We also showed that Chagas' disease patients with minimal baseline wall motion abnormalities detected by two-dimensional echocardiography have more intense contractility impairment when compared to patients with the indeterminate form of the disease without this abnormality. The prognostic implications of these findings have not been established. We evaluated 59 patients (37-76 years, mean = 55 years) with different clinical forms of Chagas' disease, who had normal left ventricular global systolic function at baseline (57.6 ± 6.9%) and who had at least one additional echo during clinical follow-up (0.4-17.6; mean 4.6 years). Group 1 consisted of 14 patients with minor baseline left ventricle wall motion abnormalities and group 2 consisted of 45 patients without these abnormalities. During follow-up, global left ventricle systolic function deterioration was observed in 10 group 1 patients (71.4%) and in only 10 group 2 patients (22.2%; P < 0.005). Age and duration of follow-up were not independent determinants of left ventricular function deterioration in these patients. The present data indicate that mild segmental left ventricular wall motion abnormalities are associated with worsening of systolic function in Chagas' disease patients who have normal baseline global systolic performance.

Bioeffects of albumin-encapsulated microbubbles and real-time myocardial contrast echocardiography in an experimental canine model

Myocardial contrast echocardiography has been used for assessing myocardial perfusion. Some concerns regarding its safety still remain, mainly regarding the induction of microvascular alterations. We sought to determine the bioeffects of microbubbles and real-time myocardial contrast echocardiography (RTMCE) in a closed-chest canine model. Eighteen mongrel dogs were randomly assigned to two groups. Nine were submitted to continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) plus continuous imaging using power pulse inversion RTMCE for 180 min, associated with manually deflagrated high-mechanical index impulses. The control group consisted of 3 dogs submitted to continuous imaging using RTMCE without PESDA, 3 dogs received PESDA alone, and 3 dogs were sham-operated. Hemodynamics and cardiac rhythm were monitored continuously. Histological analysis was performed on cardiac and pulmonary tissues. No hemodynamic changes or cardiac arrhythmias were observed in any group. Normal left ventricular ejection fraction and myocardial perfusion were maintained throughout the protocol. Frequency of mild and focal microhemorrhage areas in myocardial and pulmonary tissue was similar in PESDA plus RTMCE and control groups. The percentages of positive microscopical fields in the myocardium were 0.4 and 0.7% (P = NS) in the PESDA plus RTMCE and control groups, respectively, and in the lungs they were 2.1 and 1.1%, respectively (P = NS). In this canine model, myocardial perfusion imaging obtained with PESDA and RTMCE was safe, with no alteration in cardiac rhythm or left ventricular function. Mild and focal myocardial and pulmonary microhemorrhages were observed in both groups, and may be attributed to surgical tissue manipulation.