DNA damage and aging on hematopoietic stem cells: Impact of oxidative stress in ApoE-/- mice.

The effects of aging on ROS production and DNA damage were assessed in hematopoietic stem cells (HSCs) from apolipoprotein E-deficient (ApoE-/-) mice (2-, 12- and 24-month-old), a traditional experimental model of atherogenic dyslipidemia. HSCs from aged ApoE-/- mice were associated with increased ROS levels, leading to loss quiescence, DNA damage, apoptosis and telomere shortening. The concurrence of lack of ApoE and aging result in exhaustion and senescence of HSCs accompanied by increased oxidative stress and inflammation. Therefore, our data open avenues to a better understanding of age-related changes and genetic factors, which may synergistically compromise the efficacy of aged HSC recovery and/or transplantation.

Chronic Cadmium Exposure Accelerates the Development of Atherosclerosis and Induces Vascular Dysfunction in the Aorta of ApoE-/- Mice.

Cadmium exposure is related to cardiovascular diseases, including hypertension, atherosclerosis, increased oxidative stress, endothelial dysfunction, and specific biochemical changes induced by this metal. Thus, we aimed to investigate whether cadmium exposure induces endothelial dysfunction, accelerates atherosclerotic plaque formation in the aorta, and enhances oxidative stress in apolipoprotein E knockout (ApoE-/-) mice. Experiments were performed in 14-week-old male wild-type and ApoE-/- mice. ApoE-/- mice received cadmium (CdCl2 100 mg/L in drinking water for 28 days) or vehicle (distilled water). After treatment, vascular reactivity to phenylephrine, acetylcholine, and sodium nitroprusside was analyzed using isolated aorta. Bone marrow cells were isolated to assess the production of nitric oxide and reactive oxygen and nitrogen species. ApoE-/- cadmium-treated mice had higher cholesterol levels than non-exposed mice. Cadmium exposure decreased the vasodilatation response to acetylcholine in aortic ring of ApoE-/- mice, though no changes in phenylephrine or sodium nitroprusside responses were observed. L-NAME reduced vasodilator responses to acetylcholine; this effect was lower in ApoE-/- cadmium-treated mice, suggesting reduction in nitric oxide (NO) bioavailability. Moreover, in bone marrow cells, cadmium decreased cytoplasmic levels of NO and increased superoxide anions, hydrogen peroxide, and peroxynitrite in ApoE-/- mice. Morphological analysis showed that cadmium exposure increased plaque deposition in the aorta by approximately 3-fold. Our results suggest that cadmium exposure induces endothelial dysfunction in ApoE-/- mice. Moreover, cadmium increased total cholesterol levels, which may promote the early development of atherosclerosis in the aorta of ApoE-/- mice. Our findings support the hypothesis that cadmium exposure might increase the risk of atherosclerosis.

Gender-dependent effects of aging on the kidney.

It is well known that the kidney plays an important role in the development of cardiovascular diseases such as hypertension. The normal aging process leads to changes in kidney morphology, hemodynamics and function, which increase the incidence of cardiovascular events in the elderly population. These disturbances are influenced by several factors, including gender. In general, females are protected by the effects of estrogens on the cardiorenal system. Several studies have demonstrated the beneficial effects of estrogens on renal function in the elderly; however, the relationships between androgens and kidney health during one's lifetime are not well understood. Sex steroids have many complex actions, and the decline in their levels during aging clearly influences kidney function, decreases the renal reserve and facilitates the development of cardiovascular disorders. Therefore, in this review, we discuss the cellular, biochemical, and molecular mechanisms by which sex hormones may influence renal function during the aging process.

Gender-dependent effects of aging on the kidney

It is well known that the kidney plays an important role in the development of cardiovascular diseases such as hypertension. The normal aging process leads to changes in kidney morphology, hemodynamics and function, which increase the incidence of cardiovascular events in the elderly population. These disturbances are influenced by several factors, including gender. In general, females are protected by the effects of estrogens on the cardiorenal system. Several studies have demonstrated the beneficial effects of estrogens on renal function in the elderly; however, the relationships between androgens and kidney health during one’s lifetime are not well understood. Sex steroids have many complex actions, and the decline in their levels during aging clearly influences kidney function, decreases the renal reserve and facilitates the development of cardiovascular disorders. Therefore, in this review, we discuss the cellular, biochemical, and molecular mechanisms by which sex hormones may influence renal function during the aging process.

Oral rapamycin attenuates atherosclerosis without affecting the arterial responsiveness of resistance vessels in apolipoprotein E-deficient mice

The objective of the present study was to assess the effects of the immunosuppressant rapamycin (Rapamune®, Sirolimus) on both resistance vessel responsiveness and atherosclerosis in apolipoprotein E-deficient 8-week-old male mice fed a normal rodent diet. Norepinephrine (NE)-induced vasoconstriction, acetylcholine (ACh)- and sodium nitroprusside (SNP)-induced vasorelaxation of isolated mesenteric bed, and atherosclerotic lesions were evaluated. After 12 weeks of orally administered rapamycin (5 mg·kg-1·day-1, N = 9) and compared with untreated (control, N = 9) animals, rapamycin treatment did not modify either NE-induced vasoconstriction (maximal response: 114 ± 4 vs 124 ± 10 mmHg, respectively) or ACh- (maximal response: 51 ± 8 vs 53 ± 5 percent, respectively) and SNP-induced vasorelaxation (maximal response: 73 ± 6 vs 74 ± 6 percent, respectively) of the isolated vascular mesenteric bed. Despite increased total cholesterol in treated mice (982 ± 59 vs 722 ± 49 mg/dL, P < 0.01), lipid deposition on the aorta wall vessel was significantly less in rapamycin-treated animals (37 ± 12 vs 68 ± 8 µm² x 10³). These results indicate that orally administered rapamycin is effective in attenuating the progression of atherosclerotic plaque without affecting the responsiveness of resistance vessels, supporting the idea that this immunosuppressant agent might be of potential benefit against atherosclerosis in patients undergoing therapy.

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan (10 mg·kg-1·day-1; N = 5) or the superoxide scavenger tempol (0.2 mmol·kg-1·day-1; N = 5), which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system.

The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin.

Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice.

Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

Gene transfer to carotid sinus in vivo: a novel approach to investigation of baroreceptors.

Baroreceptor nerve endings are located in the adventitia of the carotid sinuses and aortic arch. The goal of the present study was to develop a method for gene transfer to the carotid sinus adventitia. Replication-deficient adenovirus containing the gene for Escherichia coli beta-galactosidase (beta-Gal) was applied topically to the carotid sinuses of anesthetized rabbits. Transgene expression was localized by histochemical staining and quantified by chemiluminescence assay (Galacto-Light). Possible effects of adenovirus on baroreceptor sensitivity were investigated by recording baroreceptor activity from the vascularly isolated carotid sinus over a pressure range of 0 to 160 mm Hg. Beta-Gal expression in carotid sinus was evident 1 day after virus application, was dose dependent, and was markedly enhanced after 4 days. Expression was restricted to the adventitia of the vessel wall and was not present in vehicle-treated carotid sinuses. Baroreceptor sensitivity measured from carotid sinuses exposed to adenovirus 4 to 5 days beforehand was not altered compared with that measured from control carotid sinuses. In summary, topical application of adenoviral vectors to the carotid sinus provides transgene expression restricted to the region of baroreceptor innervation. The technique provides a novel approach to delineate mechanisms involved in baroreceptor activation and to deliver neuroactive gene products to the baroreceptors.

Neural reflex regulation of arterial pressure in pathophysiological conditions: interplay among the baroreflex, the cardiopulmonary reflexes and the chemoreflex.

The maintenance of arterial pressure at levels adequate to perfuse the tissues is a basic requirement for the constancy of the internal environment and survival. The objective of the present review was to provide information about the basic reflex mechanisms that are responsible for the moment-to-moment regulation of the cardiovascular system. We demonstrate that this control is largely provided by the action of arterial and non-arterial reflexes that detect and correct changes in arterial pressure (baroreflex), blood volume or chemical composition (mechano- and chemosensitive cardiopulmonary reflexes), and changes in blood-gas composition (chemoreceptor reflex). The importance of the integration of these cardiovascular reflexes is well understood and it is clear that processing mainly occurs in the nucleus tractus solitarii, although the mechanism is poorly understood. There are several indications that the interactions of baroreflex, chemoreflex and Bezold-Jarisch reflex inputs, and the central nervous system control the activity of autonomic preganglionic neurons through parallel afferent and efferent pathways to achieve cardiovascular homeostasis. It is surprising that so little appears in the literature about the integration of these neural reflexes in cardiovascular function. Thus, our purpose was to review the interplay between peripheral neural reflex mechanisms of arterial blood pressure and blood volume regulation in physiological and pathophysiological states. Special emphasis is placed on the experimental model of arterial hypertension induced by N-nitro-L-arginine methyl ester (L-NAME) in which the interplay of these three reflexes is demonstrable.

Neural reflex regulation of arterial pressure in pathophysiological conditions: interplay among the baroreflex, the cardiopulmonary reflexes and the chemoreflex

The maintenance of arterial pressure at levels adequate to perfuse the tissues is a basic requirement for the constancy of the internal environment and survival.The objective of the present review was to provide information about the basic relfex mechanisms that are responsible for the moment-to-moment regulation of the cardiovascular system. We demonstrate that this control is largely provided by the action of arterial and non-arterial reflexes that detect and correct changes in arterial pressure (baroreflex), blood volume or chemical composition (mechano-and chemosensitive cardiopulmonary reflexes), and changes in bloodgas composition (chemoreceptor reflex). The importance of the integration of these cardiovascular reflexes is well understood and it is clear that processing mainly occurs in the nucleus tractus solitarii, although the mechanism is poorly understood.There are several indications that the interactions of baroreflex, chemoreflex and Bezold-Jarisch reflex inputs, and the central nervous system control the activity of autonomic preganglionic neurons through parallel afferent and efferent pathways to achieve cardiovascular homeostasis. It is surprising that so little appears in the literature about the integration of these neural reflexes in cardiovascular function. Thus, our purpose was to review the interplay between peripheral neural reflex mechanisms of arterial blood pressure and blood volume regulation in physiological and pathophysiological states. Special emphasis is placed on the experimental model or arterial hypertension induced by N-nitro-L-arginine methyl ester (L-NAME) in which the interplay of these three reflexes is demonstrable.

Adenovirus-mediated gene transfer to cultured nodose sensory neurons.

Recent advances have enabled transfer of genes to various types of cells and tissues. The goals of the present study were to transfer genes to nodose sensory neurons using replication-deficient adenovirus vectors and to define the conditions needed to optimize the gene transfer. Neurons were dissociated from rat nodose ganglia and maintained in culture. Cultures were exposed for 30 min to vectors containing the beta-galactosidase gene lacZ driven by either the Rous sarcoma virus (RSV) or the cytomegalovirus (CMV) promoter. Cultures were fixed and treated with X-gal to evaluate lacZ expression 1-7 days after exposure to virus. Increasing concentrations of virus led to dose-related increases in the number of neurons expressing lacZ. LacZ was expressed in 8 +/- 2, 39 +/- 6, and 82 +/- 3% of neurons 1 day after exposure to 10(7), 10(8), and 10(9) pfu/ml of AdRSVlacZ, respectively (P < 0.05). The same doses of AdCMVlacZ led to expression in 41 +/- 9, 60 +/- 10, and 86 +/- 4% of neurons. Expression driven by the CMV promoter was essentially maximal within 1 day and remained stable for at least 7 days. In contrast, expression driven by the RSV promoter was less on day 1 but increased over time (1-7 days). There was no lacZ expression in vehicle-treated cultures and exposure to the adenovirus vectors did not adversely influence cell viability. Exposure of the neuronal cultures to an adenovirus vector containing the gene for green fluorescent protein (AdRSVgfp, 10(9) pfu/ml) enabled visualization of successful gene transfer in living neurons. The results indicate that gene transfer to cultured nodose neurons can be accomplished using adenovirus vectors. The expression of the transferred gene persists for at least 7 days, occurs more rapidly when expression is driven by the CMV compared with the RSV promoter, and occurs without adversely affecting cell viability.

[Assessment of the Bezold-Jarisch reflex in experimental myocardial infarction]. / Avaliação do reflexo Bezold-Jarisch no infarto experimental do miocárdio.

PURPOSE: To evaluate the cardiogenic depressor Bezold-Jarisch reflex in rats with chronic myocardial infarction. METHODS: Adult Wistar rats were submitted to ligation of the anterior descending coronary artery of the left ventricle (group INF, n = 15) and compared with rats submitted to sham-operation (group Sham, n = 15). Thirty days after the surgery, without influence of anesthetics, the basal mean arterial pressure (MAP) and heart rate (HR) were measured. Immediately after, the Bezold-Jarisch reflex was evaluated measuring the falls in diastolic arterial pressure (DAP) and the simultaneous bradycardia induced by injections of 5-hydroxytryptamine (5-HT, 4 to 32 micrograms/kg, i.v.). RESULTS: The INF group showed significantly lower basal MAP and HR values (103 +/- 3 mmHg and 328 +/- 6 bpm) when compared to the Sham group (110 +/- 2 mmHg and 348 +/- 7 bpm). The Bezold-Jarisch reflex was significantly attenuated in the INF group (falls of DAP from 2 +/- 2 to 31 +/- 3 mmHg and HR from 8 +/- 5 to 204 +/- 15 bpm), when compared to the Sham group (falls of DAP from 10 +/- 3 to 41 +/- 3 mmHg and HR from 58 +/- 12 to 276 +/- 16 bpm). The morphological analysis showed a myocardial infarction mainly located at the anterolateral portion of the left ventricle with a maximal extension of 35% of the left ventricle circumference. The INF group showed right ventricular and left atrial hypertrophy when compared to the Sham group. CONCLUSION: The experimental chronic myocardial infarction in rats is followed by significant attenuation of the Bezold-Jarisch reflex, probably as result of a heart failure and, consequently, of functional alterations in the chemosensitive receptors of cardiac unmyelinated vagal afferents.

Impairment of the Bezold-Jarisch reflex in conscious rats with myocardial hypertrophy

The aim of the present investigation was to study the Bezold-Jarisch reflex in catecholamine-induced myocardial hypertrophy. Ten conscious male albino rats (260-300 g) were treated for 15 days with isoproterenol (IR), 0.3 mg/kg injected im once a day, and compared to 10 control rats (CR) similarly injected with vehicle (0.25 ml). No significant changes in body weight, resting mean arterial pressure or heart rate were observed in the IR group. Left and right ventricular hypertrophy was observed in IR animals (27 and 28, respectively, P < 0.01) when compared to CR. The Bezold-Jarisch reflex was tested by injecting 5-hydroxytryptamine (4-32 micrograms/kg, iv) and was characterized by a simultaneous fall in diastolic arterial pressure (for example: 91 +/- 4 to 61 +/- 3 mm Hg, 16 micrograms/kg) and bradycardia (for example: 330 +/- 10 to 177 +/- 25 bpm, 16 micrograms/kg). This reflex was significantly attenuated in the IR when compared to the CR group. Our data suggest that ventricular hypertrophy without changes in arterial pressure can lead to a reduction of the Bezold-Jarisch reflex. The results reported here are in agreement with other studies showing that the impairment of cardiopulmonary reflex in hypertensive animals and humans occurs exclusively when the hypertension is accompanied by ventricular hypertrophy.