Rolando J. Ramirez

Relaxin is essential for renal vasodilation during pregnancy in conscious rats

Marked vasodilation in the kidney and other nonreproductive organs is one of the earliest maternal adaptations to occur during pregnancy. Despite the recognition of this extraordinary physiology for over four decades, the gestational hormone responsible has remained elusive. Here we demonstrate a key role for relaxin, a member of the IGF family that is secreted by the corpus luteum in humans and rodents. Using a gravid rodent model, we employ two approaches to eliminate relaxin or its biological activity from the circulation: ovariectomy and administration of neutralizing antibodies. Both abrogate the gestational elevation in renal perfusion and glomerular filtration, as well as preventing the reduction in myogenic reactivity of isolated, small renal arteries. Osmoregulatory changes, another pregnancy adaptation, are also abolished. Our results indicate that relaxin mediates the renal vasodilatory responses to pregnancy and thus may be important for maternal and fetal health. They also raise the likelihood of a role for relaxin in other cardiovascular changes of pregnancy, and they suggest that, like estrogen, relaxin should be considered a regulator of cardiovascular function.

Uteroplacental insufficiency lowers the threshold towards hypoxia-induced cerebral apoptosis in growth-retarded fetal rats.

Infants suffering uteroplacental insufficiency and hypoxic ischemic injury often demonstrate cerebral apoptosis. Our objective was to determine the global effects of uteroplacental insufficiency upon cerebral gene expression of the apoptosis related proteins Bcl-2 and Bax and their role in increasing vulnerability to hypoxia-induced cerebral apoptosis. We therefore caused uteroplacental insufficiency and growth retardation by performing bilateral uterine artery ligation upon pregnant rats 2 days prior to term delivery and elicited further perinatal fetal hypoxia by placing maternal rats in 14% FiO(2) 3 h prior to delivery. We quantified cerebral levels of Bcl-2 and Bax mRNA, lipid peroxidation, caspase-3 activity, and cAMP in control and growth retarded term rat pups that experienced either normoxia or hypoxia. Uteroplacental insufficiency alone caused a significant decrease in cerebral Bcl-2 mRNA levels without altering cerebral Bax mRNA levels, malondialdehyde levels, or caspase-3 activity. In contrast, uteroplacental insufficiency and subsequent fetal hypoxia significantly increased cerebral Bax mRNA levels, lipid peroxidation and caspase-3 activity; Bcl-2 mRNA levels continued to be decreased. Hypoxia alone increased cerebral cAMP levels, whereas uteroplacental insufficiency and subsequent hypoxia decreased cerebral cAMP levels. We speculate that the decrease in Bcl-2 gene expression increases the vulnerability towards cerebral apoptosis in fetal rats exposed initially to uteroplacental insufficiency and subsequent hypoxic stress.

Alterations in vasomotor systems and mechanics of resistance-sized mesenteric arteries from SHR and WKY male rats following in vivo testosterone manipulation

BackgroundTestosterone (T) and the sympathetic nervous system each contribute to the pathology of hypertension. Altered blood vessel reactivity is also associated with the pathology of high blood pressure. The purpose of this study was to examine the effects of T manipulation in the regulation of resistance-sized blood vessel reactivity.MethodsAdult spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) male rats at 8 weeks of age were used. The rats were divided into groups consisting of gonadally intact controls (CONT), castrate with sham implant (CAST) and castrate with T implant (CAST + T) (n = 6 to 12 per group). Following a short-term period of T treatment (approximately 4 weeks), plasma norepinephrine (NE) and plasma T were assessed by performing high-performance liquid chromatography and RIA, respectively. Resistance-sized mesenteric artery reactivity was assessed on a pressurized arteriograph for myogenic reactivity (MYO), phenylephrine (PE) responsiveness and passive structural mechanics.ResultsSHR and WKY males exhibited similar physiological trends in T manipulation, with castration significantly lowering plasma T and NE and T replacement significantly increasing plasma T and NE. T manipulation in general resulted in significant alterations in MYO of second-order mesenteric arteries, with T replacement decreasing MYO in SHR (P < 0.05) compared to CONT, T replacement increasing MYO, and CAST decreasing MYO in WKY rats (P < 0.001) compared to CONT rats. Additionally, PE-induced constriction was significantly altered in both strains following T treatment, with the effective concentration of PE to constrict the vessel to 50% of the total diameter significantly increased in the CAST + T SHR compared to CONT (P < 0.05). Comparisons of passive structural mechanics between SHR and WKY treatment groups indicated in SHR a significantly increased wall-to-lumen ratio and decreased circumferential wall stress compared to WKY treatment groups.ConclusionsThese data suggest that T and NE are involved in a complex interaction with both myogenic reactivity and structural alterations of resistance-sized blood vessels and that these factors likely contribute to the development and maintenance of hypertension.

Relaxin mediates uterine artery compliance during pregnancy and increases uterine blood flow

Normal pregnancy involves dramatic remodeling of the uterine vasculature, with abnormal vascular adaptations contributing to pregnancy diseases such as preeclampsia. The peptide hormone relaxin is important for the renal and systemic hemodynamic adaptations to pregnancy, and has been shown to increase arterial compliance and outward hypertrophic remodeling. Therefore, we investigated the possibility that relaxin acts on its receptor, RXFP1, to mediate uterine artery compliance in late pregnancy and increase uterine blood flow velocity in rats. RXFP1 was predominantly localized to the tunica media vascular smooth muscle cells in the uterine artery, although receptors were also detected in endothelial cells. Highest expression of Rxfp1 in the uterine artery occurred in estrus and early pregnancy. Isolated uterine arteries from late pregnant rats treated with a monoclonal antibody against circulating relaxin (MCA1) had significantly increased vessel wall stiffness compared with controls, with no reduction in wall thickness. Chronic infusion of relaxin (4 μg/h, osmotic minipump) for 5 d in nonpregnant rats significantly increased uterine artery blood flow velocity. Overall, these data demonstrate a functional role for relaxin in mediating uterine artery compliance in pregnant rats, which may be necessary to maintain adequate uterine blood flow to the uterus and placenta.—Vodstrcil, L. A., Tare, M., Novak, J., Dragomir, N., Ramirez, R. J., Wlodek, M. E., Conrad, K. P., Parry, L. J. Relaxin mediates uterine artery compliance during pregnancy and increases uterine blood flow. FASEB J. 26, 4035–4044 (2012). www.fasebj.org

Moderate Ascorbate Deficiency Increases Myogenic Tone of Arteries From Pregnant but Not Virgin Ascorbate–Dependent Rats

Plasma ascorbic acid is decreased in women with the pregnancy disorder preeclampsia. We used a mutant strain of rats (Osteogenic Disorder Shionogi), dependent on dietary sources of vitamin C, to investigate whether reduced intake of the vitamin would differentially affect vascular function in late-pregnant (day 19) and age-matched virgin rats. The animals were given either 1 mg/mL of ascorbic acid ad libitum in drinking water [fully supplemented (FS)] or 0.25 mg/mL [marginally supplemented (MS)]. Fetal weights were 21% lower in MS than FS rats, whereas mean maternal weights and pup numbers did not differ. Small mesenteric arteries (diameter, 268±7 &mgr;m) were mounted in a pressurized arteriograph. Myogenic reactivity (contractile response to step increases in intraluminal pressure) was increased in arteries from MS pregnant compared with FS pregnant rats to levels observed in virgin rats. Ascorbic acid intake did not affect myogenic responses of arteries from virgin rats. Hence, the normal pregnancy-induced reduction in myogenic reactivity was abrogated in MS pregnant animals. Inhibition of nitric oxide synthase had no effect on the myogenicity of arteries from virgin or MS pregnant rats but increased myogenicity of FS pregnant rats to the level of MS pregnant rats. Free radical scavengers reversed the accentuated myogenicity of MS pregnant rats without affecting FS pregnant or virgin rat arteries. These data indicate that moderate ascorbate deprivation increases mesenteric artery myogenic responsiveness during pregnancy. This increase may result from a decrease in nitric oxide–mediated modulation of the myogenic contractile response.

Colony social stress differentially alters blood pressure and resistance-sized mesenteric artery reactivity in SHR/y and WKY male rats.

Increased sympathetic nervous system (SNS) activity, testosterone, and spontaneously hypertensive rat Y chromosome (SHR Yc) play a role in a genetic model of hypertension. Male rats with the SHR Yc and Wistar-Kyoto (WKY) autosomes (denoted SHR/y) exhibit these characteristics when compared to rats with the WKY Yc and WKY autosomes (denoted WKY). We hypothesized that chronic social stress will increase blood pressure and SNS activity more in SHR/y males compared to WKY males, resulting in increased myogenic reactivity along with decreased vasoconstriction of small mesenteric arteries. SHR/y and WKY males were housed in strain- specific colonies (10 males with 10 females) or as controls (10 males). Systolic blood pressure (SBP) and blood samples were collected prior to termination. Second-order mesenteric arteries were studied using a pressure arteriograph in which myogenic reactivity and phenylephrine (PE) responsiveness were measured. SHR/y colony SBP, and circulating norepinephrine and testosterone concentrations were elevated compared to control and WKY colony males (p < 0.05). Mesenteric artery myogenic reactivity was increased in SHR/y colony males (p < 0.001). Mesenteric arteries from SHR/y colony males exhibited a significant decrease in PE-induced constriction. Colony social stress elevated both SNS activity and testosterone level which may be responsible for the increased mesenteric artery myogenic reactivity, and SBP as noted in SHR/y males.

Increased Myogenic Responses of Resistance-Sized Mesenteric Arteries After Reduced Uterine Perfusion Pressure in Pregnant Rats

Objective. A central component of preeclampsia is a reduction in utero-placental perfusion. We tested the hypothesis that vascular reactivity of second – order mesenteric arteries would be increased in a pregnant rat with reduced uterine perfusion pressure (RUPP). Methods. Pregnant 10–12 week old Sprague-Dawley rats underwent RUPP surgery on gestational day 14, and experiments were carried out on day 20. SHAM operated animals were used as controls. Resistance caliber mesenteric arteries (200–250 μm) were isolated, myogenic reactivity and responses to vasoconstrictor and vasodilator agonists were assessed utilizing a pressurized arteriograph system. Results. RUPP resulted in maternal hypertension and reductions in fetal number and weight. Resistance caliber mesenteric arteries (200–250 μm) were isolated, myogenic reactivity and responses to vasoconstrictor and vasodilator agonists were assessed utilizing a pressurized arteriograph system. Myogenic reactivity responses were normalized as a percent change in vessel diameter from an initial diameter at 20 mmHg. The mesenteric arteries from RUPP animals exhibited a significant increase in myogenic reactivity compared to SHAM controls (p < 0.05). This increased myogenicity was reversed with prostaglandin inhibition, suggesting a role for a vasoconstrictor prostaglandin. In addition to alterations in myogenic reactivity, resistance-sized arteries from RUPP animals have decreased responses to nitric oxide (NO) as evidenced by decreases in responses to methacholine (ME; P < 0.05) and no change in myogenic reactivity after NO synthase blockade. Conclusion. RUPP alters the behavior of resistance caliber arteries to favor a more contractile phenotype with decreased in NO responses, which is similar to what is seen in preeclampsia.

Androgens alter brain catecholamine content and blood pressure in the testicular feminized male rat.

Abstract Androgens interact with catecholamines in the central nervous system (CNS) to regulate many physiological processes including blood pressure (BP). To test the hypothesis that testosterone (T) and 5a-dihydrotestosterone (DHT) modulate CNS catecholamines and BP through androgen receptor (AR)-dependent and independent mechanisms, we used the testicular feminized male (Tfm) rat. Females that carry the AR mutation (Tfm mutation) on the X chromosome were bred with spontaneously hypertensive rat (SHR) males. The normal AR male and Tfm offspring were divided into groups: control, castrated, castrated, and T or (DHT) replacement. In both AR normal and Tfm males, BP was reduced by castration, but T restored BP in both groups. In the amygdale, castration decreased dopamine (DA) in both strains and both T and DHT restored it. In the bed nucleus of the stria terminalis castration increased DA which was further increased by DHT and reduced to normal by T in both strains. In the frontal cortex, castration reduced DA content in both strains but only T restored it to normal in SHR but not in Tfm. Brain norepinephrine (NE) content showed a significant strain effect for the preoptic area (POA), but no treatment effect. Although castration did not change NE in the amygdala or POA in either strain, both T and DHT increased NE in the Tfm castrates. Blood pressure was influenced by T manipulation and correlated most significantly with DA content in the amygdala, frontal cortex, and stria terminalis. These data demonstrate an action of androgen on brain catecholamines and BP, which is independent of the classic androgen receptor.

Hypertension Induced by Episodic Reductions in Uteroplacental Blood Flow in Gravid Rat

Background. The etiology of preeclampsia remains unclear. Animal modeling of preeclampsia has been useful; however, no model to date represents episodic changes in uteroplacental blood flow that may occur in preeclampsia. Objective. To develop a gravid rat model characterized by episodic reductions in uteroplacental blood flow. Method. Pregnant Sprague Dawley rats were used and subjected to SHAM, reduced uterine perfusion pressure (RUPP), or aortic occlusion on gestational Day 14. Aortic occlusion surgery consisted of implantation of a silastic vascular occluder around the abdominal aorta and silver clips around the uterine–ovarian arteries. Aortic occlusion animals were subjected to five consecutive days of occlusion (40% reduction) each session lasting 1 h. On Day 21, maternal mean arterial pressure (MAP) and fetal morphology were assessed. For isolated blood vessels, resistance-sized mesenteric arteries were harvested and mounted on a pressure arteriograph. Result. Occluder animals experienced a 10 mmHg rise in MAP as compared to SHAM (p < 0.05), and RUPP MAP was significantly increased as compared to control subjects (p < 0.05). Pups from Occluder animals exhibited a decrease in fetal weight as compared to SHAM (p < 0.05), but an increase in fetal weight as compared to RUPP (p < 0.05). Myogenic reactivity of second-order mesenteric arteries increased in Occluder animals as compared to SHAM (p < 0.05), but were similar to that of RUPP. Conclusion. Episodic reductions in uteroplacental blood flow play a crucial role in the altered vascular reactivity seen in Occluder animals and may represent a new model to investigate the mechanisms associated with episodic reductions in uteroplacental blood flow in pathological pregnancies.

Testosterone increases myogenic reactivity of second-order mesenteric arteries in both defective and normal androgen receptor adult male rats.

BACKGROUND Testosterone (T) and the androgen receptor (AR) are involved in mechanisms associated with hypertension and vessel reactivity. OBJECTIVE To investigate T and the AR on blood vessel reactivity, testicular feminized male (TFM; AR deficient males) and normal androgen receptor (NAR) male rats were used. Therefore, if the functional AR is necessary for plasma T to regulate vessel responsiveness, TFM males will exhibit altered vessel function compared to NAR males. METHODS Adult (16 weeks of age) TFM or NAR males were assigned to the following treatment groups: gonadal intact controls (CONT), castrate (CAST), or castrate with T replacement (CAST+T) with (n=8-10/group). RESULTS Plasma T followed a consistent pattern with CAST+T elevated compared to CONT and CAST TFM and NAR males. In addition, CAST plasma T was significantly decreased compared to CONT and CAST+T in TFM and NAR males. In a similar manner for systolic blood pressure (SBP), CAST lowered SBP compared to CONT in both NAR and TFM. Following 8 weeks of treatment, second-order mesenteric artery responses to changes in intraluminal pressure (myogenic reactivity) were analyzed using a pressure arteriograph system. Both TFM (P < 0.05) and NAR (P < 0.05) CAST groups revealed a decrease in myogenic reactivity compared to CONT. Following T treatment the TFM CAST+T myogenic reactivity returned to CONT levels, whereas the NAR CAST+T myogenic reactivity increased a further 10%. CONCLUSION The results of this study indicate that T differentially regulates mesenteric artery reactivity in TFM and NAR males. Our data also demonstrate that both AR and/or non-AR mediated mechanisms may partially contribute to SBP regulation.