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This study aimed to investigate whether hydralazine could reduce cardiac ischemia/reperfusion (I/R) injury in rats. Anesthetized male Sprague-Dawley rats underwent myocardial I/R injury. Saline, hydralazine (HYD, 10-30 mg/kg) was administered intraperitoneally 10 min before reperfusion. After 30 min of ischemia and 24 h of reperfusion, the myocardial infarct size was determined using TTC staining. Heart function and oxidative stress were determined through biochemical assay and DHE staining. HE staining was used for histopathological evaluation. Additionally, the cardiomyocytes apoptosis and protein expression of PI3K-Akt-eNOS pathway marker were detected by TUNEL and Western blotting. The serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and reactive oxygen species were significantly elevated in cardiac I/R group, but the superoxide dismutase (SOD) level was suppressed. However, intraperitoneal pretreatment with hydralazine at a dose of 10-30 mg/kg before cardiac I/R significantly limited the increase in CK-MB, LDH, oxidative stress, inflammatory factors, histological damage and apoptosis in the hearts. In addition, hydralazine also increased p-PI3K, p-AKT, p-eNOS expression and decreased Cleaved Caspase-3, Cleaved Caspase-9 expression in the hearts. Our results suggest that the cardioprotective effect of hydralazine against I/R injury might be a cooperation of the inhibition of oxidative stress, inflammatory response, apoptosis with the motivation of eNOS phosphorylation via activating the PI3K/AKT signal pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It has been reported that the serine protease kallikrein stimulates and that aprotinin, a protease inhibitor, inhibits renal renin secretion. Since direct stimulation of the protease-activated receptor (PAR) 2 increases renin secretion in isolated perfused mouse kidneys, we hypothesized that activation of PAR2 receptors by serine proteases could be involved in the synthesis and secretion of renin in vivo. We therefore determined the response of plasma renin concentration (PRC) to acute intraperitoneal administration of the PAR2 agonist SLIGRL, isoproterenol, hydralazine, furosemide, losartan, or lipopolysaccharide in conscious wild-type (WT) and Par2-deficient mice. Basal PRC was not different in Par2-deficient mice compared with WT mice. All six acute treatments caused significant increases of PRC in both WT and Par2-deficient mice. The response was significantly lower only in endotoxin-treated Par2-deficient mice. Chronic treatment with losartan, low salt intake, the combination of both, or furosemide caused an increase of PRC and renin mRNA in WT mice, whereas a high salt intake caused a decrease. Alterations in PRC and renal renin mRNA expression were not different between WT and Par2 -/- mice in response to chronic treatments. Par2-deficiency did not alter furosemide-induced diuresis and natriuresis. Systolic blood pressure responses to chronic treatments were not different between WT and Par2 -/- mice. In conclusion, deficiency of Par2 receptors does not alter renin secretion and renin gene expression modulated by a variety of typical maneuvers. However, activation of Par2 receptors by serine proteases seems to be of importance for renin secretion in the context of inflammation.

Many studies have suggested that renal T cell infiltration contributes to the pathogenesis of salt-sensitive hypertension. To investigate this mechanism further, we determined T cell profiles in the kidney and lymphoid tissues as a function of blood pressure in the female Envigo Dahl salt-sensitive (SS) rat maintained on low-Na + (LS) diet. Mean arterial pressure and heart rate were measured by telemetry in SS rats from 1 mo old (juvenile) to 4 mo old. Normotensive salt-resistant (SR) rats were included as controls. Frequencies of T helper (CD4 + ) cells were greater in the kidney, lymph nodes, and spleen in 4-mo-old hypertensive SS rats compared with normotensive SR animals and SS juvenile rats, suggesting that renal T cell infiltration contributes to hypertension in the SS rat on a LS diet. At 1.5 mo, half of the SS rats were treated with vehicle (Veh), and the rest received hydralazine (HDZ; 25 mg·kg -1 ·day -1 ) for 11 wk. HDZ impeded the development of hypertension compared with Veh-treated control rats [mean arterial pressure: 157 ± 4 mmHg in the Veh-treated group ( n = 6) vs. 133 ± 3 mmHg in the HDZ-treated group ( n = 7), P < 0.001] without impacting T helper cell frequencies in the tissues, suggesting that HDZ can overcome mechanisms of hypertension driven by renal T cell infiltration under the LS diet. Renal frequencies of CD4 + CD25 + and CD4 + CD25 + FoxP3 + regulatory T cells were significantly higher in 4-mo-old hypertensive rats compared with normotensive SR rats and SS juvenile rats, suggesting that these T cell subpopulations play a compensatory role in the development of hypertension. Greater understanding of these T cell populations could lead to new therapeutic targets for treating inflammatory diseases associated with hypertension.

Vascular endothelial growth inhibitor (VEGI; also referred to as TNFSF15 or TL1A) is involved in the modulation of vascular homeostasis. VEGI is known to operate via two receptors: Death receptor‑3 (DR3) and decoy receptor‑3 (DcR3). DR3, which is thus far the only known functional receptor for VEGI, contains a death domain and induces cell apoptosis. DcR3 is secreted as a soluble protein and antagonizes VEGI/DR3 interaction. Overexpression of DcR3 and downregulation of VEGI have been detected in a number of cancers. The aim of the present study was to investigate the effects of sodium valproate (VPA), a histone deacetylase inhibitor, in combination with hydralazine hydrochloride (Hy), a DNA methylation inhibitor, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Combination treatment with Hy and VPA synergistically induced the expression of VEGI and DR3 in both OS and HMVE cells, without inducing DcR3 secretion. In addition, it was observed that the combination of VPA and Hy significantly enhanced the inhibitory effect on vascular tube formation by VEGI/DR3 autocrine and paracrine pathways. Furthermore, the VEGI/VEGF‑A immune complex was pulled down by immunoprecipitation. Taken together, these findings suggest that DNA methyltransferase and histone deacetylase inhibitors not only have the potential to induce the re‑expression of tumor suppressor genes in cancer cells, but also exert anti‑angiogenic effects, via enhancement of the VEGI/DR3 pathway and VEGI/VEGF‑A interference.

Women with Marfan syndrome (MFS) are at high risk for pregnancy-associated aortic dissection. Pathogenic models that singularly invoke hemodynamic stress are difficult to reconcile with predominant postnatal occurrence of aortic tear, often occurring weeks to months after delivery. In consideration of events that peak at term, are sustained after delivery, and might synergize with previously defined signaling pathways implicated in aneurysm progression, we examined the hormone oxytocin, which initiates uterine contraction and milk letdown for the duration of lactation through phosphorylation of extracellular signal-regulated kinase (ERK). In a mouse model of MFS that shows highly penetrant postnatal aortic dissection, risk was strongly attenuated by preventing lactation or use of an oxytocin receptor antagonist. Survival correlated inversely with the extent of ERK activation in the aortic wall, and strong protection was observed upon attenuation of ERK phosphorylation using an inhibitor of ERK kinase (MEK) or the U.S. Food and Drug Administration-approved medication hydralazine, offering potential therapeutic strategies for pregnancy-associated vascular catastrophe in the setting of MFS., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Hydralazine has been reported as a selective mechanism-based inactivator of aldehyde oxidase (AO) and it is widely used in the pharmaceutical industry for reaction phenotyping to estimate fraction metabolized by AO and to identify AO substrates. In this study, however, hydralazine was found to inhibit CYP1A2, 2B6, 2D6, and 3A in human suspension hepatocytes under reaction phenotyping assay conditions, at concentrations that chemically knocked out most of the AO activities (≥50 μM). Furthermore, hydralazine is a time-dependent inhibitor of CYP1A2. Based on these findings, precautions need to be taken when using hydralazine as an AO inhibitor for in vitro studies because fraction metabolized by AO is likely to be overestimated and the likelihood of false positives in identifying AO substrates increases., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Purpose: Radiation therapy (RT) offers an important and curative approach to treating prostate cancer, but it is associated with a high incidence of erectile dysfunction (ED). It is not clear whether the etiology of radiation-induced ED (RI-ED) is driven by RT-mediated injury to the vasculature, the nerves, or both. This pilot study sought to distinguish the effects of vascular and nerve injury in RI-ED by applying a vascular radioprotectant in a rat model of prostate RT., Methods: A single dose of the thrombopoietin mimetic (TPOm; RWJ-800088), previously shown to mitigate radiation-induced vascular injury, was administered 10 minutes after single-fraction conformal prostate RT. Nine weeks after RT, rats were assessed for erectile and arterial function. Nerve markers were quantified with reverse transcriptase polymerase chain reaction. Immunofluorescent microscopy further characterized vascular effects of RT and TPOm., Results: Sham animals and animals that received RT and TPOm showed significant arterial vasodilation in response to systemic hydralazine (24.1% ± 7.3% increase; P = .03 in paired t test). However, animals that received RT and vehicle were unable to mount a vasodilatory response (-7.4% ± 9.9% increase; P = .44 in paired t test). TPOm prevented RT-induced change in the penile artery cross-sectional area (P = .036), but it did not ameliorate cavernous nerve injury as evaluated by gene expression of neuronal injury markers. Despite significant structural and functional vascular protective effects and some trends for differences in nerve injury/recovery markers, TPOm did not prevent RI-ED at 9 weeks, as assessed by intracavernous pressure monitoring after cavernous nerve stimulation., Conclusions: These data suggest that vascular protection alone is not sufficient to prevent RI-ED and that cavernous nerve injury plays a key role in RI-ED. Further research is required to delineate the multifactorial nature of RI-ED and to determine if TPOm with modified dosing regimens can mitigate against nerve injury either through direct or vascular protective effects., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Blood pressure variability (BPV) is an independent predictor for early hematoma expansion, neurologic deterioration, and mortality. There are no studies on the effect of intravenous (IV) antihypertensive drugs on BPV. We sought to determine whether patients have more BPV with certain antihypertensive agents, in particular the effect of IV nicardipine., Methods: We conducted a single-center, retrospective chart review of individuals diagnosed with spontaneous intracerebral hemorrhage (ICH) receiving labetalol, hydralazine, and/or nicardipine within 24 h of hospital admission to assess the primary endpoint of BPV, defined as the standard deviation of systolic BP, with labetalol and/or hydralazine compared to nicardipine ± labetalol and/or hydralazine. Repeated measures linear regression was performed to compare BPV over 24 h between regimens, and Cox proportional hazards regression was used to compare the time to goal SBP between regimens., Results: Of the 1330 patients screened, 272 were included in our analysis; those included had a mean age of 69 years with 87.9% of Caucasian race. A total of 164 patients received IV bolus antihypertensives alone (labetalol, hydralazine or both), and 108 patients received IV nicardipine with or without additional IV boluses (labetalol, hydralazine, or both). Those who had IV nicardipine had significantly less BPV (p = 0.04) and was more likely to attain an SBP goal < 140 mmHg (p < 0.01)., Conclusion: Our study suggests patients with ICH who do not receive a nicardipine-based antihypertensive regimen have more BPV, which has been associated with poor clinical outcomes. Prospective, randomized, controlled trials are needed to determine the impact of specific antihypertensive regimens on clinical outcomes.

In this study, we investigated whether hydralazine could reduce renal ischemia and reperfusion (I/R) injury in rats. Renal I/R was induced by a 70-min occlusion of the bilateral renal arteries and a 24-h reperfusion, which was confirmed by the increased the mortality, the levels of blood urea nitrogen (BUN), blood creatinine (Cr), renal tissue NO and the visible histological damage of the kidneys. Apoptosis was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) staining. Furthermore, the serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly elevated in renal I/R group, while the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) levels were suppressed. However, intragastric pretreatment with hydralazine at doses of 7.5-30 mg/kg before renal I/R significantly limited the increase in mortality, BUN, Cr, oxidative stress, inflammatory factors, histological damage and apoptosis in the kidneys. In addition, hydralazine also increased p-AKT, Bcl-2 expression and decreased iNOS, Bax, cleaved caspase-3 expression in the kidneys. In conclusion, hydralazine reduced renal I/R injury probably via inhibiting NO production by iNOS/NO pathway, inhibiting oxidative stress, inflammatory response and apoptosis by a mitochondrial-dependent pathway., (Copyright © 2018. Published by Elsevier B.V.)

During cellular metabolism, spontaneous oxidative damage to unsaturated lipids generates many electrophilic carbonyl compounds that readily attack cell macromolecules, forming adducts that are potential drivers of tissue dysfunction. Since such damage is heightened in many degenerative conditions, researchers have assessed the efficacy of nucleophilic carbonyl-trapping drugs in animal models of such disorders, anticipating that they will protect tissues by intercepting toxic lipid-derived electrophiles (LDEs) within cells. This Commentary explores recent animal evidence for carbonyl scavenger efficacy in two disparate yet significant conditions known to involve LDE production, namely spinal cord injury (SCI) and alcoholic liver disease (ALD). Primary emphasis is placed on studies that utilised hydralazine, a clinically-approved "broad-spectrum" scavenger known to trap multiple LDEs. In addition to reviewing recent studies of hydralazine efficacy in animal SCI and ALD models, the Commentary reviews new insights concerning novel lifespan- and healthspan-extending properties of hydralazine obtained during studies in model invertebrate organisms, since the mechanisms involved seem of likely benefit during the treatment of degenerative disease. Finally, noting that human translation of the histoprotective properties of hydralazine have been limited, the final section of the Commentary will address two obstacles that hamper clinical translation of LDE-trapping therapies while also suggesting potential strategies for overcoming these problems., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Posterior reversible encephalopathy syndrome (PRES) is a newly defined syndrome; therefore, this transient clinical condition is not well known and probably underdiagnosed. It develops quickly with symptoms that are usually indistinguishable from eclampsia. Nurses need to be knowledgeable and aware of identifying symptoms and appropriate treatment. The condition is thought to share pathophysiology with eclampsia, and it is suggested that endothelial dysfunction combined with hypertension causes disruption in the blood brain barrier resulting in cerebral edema. Seizures develop secondary to cerebral edema, and mark later stages of the disease. Treatment is aimed at reducing blood pressure (BP) with antihypertensive therapy and seizure control with magnesium sulfate. When PRES is treated early, symptoms typically disappear within a few days and imaging studies normalize in several weeks. Permanent brain damage can occur if diagnosis and treatment are delayed. If PRES is suspected, thorough focused assessments and increased communication among the healthcare team are essential for patient care. When pregnant or postpartum women present with elevated BP accompanied with neurologic symptoms, imaging studies should be considered. An exemplar case is presented of a woman with normal prenatal course that is complicated by rapidly developing preeclampsia, eclampsia, and PRES.

Previous research has characterized the important role of aldehyde oxidases (AOX) in biotransformation of N-heterocyclic therapeutic drugs and environmental contaminants in mammals. Research pertaining to AOX activity in non-mammalian vertebrates, however, is scarce, despite its biological role as a potentially important metabolic pathway for xenobiotics. One of the limiting factors of research on AOX is that available photometric methods are relatively insensitive, limited in throughput, and prone to cross-reactivity from other enzymes. Therefore, this study aimed to develop a novel and improved fluorometric AOX assay. This assay is based on the conversion of the exogenous aldehyde substrate 4-(dimethyl)amino cinnamaldehyde to its corresponding fluorescent acid by AOX, and was evaluated using partially purified hepatic cytosol from rat, human, and rainbow trout. Purification of native cytosol by heat treatment and ammonium sulfate precipitation resulted in increased specific activity of AOX. Michaelis-Menten kinetic parameters (K m and V max ) were comparable to values previously generated by photometric methods. Furthermore, effects of the inhibitor hydralazine on AOX activity revealed half maximal inhibitory concentrations comparable to those generated using conventional methods. Product identity was confirmed by liquid chromatography and mass spectrometry. In summary, this study successfully developed a rapid and sensitive assay for determination of AOX activity in across different vertebrate species that is 4- to 10-fold more sensitive compared to conventional absorbance-based methods. It can be applied in environmental, toxicological, and pharmacological studies relating to identification of AOX substrates, as well as the induction of AOX expression through drugs and environmental contaminants., (Copyright © 2018. Published by Elsevier Inc.)

The use of direct acting vasodilators (the combination of hydralazine and isosorbide dinitrate -Hy+ISDN-) in heart failure with reduced ejection fraction (HFrEF) is supported by evidence, but rarely used.However, treatment with Hy+ISDN is guideline-recommended for HFrEF patients who cannot receive either angiotensin-converting enzyme inhibitors or angiotensin receptor blockers due to intolerance or contraindication, and in self-identified African-American HFrEF patients who are symptomatic despite optimal neurohumoral therapy.The Hy+ISDN combination has arterial and venous vasodilating properties. It can decrease preload and afterload, decrease left ventricular end-diastolic diameter and the volume of mitral regurgitation, reduce left atrial and left ventricular wall tension, decrease pulmonary artery pressure and pulmonary arterial wedge pressure, increase stroke volume, and improve left ventricular ejection fraction, as well as induce left ventricular reverse remodelling. Furthermore, Hy+ISDN combination has antioxidant property, it affects endothelial dysfunction beneficially and improves NO bioavailability. Because of these benefits, this combination can improve the signs and symptoms of heart failure, exercise capacity and quality of life, and, most importantly, reduce morbidity and mortality in well-defined subgroups of HFrEF patients.Accordingly, this therapeutic option can in many cases play an essential role in the treatment of HFrEF.

Nuclear factor (erythroid-derived 2)-like 2 and its Caenorhabditis elegans ortholog, SKN-1, are transcription factors that have a pivotal role in the oxidative stress response, cellular homeostasis, and organismal lifespan. Similar to other defense systems, the NRF2-mediated stress response is compromised in aging and neurodegenerative diseases. Here, we report that the FDA approved drug hydralazine is a bona fide activator of the NRF2/SKN-1 signaling pathway. We demonstrate that hydralazine extends healthy lifespan (~25%) in wild type and tauopathy model C. elegans at least as effectively as other anti-aging compounds, such as curcumin and metformin. We show that hydralazine-mediated lifespan extension is SKN-1 dependent, with a mechanism most likely mimicking calorie restriction. Using both in vitro and in vivo models, we go on to demonstrate that hydralazine has neuroprotective properties against endogenous and exogenous stressors. Our data suggest that hydralazine may be a viable candidate for the treatment of age-related disorders.

Aldehyde oxidase (AOX) is a cytosolic enzyme responsible for the metabolism of some drugs and drug candidates. AOX catalyzes the oxidative hydroxylation of substrates including several aliphatic and aromatic aldehydes, and nitrogen-containing heterocyclic compounds. AOX is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite and hydroxamic acids. These reductive transformations are not well understood and are generally believed to only occur at low oxygen concentrations. In this study, we used 5-nitroquinoline (5NQ) as a substrate to further understand both the oxidative and the reductive transformations catalyzed by AOX. In vitro reaction of 5NQ with AOX under aerobic conditions generated the oxidized (2-oxo-5-nitroquinoline, 2-oxo-5NQ), the reduced (5-aminoquinoline, 5AQ) and the oxidized/reduced (2-oxo-5-aminoquinoline, 2-oxo-5AQ) metabolites. Interestingly, in human liver cytosol, co-incubation of 5NQ and known AOX oxidative substrates DACA and phthalazine significantly increased the yield of the reduced metabolite, while oxidized metabolites production decreased. These data indicate that 5NQ can be reduced at atmospheric oxygen concentrations and that the reductive transformation occurs at a second site that is kinetically distinct from the oxidative site., (Copyright © 2017. Published by Elsevier Inc.)

Diabetes-induced neuropathic pain (DNP) substantially influences people's life qualities. Hyperglycemia-induced excess free radicals have been considered as the most critical mechanisms underlying DNP. As an unsaturated aldehyde and a reactive product of lipid peroxidation, acrolein plays critical roles in diabetic nephropathy and inflammatory pain. We sought to determine whether acrolein is involved in DNP in this study. Diabetes was induced by a single intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin (STZ). An acrolein scavenger hydralazine (5 mg/kg) was administered through a daily injection for 4 weeks, starting immediately within 30 min after STZ injection. Western blot showed that hydralazine could effectively inhibit STZ-induced upregulation of acrolein in the spinal dorsal horn on day 7-28 after STZ injection. Behavioral tests showed that STZ injection induced significant mechanical allodynia and thermal hyperalgesia, which could be alleviated by hydralazine. Immunofluorescent histochemistry and Western blot showed that STZ induced significant microglial activation. ELISA data indicated upregulation of inflammatory cytokines IL-1β and TNF-α expression in the spinal dorsal horn. Furthermore, hydralazine effectively attenuated microglial activation and expression of inflammatory mediators. Our data indicate that acrolein might be involved in the development of neuroinflammation and behavioral consequences of DNP. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1858-1864, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

This study investigated the effect of epinephrine (a vasoconstrictor) and hydralazine (a vasodilator) on the hepatic disposition of 5-fluorouracil (5-FU) after application to the surface of the liver in rats. Normal livers were compared with a Walker 256 carcinoma cell tumor model. A cylindrical diffusion cell was attached to the liver surface. 5-Fluorouracil was added into the diffusion cell in combination with vasomodulators or after pretreatment with epinephrine. After selected treatment times, the 5-FU concentrations were assayed at three sites in the excised livers. The 5-FU concentration in the region under the cell attachment site (site 1) was significantly higher after concomitant application of 5-FU and epinephrine, compared with 5-FU alone, and increased in an epinephrine dose-dependent manner. On the other hand, preferential distribution of 5-FU at site 1 was not seen when applied in combination with hydralazine. After 10 min of epinephrine pretreatment, the concentration of 5-FU at site 1 was approximately two times higher than that for the control. Furthermore, the 5-FU concentration at site 1 of the tumor model was greatly increased compared with the normal liver. These results suggest that application of epinephrine to the liver surface might enhance the accumulation of 5-FU at the desired target site., (Copyright © 2017 John Wiley & Sons, Ltd.)

Previous studies have demonstrated that a range of stimuli activate neurons, including catecholaminergic neurons, in the ventrolateral medulla. Not all catecholaminergic neurons are activated and other neurochemical content is largely unknown hence whether stimulus specific populations exist is unclear. Here we determine the neurochemistry (using in situ hybridization) of catecholaminergic and noncatecholaminergic neurons which express c-Fos immunoreactivity throughout the rostrocaudal extent of the ventrolateral medulla, in Sprague Dawley rats treated with hydralazine or saline. Distinct neuronal populations containing PPCART, PPPACAP, and PPNPY mRNAs, which were largely catecholaminergic, were activated by hydralazine but not saline. Both catecholaminergic and noncatecholaminergic neurons containing preprotachykinin and prepro-enkephalin (PPE) mRNAs were also activated, with the noncatecholaminergic population located in the rostral C1 region. Few GlyT2 neurons were activated. A subset of these data was then used to compare the neuronal populations activated by 2-deoxyglucose evoked glucoprivation (Brain Structure and Function (2015) 220:117). Hydralazine activated more neurons than 2-deoxyglucose but similar numbers of catecholaminergic neurons. Commonly activated populations expressing PPNPY and PPE mRNAs were defined. These likely include PPNPY expressing catecholaminergic neurons projecting to vasopressinergic and corticotrophin releasing factor neurons in the paraventricular nucleus, which when activated result in elevated plasma vasopressin and corticosterone. Stimulus specific neurons included noncatecholaminergic neurons and a few PPE positive catecholaminergic neuron but neurochemical codes were largely unidentified. Reasons for the lack of identification of stimulus specific neurons, readily detectable using electrophysiology in anaesthetized preparations and for which neural circuits can be defined, are discussed., (© 2017 Wiley Periodicals, Inc.)

Objective: Sympathoexcitation plays an important role in the pathogenesis of hypertension in patients with chronic kidney disease (CKD). The paraventricular nucleus of the hypothalamus (PVN) in the brain controls sympathetic outflow through γ-amino butyric acid (GABA)-ergic mechanisms. Renal denervation (RDN) exerts a long-term antihypertensive effect in hypertension with CKD; however, the effects of RDN on sympathetic nerve activity and GABA-ergic modulation in the PVN are not clear. We aimed to elucidate whether RDN modulates sympathetic outflow through GABA-ergic mechanisms in the PVN in hypertensive mice with CKD., Methods and Results: In 5/6-nephrectomized male Institute of Cancer Research mice (Nx) at 4 weeks after nephrectomy, systolic blood pressure (SBP) was significantly increased, accompanied by sympathoexcitation. The Nx-mice underwent RDN or sham operation, and the mice were divided into three groups (Control, Nx-Sham, and Nx-RDN). At 2 weeks after RDN, SBP was significantly decreased and urinary sodium excretion was increased in Nx-RDN compared with Nx-Sham. Urinary norepinephrine excretion (uNE) levels did not differ significantly between Nx-RDN and Nx-Sham. At 6 weeks after RDN, SBP continued to decrease and uNE levels also decreased in Nx-RDN compared with Nx-Sham. Bicuculline microinjection into the PVN increased mean arterial pressure and lumbar sympathetic nerve activity in all groups. The pressor responses and change in lumbar sympathetic nerve activity were significantly attenuated in Nx-Sham, but were enhanced in Nx-RDN at 6 weeks after RDN., Conclusions: The findings from the present study indicate that RDN has a prolonged antihypertensive effect and, at least in the late phase, decreases sympathetic nerve activity in association with enhanced GABA-ergic input into the PVN in mice with CKD., (Copyright © 2016 Elsevier B.V. All rights reserved.)

It has been well established that chronic pressure overload resulting from hypertension leads to ventricular hypertrophy and electrophysiological remodeling. The transient outward potassium current (I to ) reduction described in hypertensive animals delays ventricular repolarization, leading to complex ventricular arrhythmias and sudden death. Antihypertensive drugs, as angiotensin-converting enzyme inhibitors (ACEi), can restore I to and reduce the incidence of arrhythmic events. The purpose of this study was to evaluate the differential effects of long-term treatment with ACEi or direct-acting smooth muscle relaxant on the I to of left and right ventricle myocytes of spontaneously hypertensive rats (SHR). Animals were divided into four groups: normotensive Wistar-Kyoto rats (WKY), hypertensive (SHR), SHR treated for 6 weeks with enalapril 10 mg/kg/day (SHRE), or hydralazine 20 mg/kg/day (SHRH). Systolic blood pressure (SBP) and hypertrophy index (heart weight/body weight (HW/BW)) were determined at the end of treatment period. Cell membrane capacitance (C m ) and I to were assessed in cardiomyocytes isolated from left and right ventricles. The SHR exhibited significantly increased SBP and HW/BW when compared to the WKY. The treated groups, SHRE and SHRH, restored normal SBP but not HW/BW. The SHR group exhibited a diminished I to in the left but not the right ventricle. Both the treated groups restored I to in the left ventricle. However, in the right ventricle, only enalapril treatment modified I to . The SHRE group exhibited a significant increase in I to compared to all the other groups. These findings suggest that enalapril may increase I to by a pressure overload independent mechanism.

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor-α (TNF-α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose TNF-α (0.5 ng/mL) or TNF-α plus soluble fms-like tyrosine kinase-1 (sFlt-1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR-8/SVneo cells were co-cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF-α on eNOS mRNA expression. After pre-incubating endothelial cells with TNF-α and sFlt-1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF-α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF-α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF-α. The anti-angiogenic molecule sFlt-1 may antagonise the potential benefit of these medications by interfering with the NOS pathway., (© 2016 John Wiley & Sons Australia, Ltd.)

Hypertensive disorders of pregnancy globally affect 6-8% of gestation and remain a major cause of both foetal and maternal morbidity and mortality. However, the antihypertensive medications for the patients of this disease are strictly limited due to the teratogenic potentials. Here, we found that tele-methylhistamine (tMH) increased in response to the administration of hydralazine (Hdz), a vasodilative agent, in the pregnancy-associated hypertensive (PAH) mice. Hdz abrogated the degradation of tMH catalyzed by monoamine oxidase B (MAO-B) in vitro. These results suggested that Hdz inhibited the MAO-B activity and consequently tMH increased in the maternal circulation of PAH mice., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The p300/CBP-associated factor (PCAF) and related GCN5 bromodomain-containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine-based L-45 (dubbed L-Moses) as the first potent, selective, and cell-active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)-(-)-norephedrine furnished L-45 in enantiopure form. L-45 was shown to disrupt PCAF-Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co-crystal structure of L-45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L-45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell-permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The neovascularization of atherosclerotic lesions is involved in plaque development and may contribute to intraplaque hemorrhage and plaque fragilization and rupture. Among the various proangiogenic agents involved in the neovascularization process, proatherogenic oxidized LDLs (oxLDLs) contribute to the formation of tubes via the generation of sphingosine 1-phosphate (S1P), a major mitogenic and proangiogenic sphingolipid mediator. In this study, we investigated whether 4-hydroxynonenal (4-HNE), an aldehydic lipid oxidation product abundantly present in oxLDLs, contributes to their proangiogenic properties. Immunofluorescence analysis of human atherosclerotic lesions from carotid endarterectomy showed the colocalization of HNE-adducts with CD31, a marker of endothelial cells, suggesting a close relationship between 4-HNE and neovessel formation. In vitro, low 4-HNE concentration (0.5-1  µ M) elicited the formation of tubes by human microvascular endothelial cells (HMEC-1), whereas higher concentrations were not angiogenic. The formation of tubes by 4-HNE involved the generation of reactive oxygen species and the activation of the sphingolipid pathway, namely, the neutral type 2 sphingomyelinase and sphingosine kinase-1 (nSMase2/SK-1) pathway, indicating a role for S1P in the angiogenic signaling of 4-HNE. Carbonyl scavengers hydralazine and bisvanillyl-hydralazone inhibited the nSMase2/SK1 pathway activation and the formation of tubes on Matrigel® evoked by 4-HNE. Altogether, these results emphasize the role of 4-HNE in the angiogenic effect of oxLDLs and point out the potential interest of pharmacological carbonyl scavengers to prevent the neovascularization process.

Background/aims: Understanding cellular mechanisms induced by hypoxia is fundamental to reduce blood-brain barrier (BBB) disruption. Nevertheless, the investigation of hypoxia on cellular pathways is complex with true hypoxia because HIF-1α has a short lifetime and rapidly reverts back to a normoxic state. To overcome this difficulty, mimetic agents of the hypoxia pathway have been developed, including the gold standard CoCl2. In this study, we proposed to compare CoCl2 and hydralazine in order to determine a suitable mimetic agent of hypoxia for the study on the BBB., Methods: We studied the cytotoxicity and the impact of these molecules on the integrity of an in vitro BBB model by comparing them to hypoxia controls., Results: We showed that the impact of hypoxic stress in our in vitro BBB model is rather similar between hydralazine and CoCl2. Chemical hypoxic stress led to an increase of BBB permeability either with CoCl2 or hydralazine. Tight junction protein expressions showed that this chemical hypoxic stress decreased ZO-1 but not occluding expressions, and cells had set up a defence mechanism by increasing expression and activity of their efflux transporters., Conclusion: Our results demonstrated that hydralazine is a better mimetic agent and more suitable than CoCl2 because it had the same effect but without the cytotoxic effect on in vitro BBB cells., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background: To investigate the combined use of intravoxel incoherent motion (IVIM) diffusion-weighted (DW) and blood oxygen level dependent (BOLD) magnetic resonance imaging (MRI) to assess rat renal function using a 1.5T clinical platform., Methods: Multiple b-value DW and BOLD MR images were acquired from adult rats using a parallel clinical coil arrangement, enabling quantitation of the apparent diffusion coefficient (ADC), IVIM-derived diffusion coefficient (D), pseudodiffusion coefficient (D*) and perfusion fraction (f), and the transverse relaxation time T 2 *, for whole kidney, renal cortex, and medulla. Following the acquisition of two baseline datasets to assess measurement repeatability, images were acquired following i.v. administration of hydralazine, furosemide, or angiotensin II for up to 40 min., Results: Excellent repeatability (CoV <10 %) was observed for ADC, D, f and T 2 * measured over the whole kidney. Hydralazine induced a marked and significant (p < 0.05) reduction in whole kidney ADC, D, and T 2 *, and a significant (p < 0.05) increase in D* and f. Furosemide significantly (p < 0.05) increased whole kidney ADC, D, and T 2 *. A more variable response to angiotensin II was determined, with a significant (p < 0.05) increase in medulla D* and significant (p < 0.05) reduction in whole kidney T 2 * established., Conclusions: Multiparametric MRI, incorporating quantitation of IVIM DWI and BOLD biomarkers and performed on a clinical platform, can be used to monitor the acute effects of vascular and tubular modulating drugs on rat kidney function in vivo. Clinical adoption of such functional imaging biomarkers can potentially inform on treatment effects in patients with renal dysfunction.

Zidovudine (AZT) remains the mainstay of antiretroviral therapy against HIV in resource-poor countries; however, its use is frequently associated with hepatotoxicity. Not all HIV patients on AZT develop hepatotoxicity, and the determining factors are unclear. Alcohol consumption and cigarette smoking are known risk factors for HIV hepatotoxicity, and both are significant sources of acrolein, a highly reactive and toxic aldehyde. This study examines the potential hepatotoxic interactions between acrolein and AZT. Our data demonstrate that acrolein markedly enhanced AZT-induced transcriptionally permissive histone modifications (H3K9Ac and H3K9Me3) allowing the recruitment of transcription factor NF-kB and RNA polymerase II at the FasL gene promoter, resulting in FasL upregulation and apoptosis in hepatocytes. Notably, the acrolein scavenger, hydralazine prevented these promoter-associated epigenetic changes and inhibited FasL upregulation and apoptosis induced by the combination of AZT and acrolein, as well as AZT alone. Our data strongly suggest that acrolein enhancement of promoter histone modifications and FasL upregulation are major pathogenic mechanisms driving AZT-induced hepatotoxicity. Moreover, these data also indicate the therapeutic potential of hydralazine in mitigating AZT hepatotoxicity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Reactive carbonyl species (RCS) are endogenous or exogenous byproducts involved in the pathogenic mechanisms of different oxidative-based disorders. Detoxification of RCS by carbonyl quenchers is a promising therapeutic strategy. Among the most studied quenchers are aminoguanidine, hydralazine, pyridoxamine, and carnosine; their quenching activity towards four RCS (4-hydroxy-trans-2-nonenal, methylglyoxal, glyoxal, and malondialdehyde) was herein analyzed and compared. Their ability to prevent protein carbonylation was evaluated in vitro by using an innovative method based on high-resolution mass spectrometry (HRMS). The reactivity of the compounds was RCS dependent: carnosine efficiently quenched 4-hydroxy-trans-2-nonenal, pyridoxamine was particularly active towards malondialdehyde, aminoguanidine was active towards methylglyoxal and glyoxal, and hydralazine efficiently quenched all RCS. Reaction products were generated in vitro and were characterized by HRMS. Molecular modeling studies revealed that the reactivity was controlled by specific stereoelectronic parameters that could be used for the rational design of improved carbonyl quenchers., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Spectrofluoremetric technique was employed to study the binding behavior of hydralazine with bovine serum albumin (BSA) at different temperatures. Binding study of bovine serum albumin with hydralazine has been studied by ultraviolet-visible spectroscopy, fluorescence spectroscopy and confirmed by three-dimensional, synchronous, circular dichroism, and Raman spectroscopic methods. Effect of β-cyclodextrin on binding was studied. The experimental results showed a static quenching mechanism in the interaction of hydralazine with bovine serum albumin. The binding constant and the number of binding sites are calculated according to Stern-Volmer equation. The thermodynamic parameters ∆H(o) , ∆G(o) , ∆S(o) at different temperatures were calculated. These indicated that the hydrogen bonding and weak van der Waals forces played an important role in the interaction. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r, between the donor (BSA) and acceptor (hydralazine) was evaluated and found to be 3.95 nm. Spectral results showed that the binding of hydralazine to BSA induced conformational changes in BSA. The effect of common ions on the binding of hydralazine to BSA was also examined. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Like Chinese boxes nesting inside each other, the classification of non-small cell lung cancer (NSCLC) is subdivided into smaller and smaller subtypes on the basis of histological and molecular attributes. The latter characterizes NSCLC by its molecular alterations and the identification of inhibitors that target these cancer-specific "driver" mutations. Despite the initial promise of precision-guided therapies to inhibit a finer and finer array of molecular subcategories, despite even the curative potential of immunotherapeutic checkpoint blockade, in particular, casualties still abound and true clinical success stories are few and far between; the ever-present, if sometimes unmentioned, "elephant in the room", is the acquisition of resistance, which, sooner or later, rears its ugly head to undermine treatment success and shorten survival. Emerging data suggests that epigenetic therapies are able to reprogram the aberrant tumor-associated epigenome and 'tame the beast of resistance', thereby prolonging survival. This article reviews the role of epigenetic dysregulation in NSCLC, explores PFS2 as a possible surrogate endpoint, briefly mentions possible biomarkers and highlights combinatorial treatment epigenetic strategies to "prime" tumors and reverse resistance., Competing Interests: The authors disclose that EpicentRx, Inc. has funded research surrounding molecule RRx-001.

Understanding cellular and molecular mechanisms induced by hypoxic stress is crucial to reduce blood-brain barrier (BBB) disruption in some neurological diseases. Since the brain is a complex organ, it makes the interpretation of in vivo data difficult, so BBB studies are often investigated using in vitro models. However, the investigation of hypoxia in cellular pathways is complex with physical hypoxia because HIF-1α (factor induced by hypoxia) has a short half-life. We had set up an innovative and original method of induction of hypoxic stress by hydralazine that was more reproducible, which allowed us to study its impact on an in vitro BBB model. Our results showed that hydralazine, a mimetic agent of the hypoxia pathway, had the same effect as physical hypoxia, with few cytotoxicity effects on our cells. Hypoxic stress led to an increase of BBB permeability which corresponded to an opening of our BBB model. Study of tight junction proteins revealed that this hypoxic stress decreased ZO-1 but not occludin expression. In contrast, cells established a defence mechanism by increasing expression and activity of their efflux transporters (Pgp and MRP-1). This induction method of hypoxic stress by hydralazine is simple, reproducible, controllable and suitable to understand the cellular and molecular mechanisms involved by hypoxia on the BBB.

Enhanced renin-angiotensin activity causes hypertension and cardiac hypertrophy. The angiotensin (Ang)-converting enzyme (ACE)2/Ang(1-7)/Mas axis pathway functions against Ang II type 1 receptor (AT1R) signaling. We investigated whether olmesartan (Olm), an AT1R blocker, inhibits cardiac hypertrophy independently of blood pressure, and evaluated the potential mechanisms. The 3- to 4-month-old male mice overexpressing renin in the liver (Ren-Tg) were given Olm (5 mg/kg/d) and hydralazine (Hyd) (3.5 mg/kg/d) orally for 2 months. Systolic blood pressure was higher in the Ren-Tg mice than in wild-type littermates. Olm and Hyd treatments lowered systolic blood pressure to the same degree. However, cardiac hypertrophy, evaluated by echocardiography, heart weight, cross-sectional area of cardiomyocytes, and gene expression, was inhibited by only Olm treatment, but not by Hyd. Olm treatment reversed decreased gene expressions of ACE2 and Mas receptor of Ren-Tg mice and inhibited enhanced NADPH oxidase (Nox)4 expression and reactive oxygen species, whereas Hyd treatment had no influence on them. These findings indicate that Olm treatment inhibits cardiac hypertrophy independently of blood pressure, not only through its original AT1R blockade but partly through enhancement of ACE2/Ang(1-7)/Mas axis and suppression of Nox4 expression.

Background: Endothelial progenitor cell (EPC) functions are impaired in the presence of diabetes mellitus. Aliskiren is a direct renin inhibitor, which is expected to modify proangiogenic cells. This study aimed to investigate whether and how aliskiren could improve the function of EPCs from patients with type II diabetes (T2DM)., Materials and Methods: Endothelial progenitor cells fibronectin adhesion assay, chamber assay and in vitro tube formation assay were used to estimate the degree of EPC adhesion, migration and tube formation abilities. EPC protein and mRNA expressions were evaluated by Western blot and quantitative RT-PCR, respectively. EPC vascular endothelial growth factor (VEGF) and (pro)renin receptor ((P)RR) expression was knocked down by VEGF and (P)RR siRNA., Results: Aliskiren (0·1 or 10 μM) dose-dependently improved functions and increased both VEGF and stromal cell-derived factor-1α (SDF-1α) expression of EPCs from patients with T2DM or EPCs from healthy volunteers and treated with high glucose. Transfection with VEGF siRNA significantly reduced the aliskiren-induced SDF-1α expression. Furthermore, (P)RR siRNA transfection impaired the aliskiren-induced VEGF and SDF-1 expression., Conclusions: The results show that aliskiren improved EPC function from patients with T2DM in a dose-dependent manner probably via the (P)RR and VEGF/SDF-1α-related mechanisms., (© 2016 Stichting European Society for Clinical Investigation Journal Foundation.)

Hydralazine (HYD), an old routine clinical anti-hypertension drug, is rarely used in clinic nowadays. Since the strategy of repositioning old drugs was put forward, HYD has been reported to possess various biological activities, including antitumor efficacy and reducing intra-tumor microvessel. Here, we investigated that whether HYD had the ability of anti-angiogeneis and its underlying mechanism. Cells proliferation, wound-healing, Transwell migration and invasion, tube formation and rat aortic ring assays in vitro and chicken chorioallantoic membrane (CAM) model in vivo were designed to investigated HYD's anti-angiogenic effect. Levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assessed by enzyme-linked immune sorbent assay (ELISA). Hepatocellular carcinoma (HCC) mice model was used to evaluate HYD's effect on tumor growth and microvessel density. Our results showed that HYD not only inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, wound-healing, Transwell migration and invasion and tube formation, but also suppressed the microvessel outgrowth of rat aortic ring in vitro and the neovascularzation of CAM in vivo. Furthermore, we demonstrated that HYD attenuated tumor angiogenesis and tumor growth. In the co-culture system of Transwell migration, the secretion of VEGF and bFGF was reduced by HYD respectively. In sum, our data indicate that HYD has the pharmacological effect of ant-angiogenesis by interference with VEGF and bFGF signaling pathways in endothelial cells. These findings suggest that HYD might be a promising angiogenesis inhibitor and a potential effective therapeutic agent for cancer therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report that disturbance to the circadian rhythm of urinary angiotensinogen (AGT) excretion may lead to renal damage, hypertension and diurnal blood pressure (BP) variations. We aim to clarify the circadian rhythm of the intrarenal renin-angiotensin system (RAS) and its contribution to renal damage, hypertension and BP variations, and to evaluate whether the administration of RAS blockers influences the circadian rhythms of intrarenal RAS components. Anti-thymocyte serum (ATS) nephritis rats were used as a chronic progressive glomerulonephritis model (group A) and compared with control rats (group C). Other rats with ATS nephritis received olmesartan medoxomil (an angiotensin II (AngII) type 1 receptor (AT1R) blocker; group AO) or hydralazine (a vasodilator; group AH). The levels of intrarenal RAS components were evaluated every 6 h. The expression levels of intrarenal AGT, AngII and AT1R were increased in group A and peaked at the same time as BP and urinary protein excretion during the rest phase. The amplitude of the circadian fluctuation of these proteins was more increased in group A than in group C. The circadian fluctuation of these proteins was reduced in groups AO and AH. However, renal function, proteinuria and augmentation of intrarenal RAS components were reduced only in group AO. Intrarenal RAS components, such as AGT, AngII and AT1R proteins, were increased and the amplitude of the oscillations of these proteins was augmented in ATS nephritis rats. Interestingly, renal damage may be linked to the activation of the intrarenal RAS independent of the amplitude of its oscillations and BP.

Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect., Competing Interests: There are no potential conflicts of interest

Inflammation contributes to ANG II-associated impairment of renal autoregulation and microvascular P2X1 receptor signaling, but its role in renal autoregulation in mineralocorticoid-induced hypertension is unknown. Autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. Hypertension was induced in uninephrectomized control rats (UNx) by subcutaneous implantation of a DOCA pellet plus administration of 1% NaCl in the drinking water (DOCA-salt) for 3 wk. DOCA-salt rats developed hypertension that was unaltered by anti-inflammatory treatment with pentosan polysulfate (DOCA-salt+PPS) but was suppressed with "triple therapy" (hydrochlorothiazide, hydralazine, and reserpine; DOCA-salt+TTx). Baseline arteriolar diameters were similar across all groups. UNx rats exhibited pressure-dependent vasoconstriction with diameters declining to 69 ± 2% of control at 170 mmHg, indicating intact autoregulation. DOCA-salt treatment significantly blunted this pressure-mediated vasoconstriction. Diameters remained between 91 ± 4 and 98 ± 3% of control over 65-170 mmHg, indicating impaired autoregulation. In contrast, pressure-mediated vasoconstriction was preserved in DOCA-salt+PPS and DOCA-salt+TTx rats, reaching 77 ± 7 and 75 ± 3% of control at 170 mmHg, respectively. ATP is required for autoregulation via P2X1 receptor activation. ATP- and β,γ-methylene ATP (P2X1 receptor agonist)-mediated vasoconstriction were markedly attenuated in DOCA-salt rats compared with UNx (P < 0.05), but significantly improved by PPS or TTx (P < 0.05 vs. DOCA-salt) treatment. Arteriolar responses to adenosine and UTP (P2Y2 receptor agonist) were unaffected by DOCA-salt treatment. PPS and TTx significantly reduced MCP-1 and protein excretion in DOCA-salt rats. These results support the hypothesis that hypertension triggers inflammatory cascades but anti-inflammatory treatment preserves renal autoregulation in DOCA-salt rats, most likely by normalizing renal microvascular reactivity to P2X1 receptor activation., (Copyright © 2016 the American Physiological Society.)

The cellular mechanisms of hydralazine-induced relaxation were investigated in isolated mesenteric resistance arteries from pregnant rats. Administration of hydralazine relaxed phenylephrine-constricted mesenteric arteries with an EC50 of 3.6 ± 0.3 μM and an efficacy of 75 ± 6.2%. These vasodilatory effects were abolished by: (1) preconstriction with a potassium depolarizing solution, (2) endothelial denudation (for concentrations of hydralazine<10 μM), (3) addition of non-selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors, and (4) pretreatment with a prostacyclin receptor antagonist (R01138452). Nitric oxide synthase (NOS) inhibition did not significantly alter the sensitivity or magnitude of the vasodilatory response; surprisingly, exposure to hydralazine also did not elevate endothelial cell Ca(2+), suggesting a novel mechanism of activation. In summary, hydralazine is a potent resistance artery vasodilator that affects both endothelial and vascular smooth muscle (VSM) cells in a concentration-dependent manner. At clinically relevant concentrations (<10 μM), its effects in the splanchnic resistance vasculature are: (1) primarily endothelial in origin, require (2) hyperpolarization and (3) activation of COX, and (4) are mediated by the PGI2 (IP) receptor., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Overt systemic inflammatory response is a predisposing mechanism for infection-induced skeletal muscle damage and rhabdomyolysis. Aberrant DNA methylation plays a crucial role in the pathophysiology of excessive inflammatory response. The antiarrhythmic drug procainamide is a non-nucleoside inhibitor of DNA methyltransferase 1 (DNMT1) used to alleviate DNA hypermethylation. Therefore, we evaluated the effects of procainamide on the syndromes and complications of rhabdomyolysis rats induced by lipopolysaccharide (LPS). Rhabdomyolysis animal model was established by intravenous infusion of LPS (5 mg/kg) accompanied by procainamide therapy (50 mg/kg). During the experimental period, the changes of hemodynamics, muscle injury index, kidney function, blood gas, blood electrolytes, blood glucose, and plasma interleukin-6 (IL-6) levels were examined. Kidneys and lungs were exercised to analyze superoxide production, neutrophil infiltration, and DNMTs expression. The rats in this model showed similar clinical syndromes and complications of rhabdomyolysis including high levels of plasma creatine kinase, acute kidney injury, hyperkalemia, hypocalcemia, metabolic acidosis, hypotension, tachycardia, and hypoglycemia. The increases of lung DNMT1 expression and plasma IL-6 concentration were also observed in rhabdomyolysis animals induced by LPS. Treatment with procainamide not only inhibited the overexpression of DNMT1 but also diminished the overproduction of IL-6 in rhabdomyolysis rats. In addition, procainamide improved muscle damage, renal dysfunction, electrolytes disturbance, metabolic acidosis, hypotension, and hypoglycemia in the rats with rhabdomyolysis. Moreover, another DNMT inhibitor hydralazine mitigated hypoglycemia, muscle damage, and renal dysfunction in rhabdomyolysis rats. These findings reveal that therapeutic effects of procainamide could be based on the suppression of DNMT1 and pro-inflammatory cytokine in endotoxin-induced rhabdomyolysis.

Perforator flaps are very popular in the reconstruction of soft tissue defects. As these flaps generally depend on a single perforator, drugs that increase the perfusion of the flap and/or prevent vascular complications may increase flap survival. In this study, we compared the effects of systemically administered hydralazine (arterial vasodilator via potassium channels), nifedipine (arterial vasodilator via calcium channels), piracetam (antiplatelet and regulator of microcirculation) and alprostadil (vasodilator, antiplatelet, rheological and cytoprotective) on flap survival in a rat epigastric artery perforator flap model. The percentage of necrosis was measured on each flap and evaluated using one-way analysis of variance (Anova). Histopathological analyses were also performed. Mean flap survival area was 3.85 cm(2) in the control group. Mean flap survival area was 4.88 cm(2) in the nifedipine group, 4.69 cm(2) in the hydralazine group, 10.55 cm(2) in the piracetam group and 11.3 cm(2) in the alprostadil group. When compared with the control group, all drugs except hydralazine improved flap survival; piracetam and alprostadil yielded significantly better results than nifedipine. Only the alprostadil group showed signs of improved vascularity in the histological analysis. As far as perforator flap survival is concerned, drugs that regulate the microcirculation by a combination of different antiaggregation mechanisms appear more beneficial than single action vasodilators. Alprostadil, a synthetic PGE-1 analogue, has combined antiplatelet and vasoactive effects that further increase flap survival., (Copyright © 2016 SFCM. Published by Elsevier Masson SAS. All rights reserved.)

Objective: To determine whether the incidence of hypotension or adverse fetal heart tracing (FHT) category change differed following antepartum administration of intravenous (IV) labetalol versus hydralazine., Methods: Blood pressure and FHT categories were assessed one hour before and after medication administration. Hypotension was defined as ≥30% reduction in baseline systolic blood pressure (SBP) or SBP <90 mmHg. Changes in mean arterial pressure (MAP) were also compared. The National Institute for Child Health and Human Development (NICHD) three-tier category system was used to describe the FHT. For all category II tracings, Parer and Ikeda's system was also used., Results: Sixty-nine women received hydralazine and 31 women received labetalol during the study period. The incidence of hypotension (≥30% reduction in SBP) was similar between the labetalol (10%) and hydralazine (11%) groups (p = 0.98). No women experienced post-treatment SBP <90 mmHg. No association was observed between fetal heart rate category change and drug used. No women required emergent delivery for fetal indications., Conclusions: The incidence of maternal hypotension was low and did not differ following antepartum IV labetalol versus hydralazine use. These data should reassure providers about the use of parenteral labetalol and hydralazine for the treatment of severe hypertension.

Objective: Using noninvasive bedside impedance cardiography (ICG), we compared the effectiveness and the hemodynamic impact of intravenous labetalol versus hydralazine for the reduction of acute-onset severe hypertension to ACOG-recommended blood pressure levels (ACOG Committee Opinion 514)., Study Design: In this prospective randomized pilot study of acutely severe systolic hypertension (≥160 mmHg), pregnant women received either labetalol (L) or hydralazine (H) intravenously and underwent thoracic ICG before and after treatment. Data analysis were performed using STATA software (StataCorp LP, College Station, TX); data are expressed as mean ± SD., Results: About 29 patients completed the study. There was no significant difference in mean arterial pressure (MAP) between groups [H = 119.4 mmHg, L = 117.7 mmHg, mean difference (MD) = 1.73); the estimated MD between baseline and follow-up ICG was -9.17 (p = 0.001, 95% CI: -14.39 to -3.95). There were no significant differences in total peripheral resistance (TPR) between groups (H = 1771.3, L = 1976.97, MD = 205.62) or cardiac output (CO) between groups (H = 5.7, L = 5.1, MD = 0.64) or a significant MD between these at baseline and follow-up., Conclusion: Both drugs performed similarly to achieve ACOG-recommended initial blood pressure reduction safely without side effects or excessive acute hemodynamic profile correction toward normal pregnancy values.

Introduction: Nonclinical safety studies are increasingly incorporating cardiac safety endpoints to discover potential cardiovascular liabilities. This trend for more thorough cardiovascular nonclinical safety evaluation is prudent given the high attrition rate of potential therapeutics due to unexpected cardiovascular liabilities discovered in late-stage clinical trials or post-market approval. In particular, the causal relationship of blood pressure changes that lead to risk of major adverse cardiac events suggests hemodynamic changes should be critically evaluated in preclinical studies of novel therapeutics., Methods: Jacketed external telemetry with an implanted miniature blood pressure transmitter (JET-BP) was used to characterize the tolerability, functionality, and sensitivity of this study design in dogs. Thirty-six male or female beagles (n=6 dogs/sex/group) were administered vehicle control (reverse osmosis water) or etilefrine (1, 10mg/kg), sotalol (3, 30mg/kg), and hydralazine (1, 10mg/kg) on separate days. Telemetry data were evaluated for positive control article-related changes and retrospective power analysis was also completed. Animals were evaluated for instrumentation-related changes in clinical and anatomic pathology endpoints., Results: All three positive controls elicited the expected pharmacologic responses that were statistically different at high and low doses. Retrospective power analysis confirmed this study design was able to statistically differentiate minor (approximately 5 to 15%) changes in electrocardiography and blood pressure values. This study also demonstrated the potential advantages of combining cardiovascular data across sex when the test article exposure and pharmacodynamics were consistent. Data collection using miniature telemetry blood pressure transmitters did not result in anatomic or clinical pathology findings that would prevent their use in general toxicology studies., Discussion: This characterization study indicates that JET-BP in dogs offers a scientifically-robust method to evaluate novel therapeutics for potential cardiovascular liabilities., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mineralocorticoids have been implicated in the pathogenesis of diastolic heart failure. On the contrary, angiotensin (Ang)-(1-7) has emerged as a potential strategy for treatment of cardiac dysfunction induced by excessive mineralocorticoid receptor activation. A critical question about the cardioprotective effect of Ang-(1-7) in hypertensive models is its dependence on blood pressure (BP) reduction. Here, we addressed this question by investigating the mechanisms involved in Ang-(1-7) cardioprotection against mineralocorticoid receptor activation. Sprague-Dawley (SD) and transgenic (TG) rats that overexpress an Ang-(1-7) producing fusion protein (TG(A1-7)3292) were treated with deoxycorticosterone acetate (DOCA) for 6 weeks. After treatment, SD rats became hypertensive and developed ventricular hypertrophy. These parameters were attenuated in TG-DOCA. SD-DOCA rats developed diastolic dysfunction which was associated at the cellular level with reduced Ca(2+) transient. Oppositely, TG-DOCA myocytes presented enhanced Ca(2+) transient. Moreover, higher extracellular signal-regulated kinase phosphorylation, type 1 phosphatase, and protein kinase Cα levels were found in SD-DOCA cells. In vivo, pressor effects of DOCA can contribute to the diastolic dysfunction, raising the question of whether protection in TG was a consequence of reduced BP. To address this issue, BP in SD-DOCA was kept at TG-DOCA level by giving hydralazine or by reducing the DOCA amount given to rats (Low-DOCA). Under similar BP, diastolic dysfunction and molecular changes were still evident in DOCA-hydralazine and SD-low-DOCA, but not in TG-DOCA. In conclusion, Ang-(1-7) protective signaling against DOCA-induced diastolic dysfunction occurs independently of BP attenuation and is mediated by the activation of pathways involved in Ca(2+) handling, hypertrophy, and survival., (© 2015 American Heart Association, Inc.)

Proper perfusion is vital for maintenance of neuronal homeostasis and brain function. Changes in the function and structure of cerebral parenchymal arterioles (PAs) could impair blood flow regulation and increase the risk of cerebrovascular diseases, including dementia and stroke. Hypertension alters the structure and function of large cerebral arteries, but its effects on PAs remain unknown. We hypothesized that hypertension increases myogenic tone and induces inward remodeling in PAs; we further proposed that antihypertensive therapy or mineralocorticoid receptor (MR) blockade would reverse the effects of hypertension. PAs from 18-wk-old stroke-prone spontaneously hypertensive rats (SHRSP) were isolated and cannulated in a pressure myograph. At 50-mmHg intraluminal pressure, PAs from SHRSP showed higher myogenic tone (%tone: 39.1 ± 1.9 vs. 28.7 ± 2.5%, P < 0.01) and smaller resting luminal diameter (34.7 ± 1.9 vs. 46.2 ± 2.4 μm, P < 0.01) than those from normotensive Wistar-Kyoto rats, through a mechanism that seems to require Ca(2+) influx through L-type voltage-gated Ca(2+) channels. PAs from SHRSP showed inward remodeling (luminal diameter at 60 mmHg: 55.2 ± 1.4 vs. 75.7 ± 5.1 μm, P < 0.01) and a paradoxical increase in distensibility and compliance. Treatment of SHRSP for 6 wk with antihypertensive therapy reduced PAs' myogenic tone, increased their resting luminal diameter, and prevented inward remodeling. In contrast, treatment of SHRSP for 6 wk with an MR antagonist did not reduce blood pressure or myogenic tone, but prevented inward remodeling. Thus, while hypertensive remodeling of PAs may involve the MR, myogenic tone seems to be independent of MR activity., (Copyright © 2015 the American Physiological Society.)

Salt intake promotes progression of CKD by uncertain mechanisms. We hypothesized that a salt-induced reno-cerebral reflex activates a renin-angiotensin axis to promote CKD. Sham-operated and 5/6-nephrectomized rats received a normal-salt (0.4%), low-salt (0.02%), or high-salt (4%) diet for 2 weeks. High salt in 5/6-nephrectomized rats increased renal NADPH oxidase, inflammation, BP, and albuminuria. Furthermore, high salt activated the intrarenal and cerebral, but not the systemic, renin-angiotensin axes and increased the activity of renal sympathetic nerves and neurons in the forebrain of these rats. Renal fibrosis was increased 2.2-fold by high versus low salt, but intracerebroventricular tempol, losartan, or clonidine reduced this fibrosis by 65%, 69%, or 59%, respectively, and renal denervation or deafferentation reduced this fibrosis by 43% or 38%, respectively (all P<0.05). Salt-induced fibrosis persisted after normalization of BP with hydralazine. These data suggest that the renal and cerebral renin-angiotensin axes are interlinked by a reno-cerebral reflex that is activated by salt and promotes oxidative stress, fibrosis, and progression of CKD independent of BP., (Copyright © 2015 by the American Society of Nephrology.)

Hypotensive drugs have been used to identify central neurons that mediate compensatory baroreceptor reflex responses. Such drugs also increase blood glucose. Our aim was to identify the neurochemical phenotypes of sympathetic preganglionic neurons (SPN) and adrenal chromaffin cells activated following hydralazine (HDZ; 10mg/kg) administration in rats, and utilize this and SPN target organ destination to ascribe their function as cardiovascular or glucose regulating. Blood glucose was measured and adrenal chromaffin cell activation was assessed using c-Fos immunoreactivity (-ir) and phosphorylation of tyrosine hydroxylase, respectively. The activation and neurochemical phenotype of SPN innervating the adrenal glands and celiac ganglia were determined using the retrograde tracer cholera toxin B subunit, in combination with in situ hybridization and immunohistochemistry. Blood glucose was elevated at multiple time points following HDZ administration but little evidence of chromaffin cell activation was seen suggesting non-adrenal mechanisms contribute to the sustained hyperglycemia. 16±0.1% of T4-T11 SPN contained c-Fos and of these: 24.3±1.4% projected to adrenal glands and 29±5.5% projected to celiac ganglia with the rest innervating other targets. 62.8±1.4% of SPN innervating adrenal glands were activated and 29.9±3.3% expressed PPE mRNA whereas 53.2±8.6% of SPN innervating celiac ganglia were activated and 31.2±8.8% expressed PPE mRNA. CART-ir SPN innervating each target were also activated and did not co-express PPE mRNA. Neurochemical coding reveals that HDZ administration activates both PPE+SPN, whose activity increase glucose mobilization causing hyperglycemia, as well as CART+SPN whose activity drive vasomotor responses mediated by baroreceptor unloading to raise vascular tone and heart rate., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background and Aim: The infusion of chronic angiotensin II (Ang II) has been shown to promote renal interstitial fibrosis. To evaluate the pathophysiological significance of the natriuretic peptide-GC-A system, we infused Ang II (1.0 mg/kg/day) in GC-A-deficient mice (GC-A-KO)., Methods: We used 5 groups (Wild-Saline n = 12, Wild-Ang II n = 14, GC-A-KO-Saline n = 11, GC-A-KO-Ang II n = 13, and GC-A-KO-Ang II-Hydralazine n = 10). Saline or Ang II was infused subcutaneously using an osmotic minipump for 3 weeks. Hydralazine was administered orally (0.05 g/L in drinking water)., Results: Systolic blood pressure was significantly higher in the GC-A-KO-Saline group (130 ± 12 mmHg) than in the Wild-Saline group (105 ± 30 mmHg), and was similar to that in the Wild-Ang II (141 ± 17 mmHg) and GC-A-KO-Ang II-Hydralazine (140 ± 20 mmHg) groups. Systolic blood pressure was significantly higher in the GC-A-KO-Ang II group (159 ± 21 mmHg) than in the 4 other groups. Renal tubular atrophy and interstitial fibrosis were significantly more severe in the GC-A-KO-Ang II group (atrophy 13.4 %, fibrosis 12.0 %) than in the Wild-Saline (0, 2.0 %), Wild-Ang II (2.9, 4.4 %), and GC-A-KO-Saline (0, 2.6 %) groups. Hydralazine could not inhibit this aggravation (GC-A-KO-Ang II-Hydralazine 13.5, 11.3 %). The expression of monocyte chemotactic protein-1 in tubular cells, and F4/80 and alpha-smooth muscle actin in the interstitium was clearly detected in the Ang II-infused wild and GC-A-KO groups and was associated with renal tubular atrophy and interstitial fibrosis. The expression of E-cadherin in tubular cells was absent in the Ang II-infused wild and GC-A-KO groups and was associated with renal tubular atrophy., Conclusions: The natriuretic peptide-GC-A system may play an inhibitory role in Ang II-induced renal tubular atrophy, interstitial fibrosis, and phenotypic transformation in renal tubular cells and fibroblasts.

Background: Hypertension is a powerful risk factor of atrial fibrillation (AF). The pathophysiology of AF with hypertension is associated with sympathoexcitation or the renin-angiotensin system; however, current therapies cannot sufficiently prevent its development. We previously revealed that brain angiotensin II type 1 receptor (AT1R) blockade causes a depressor response via sympathoinhibition. Herein, we evaluated whether brain AT1R contributes to AF development in hypertensive rats., Methods: We divided the stroke-prone spontaneously hypertensive rats (SHRSP) treated with intracerebroventricular (ICV) infusion of vehicle, ICV infusion of losartan (S-LOS), or oral administration of hydralazine (S-HYD); and Wistar Kyoto rats treated with ICV S-VEH., Results: Two weeks later, systolic blood pressure was significantly lower in the S-LOS group than in the S-VEH group and was even lower in the S-HYD group. Urinary norepinephrine excretion for 24h, an indirect marker of sympathoexcitation, significantly reduced in the S-LOS group but increased in the S-HYD group despite depressor response. AF was induced by transesophageal burst pacing. AF duration was significantly shorter in the S-LOS group than in the S-VEH group (5.0±0.4 vs. 15.2±3.7 s; n = 8 each; P < 0.05). However, it was significantly longer in the S-HYD group than in the S-VEH group. Interstitial atrial fibrosis and echocardiographic parameters did not differ between the SHRSP groups., Conclusions: Brain AT1R blockade suppresses AF inducibility and maintenance independent of depressor response in hypertensive rats., (© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background/aims: The aim of our study was to investigate the effect of high-salt diet on the renal expression of renalase and the potential role of the local renin-angiotensin system in this process., Methods: Sprague-Dawley (SD) rats were divided into groups according to salt content in diet and drug treatment as follows: normal-salt diet (NS), high-salt diet (HS), high-salt intake with hydralazine (HS+H), high-salt diet with enalapril (HS+E), and high-salt diet with valsartan (HS+V). The dietary intervention and drugs were given for four weeks. Renin activity and angiotensin II type 1 receptor (AT1R) levels were detected by real-time PCR. Renalase mRNA and protein were also measured., Results: After four weeks, systolic blood pressure and proteinuria were significantly increased in the HS group with respect to the NS group. Dietary salt intake caused a dramatic decrease in renalase expression in the rat kidneys. Renal cortex renin and AT1R increased significantly in the HS and HS+H groups. Urinary protein was positively correlated with renal renin and AT1R levels. However, in the HS+E and HS+V groups, enalapril and valsartan failed to influence renal renalase expression but abolished the increase in proteinuria, renal cortex renin, and AT1R levels with respect to the HS group., Conclusion: This study indicates that high salt intake reduces renal expression, and renal RAS may be not involved in the regulation of renalase in SD rats fed with high-salt diet., (© 2015 The Author(s) Published by S. Karger AG, Basel.)