Matrine alleviates hypoxia-induced inflammation and pulmonary vascular remodelling via RPS5/NF-κB signalling pathway.

Hypoxia-induced vasoconstriction and vascular remodelling are the main pathological features of hypoxic pulmonary arterial hypertension (HPAH), and inflammation is participated in the occurrence of pulmonary vascular remodelling (PVR). Matrine is an alkaloid with the effects of anti-inflammation, antifibrosis and antitumour. But, few studies have explored the role of matrine in regulating PVR, and the related mechanisms are still unknown. In this study, we found that hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) proliferation and inhibited its apoptosis, reduced the expression of ribosomal protein s5 and activated the nuclear factor kappa-B (NF-κB) signalling. Matrine, sildenafil and NF-κB inhibitor Bay 11-7082 could reverse these changes and impel the cell cycle in phase S retardation, and reduced the expression of p50, p65, proliferating cell nuclear antigen (PCNA), Bcl-2. In addition, matrine could lower right ventricular systolic pressure and mean pulmonary artery pressure of rats, α-smooth muscle actin and PCNA expression in pulmonary artery media, the levels of tumor necrosis factor-α and interleuki-1ß, thus improved hypoxia-induced PVR. This study indicated that matrine could alleviate inflammation and improve PVR through reversing the imbalance of proliferation and apoptosis of PASMCs, thus it had a therapeutic effect on HPAH.

Single-Cell Imaging Maps Inflammatory Cell Subsets to Pulmonary Arterial Hypertension Vasculopathy.

Rationale: Unraveling immune-driven vascular pathology in pulmonary arterial hypertension (PAH) requires a comprehensive understanding of the immune cell landscape. Although patients with hereditary (H)PAH and bone morphogenetic protein receptor type 2 (BMPR2) mutations have more severe pulmonary vascular pathology, it is not known whether this is related to specific immune cell subsets. Objectives: This study aims to elucidate immune-driven vascular pathology by identifying immune cell subtypes linked to severity of pulmonary arterial lesions in PAH. Methods: We used cutting-edge multiplexed ion beam imaging by time of flight to compare pulmonary arteries (PAs) and adjacent tissue in PAH lungs (idiopathic [I]PAH and HPAH) with unused donor lungs, as controls. Measurements and Main Results: We quantified immune cells' proximity and abundance, focusing on those features linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells. Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In patients with HPAH, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving T cell immunoglobulin and mucin domain-3 (TIM-3)+ T cells. This correlated with an expanded DC subset expressing indoleamine 2,3-dioxygenase 1, TIM-3, and SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1, alongside increased neutrophils, SMCs, and alpha-smooth muscle actin (ACTA2)+ endothelial cells, reinforcing the heightened severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared with IPAH. Our findings emphasize the therapeutic potential of targeting monocyte-derived dendritic cells, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.

Treprostinil Effectiveness in Higher-Risk Pediatric Patients With Idiopathic and Heritable Pulmonary Arterial Hypertension.

BACKGROUND: Little is known about the effectiveness of treprostinil in higher-risk paediatric patients with various pulmonary arterial hypertension genotypes. This study was designed to investigate the prognosis of higher-risk paediatric patients with idiopathic or heritable pulmonary arterial hypertension (IPAH/HPAH) after treprostinil therapy. METHODS: Children with IPAH/HPAH who were stratified as higher risk and treated with treprostinil in our centre were included as the study cohort. Those who received only oral medications were included as the reference cohort. All patients in the study cohort received PAH-related genotyping. Survival was defined as no death. Event-free survival was defined as no death, Potts shunt, or atrial septostomy. RESULTS: Forty-nine children (median age 7.7 years [interquartile range (IQR) 4.2-11.5 years], 65% female) were included in the study cohort and 48 children were included in the reference cohort; 84% of the study cohort had genetic disorders after genetic testing with a dominance of BMPR2 and ACVRL1 mutations. After a median therapy duration of 5.56 months (IQR 2.66-11.12 months), all patients were alive with significant improvements in clinical characteristics. One-, 2-, and 3-year survival rates were 91%, 84%, and 69%, respectively with a median follow-up duration of 19.17 months (IQR 9.7-29.79 months), which was significantly superior to the reference cohort (P = 0.038). Multivariate Cox regression analysis identified World Health Organisation functional class after therapy as a predictor for survival. There was no significant difference in survival among patients with different genotypes. CONCLUSIONS: Treprostinil can significantly improve the prognosis in children with IPAH/HPAH who are at higher risk, despite genetic backgrounds.

4-Hydroxynonenal Contributes to Angiogenesis through a Redox-Dependent Sphingolipid Pathway: Prevention by Hydralazine Derivatives.

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 µM) 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.

Antiatherogenic effect of bisvanillyl-hydralazone, a new hydralazine derivative with antioxidant, carbonyl scavenger, and antiapoptotic properties.

Reactive oxygen species (ROS) generated within the vascular wall trigger low-density lipoprotein (LDL) oxidation, lipid peroxidation, and carbonyl stress that are involved in atherogenesis. We recently reported that the antihypertensive drug, hydralazine, exhibits carbonyl scavenger and antiatherogenic properties, but only moderate antioxidant activity, so that high concentrations are required for inhibiting LDL oxidation. We aimed to develop agents sharing both antioxidant and carbonyl scavenger properties. We have synthesized a new hydralazine derivative, the bisvanillyl-hydralazone (BVH). BVH strongly inhibited LDL oxidation induced by copper and by human endothelial cells (HMEC-1), and prevented the formation of macrophagic foam cells. BVH reduced both the extracellular generation of ROS (superoxide anion and hydrogen peroxide) induced by oxidized LDL (oxLDL), as well as intracellular oxidative stress and proteasome activation, NFkappaB activation, and oxLDL-mediated proinflammatory signaling. In parallel, BVH prevented the carbonyl stress induced by oxLDL on cellular proteins, and blocked the apoptotic cascade as assessed by the inhibition of Bid cleavage, cytochrome C release, and DEVDase activation. Lastly, BVH prevented atherogenesis and carbonyl stress in apoE(-/-) mice. In conclusion, BVH is the prototype of a new class of antioxidant and carbonyl scavenger agents designed for new therapeutical approaches in atherosclerosis.

Carbonyl-scavenging drugs & protection against carbonyl stress-associated cell injury.

This mini-review highlights the chemical and cytoprotective properties of various hydralazine analogues that block the induction of cell death by acrolein, a highly toxic contributor to "carbonyl stress" during a diverse range of human diseases. Recent work on the action of hydralazine against various carbonyl-mediated pathologies is also reviewed.

A novel mechanism for drug-induced liver failure: inhibition of histone acetylation by hydralazine derivatives.

BACKGROUND/AIMS: The aim of this study was to investigate the precise mechanism of liver failure by hydralazine derivatives, with special reference to liver regeneration failure. METHODS: Histone acetylation and proliferation of hepatocytes were evaluated by immunohistochemistry with anti-acetylated histone H4 and proliferating cell nuclear antigen (PCNA). Inhibition of histone acetylation by drugs was determined by in vitro histone acetylation assay. Mice livers fed with todralazine for 1 or 4 months were subjected to immunohistochemistry and Western blotting. Todralazine-fed mice were challenged with anti-Fas to check liver regeneration failure. RESULTS: On immunohistochemistry, histone acetylation in the hepatocytes was significantly impaired in patients with hydralazine derivatives. In an in vitro acetyl transferase assay, histone acetylation was inhibited by hydralazine derivatives in a dose-dependent manner. Mice fed with todralazine (3mg/day) for 4 months showed impairment of histone acetylation in hepatocytes whereas no inhibition was observed in mice fed with todralazine for 1 month. Anti-Fas challenge to todralazine-fed mice resulted in impairment of liver regeneration in respect of liver weight loss with impairment of histone acetylation in hepatocytes. CONCLUSIONS: Todralazine could inhibit catalysis of histone acetyltransferase and long-term administration of todralazine may impair histone acetylation of the hepatocytes, resulting in liver regeneration failure.

Transformation of lupus-inducing drugs to cytotoxic products by activated neutrophils.

Drug-induced lupus is a serious side effect of certain medications, but the chemical features that confer this property and the underlying pathogenesis are puzzling. Prototypes of all six therapeutic classes of lupus-inducing drugs were highly cytotoxic only in the presence of activated neutrophils. Removal of extracellular hydrogen peroxide before, but not after, exposure of the drug to activated neutrophils prevented cytotoxicity. Neutrophil-dependent cytotoxicity required the enzymatic action of myeloperoxidase, resulting in the chemical transformation of the drug to a reactive product. The capacity of drugs to serve as myeloperoxidase substrates in vitro was associated with the ability to induce lupus in vivo.

Mutagenicity of B(a)P in the presence of some hydralazine derivatives.

Hydralazine, dihydralazine and todralazine were tested in the aspect of their mutagenic potency, and the influence upon the mutagenicity of standard promutagen--B(a)P. Hydralazine exhibited strong mutagenic activity in the Ames test while mutagenic activity of dihydralazine was relatively weak. Todralazine had no mutagenic activity, and significantly decreased mutagenicity of B(a)P. It was concluded that todralazine could be a good antimutagenic substance.

Investigations into a new antiarrhythmic substance Z-2-amino-5-chlor-benzophenon-amidin-hydralazin in humans.

Z-2-amino-5-chlor-benzophenon-amidin-hydralazin is a new chemical potential antiarrhythmic substance. The blood levels were investigated after different dosages and forms of administration in patients and volunteers. The determination of the substance can be performed by high-pressure liquid chromatography. In addition the pharmacokinetic parameters were calculated. The pharmacokinetic profile is similar in humans and animals.

The anti-tachycardic mechanism of a direct-acting vasodilator, budralazine, in rats.

The present study was undertaken to elucidate the anti-tachycardic effect of a direct-acting vasodilator, budralazine, using an electrophysiological technique. Normotensive male Wistar rats were used. Rats were anesthetized intraperitoneally with urethane and alpha-chloralose. Intravenous administration of budralazine (0.5-5.0 mg/kg) produced a dose-dependent reduction of mean arterial pressure in anesthetized rats. At doses of 0.5 and 1.0 mg/kg, budralazine induced bradycardia accompanied with a decrease in cardiac sympathetic nerve activity (ICNA). Preganglionic adrenal sympathetic nerve activity (ASNA) was also reduced by budralazine (1.0 mg/kg). A 0.5 mg/kg of budralazine neither influenced carotid sinus nerve activity nor augmented aortic depressor nerve activity (ADNA). On the contrary, at dose of 5.0 mg/kg, budralazine produced a tachycardia accompanied with increases in both ICNA and ASNA. The ADNA was decreased by budralazine (5.0 mg/kg) significantly. These findings suggest that the central sympathoinhibitory action of budralazine may be responsible for the anti-tachycardic effect of budralazine and baroreceptor-mediated tachycardia occurred after high dose of budralazine.

Effects of antihypertensive drugs on experimental cerebral ischemia in spontaneously hypertensive rats.

The effects of antihypertensive drugs on ischemic cerebral damage were investigated using the bilateral carotid artery occlusion (BCAO) model in SHR. Oral budralazine and nifedipine, at doses that increased cerebral blood flow (CBF) in SHR in our previous study (Tanaka, S. et al., Folia Pharmacol, Japan, 87, 1986), significantly improved cerebral energy failure after the BCAO, but prazosin which does not increase CBF had no effect on the energy failure. These results suggest that the amelioration by these antihypertensive drugs of the energy failure after the BCAO results from its CBF-increasing effects in SHR.

A randomized comparison of early with conservative use of antihypertensive drugs in the management of pregnancy-induced hypertension.

Two treatment strategies were compared in 155 women with pregnancy-induced hypertension who were also given comprehensive non-pharmacological care. The mean gestation at entry was 28 weeks. As long as the diastolic blood pressure (DBP) remained below 106 mmHg, oxprenolol, or oxprenolol plus dihydralazine, were given to the early treatment group, and matching placebos to the control group. Open antihypertensive treatment was provided for patients whose DBP rose above 105 mmHg. Proteinuria occurred in seven women in each group. In the early treatment group, 13 of the 78 women were delivered by caesarean section; the corresponding numbers in the control group were 27 of 76 (17 vs 36%, 95% confidence interval (CI) of difference: 5-33%); the sections included seven and 16 in the early treatment and control groups, respectively, for severe hypertension and/or fetal distress. There were five perinatal deaths, two in the early treatment group and three in the control group. Early treatment did not influence gestational age at birth or birthweight. Respiratory distress syndrome occurred in four infants in the early treated group and in 10 in the control group; 14 infants in the former group and 26 in the latter were in hospital for more than 10 days (18 vs 35%; 95% CI of difference 4-32%). These results indicate that early antihypertensive treatment with oxprenolol is safe for the fetus and newborn in pregnancy-induced hypertension, but has no advantage over non-pharmacological care in terms of fetal growth. However, it may prevent acute hypertension in late pregnancy and associated fetal distress, and thus reduce the number of caesarean sections.

Comparative hemodynamic studies of isradipine and dihydralazine in atherosclerotic and normal rabbits.

The hemodynamic effects of the calcium antagonist isradipine (code name PN 200-110) and the arteriolar vasodilator dihydralazine were compared in atherosclerotic (cholesterol-fed) and normal conscious rabbits with implanted catheters. Regional blood flows were measured using the microsphere technique. Cardiac output and blood flow to several organs, especially to the gastrointestinal system, but not to the heart and brain, were lower in atherosclerotic rabbits than in normal ones. Intravenous isradipine (10 and 30 micrograms/kg) increased heart rate less in atherosclerotic than in normal rabbits. Isradipine had no effect on the surface electrocardiogram (ECG). In contrast, 0.4 mg/kg dihydralazine caused depression of the ST segment while decreasing blood pressure similarly. This was explained partly by an intramyocardial maldistribution of coronary blood flow (using microspheres). Isradipine increased and dihydralazine decreased flow to the brain. Isradipine redistributed cardiac output in atherosclerotic and normal animals in favor of the heart, brain, and skeletal muscle. However, in atherosclerotic animals, the high dose was less effective than the low dose in some vascular beds. Thus, isradipine is not a general vasodilator in either atherosclerotic or in normal animals, but favors the vital organs. This redistribution of cardiac output contrasts favorably with that induced by dihydralazine.

Influence of antihypertensive treatment with budralazine on autoregulation of cerebral blood flow in spontaneously hypertensive rats.

We studied the effect of chronic antihypertensive treatment with budralazine on the lower blood pressure limit of cerebral blood flow autoregulation using spontaneously hypertensive rats. Cerebral blood flow in the parietal cortex and caudate nucleus was measured to determine the lower limit using the hydrogen clearance method. The lower limit in both cerebral regions was significantly higher in 10 untreated spontaneously hypertensive rats than in 10 Wistar-Kyoto rats. The upward-shifted lower limit was restored to close to normal in the caudate nucleus and was partially restored in the parietal cortex of nine rats by 9 weeks of treatment with the high dose (50-68 mg/kg/day) of budralazine, which kept blood pressure constant at approximately normotension during the treatment period; the lower limit was slightly restored in both cerebral regions of seven rats by 4 weeks of treatment with the high dose. However, 9 weeks of treatment with the low dose (19-27 mg/kg/day) of budralazine, which produced moderate continuous hypotension in nine rats, did not apparently influence the lower limit. Our results suggest that long-term antihypertensive therapy with budralazine reduces the upward-shifted lower blood pressure limit of cerebral blood flow autoregulation toward normal and that the restoration induced by budralazine depends on the degree of blood pressure reduction as well as on the duration of the therapeutic period.

Pharmacokinetics and biotransformation of hydralazine acetone hydrazone, a metabolite of hydralazine, in the rat.

The pharmacokinetics of hydralazine acetone hydrazone (HAH), which is a metabolite of hydralazine (HP), was investigated after iv administration to rats. Plasma concentrations of HAH, HP, and hydralazine pyruvic acid hydrazone (HPH) were simultaneously determined by a specific HPLC method. A five-compartment pharmacokinetic model was presented to elucidate the disposition of HAH and two products, HP and HPH. The parameters used in the model were obtained by administering each of the three compounds (10 mg/kg) separately. The proposed model described the experimental data well and the model parameters were close to the model-independent values. After HP administration, HPH appeared rapidly in plasma, but the HPH availability from HP amounted to only 17.8 +/- 3.7%, based on the comparison between the area under the plasma concentration curves of formed and iv HPH. The formation of HP from HAH in the systemic circulation was demonstrated, but formed HP disappeared rapidly. The fraction of HAH available to the systemic circulation as HPH was extremely low (7.8 +/- 2.2%), indicating that the conversion of HAH to HP was not so extensive. The present results support the hypotheses that HPH is formed via the direct reaction of HAH with pyruvic acid and that the secondary formation is mediated by conversion to HP.

Effects of clonidine and dihydralazine on atrial natriuretic factor and cGMP in humans.

The effects of a 1-wk treatment with clonidine (75 micrograms/day twice a day) and dihydralazine (25 mg/day twice a day) on base-line levels of plasma atrial natriuretic factor (ANF) and plasma and urinary guanosine 3',5'-cyclic monophosphate (cGMP) and their changes by acute saline infusion (2 liters) in eight normal subjects were evaluated. Basal ANF was decreased to 65% in the clonidine group compared with both the control and dihydralazine groups. Volume loading increased plasma ANF levels by 30-40% of base-line values in the control and the dihydralazine groups and by 15% in the clonidine group. Basal plasma and urinary cGMP levels were raised by 30 and 90% in the dihydralazine group compared with both other groups. Volume loading increased plasma cGMP levels by 40% in the control and clonidine-treated groups and by 25% in the dihydralazine-treated group. It is concluded that ANF may contribute to hemodynamic effects of clonidine but not to those of dihydralazine. Dihydralazine increases plasma and urinary cGMP, supposedly by direct activation of the soluble guanylate cyclase.

[The effect of an antihypertensive drug budralazine on cerebrovascular lesions in salt-loaded SHR].

Budralazine was evaluated for its protective effect on the onset of cerebrovascular lesions in SHR given 1.5% NaCl as drinking water. The salt-loading for 67 days rapidly accelerated the development of hypertension in SHR (from 180 to over 250 mmHg, 40 days after the loading). The acceleration of hypertension was accompanied by an increase in the incidence of brain softening, cerebral infarct, angionecrosis and hemorrhage by 30-60% following the thrombosis and necrosis of cerebral arterioles. Renal angionecrosis associated with the interstitial nephrosis was also observed by 90% in the animals. Throughout the salt-loading period, oral administration of budralazine (1, 4 and 15 mg/kg/day) resulted in a dose-dependent inhibition of the accelerated hypertension. At larger doses (4 and 15 mg/kg/day), budralazine almost completely ameliorated the cerebral and renal lesions and significantly attenuated the rise of weight in the brain and heart observed in the salt-loaded control rats. Changes in the serum biochemical findings were also inhibited by this drug. In some of the parameters measured, budralazine appeared to be more efficacious than hydralazine (1, 4 and 15 mg/kg/day, p.o.). These results suggest that budralazine attenuates the serious development of hypertension and reduces the incidence and severity of stroke in salt-loaded SHR.

Haemodynamic changes in gestational proteinuric hypertension: the effects of rapid volume expansion and vasodilator therapy.

Ten patients with gestational proteinuric hypertension were studied with a Swan-Ganz thermodilution haemodynamic catheter before, during and after plasma volume expansion. Five patients were treated with dihydralazine before volume expansion and five after volume expansion. Before treatment all patients had a low pulmonary capillary wedge pressure (PCWP), low cardiac index (CI) and high systemic vascular resistance (SVR). Following volume expansion the PCWP and CI increased, the SVR decreased but the blood pressure (BP) was unchanged. Administration of dihydralazine following volume expansion led to a decrease in PCWP, an increase in CI and a decrease in SVR and BP. Dihydralazine alone caused an increase in heart rate, PCWP, and CI, and a decrease in SVR and BP. Volume expansion, by increasing CI and decreasing SVR, may be of therapeutic benefit in the severely hypertensive pregnant patient with a low cardiac index.