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.

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.

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.

Genotoxicity of the antihypertensive drugs hydralazine and dihydralazine.

The genotoxicity of the antihypertensive agents hydralazine and dihydralazine was tested in mammalian cells and bacteria. Both drugs elicited DNA repair in rat hepatocyte primary cultures. In the Ames test, both with and without an S-9 fraction, hydralazine was mutagenic in strains TA100 and TA1537, whereas dihydralazine was weakly mutagenic in strain TA1537. These findings support the observation that hydralazine is carcinogenic in mice. The carcinogenicity of many chemicals results from interaction with DNA. Since these studies demonstrate that hydralazine and dihydralazine damage DNA in mammalian cells, these drugs should be viewed as potential human carcinogens.

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.

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.

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.

Blood pressure response and renin release following 4 days of treatment with dihydralazine and urapidil in conscious dogs.

The magnitude and the persistence of blood pressure reduction by dihydralazine and urapidil were investigated following treatment over a period of 4 days. The experiments were performed on six normotensive dogs, trained to submit to puncture of the femoral artery and to stand quietly in a special frame. The first-dose effects of orally administered dihydralazine (1.42, 7.1 mg X kg-1) and urapidil (2.0, 10.0 mg X kg-1) on heart rate, arterial blood pressure and plasma renin activity (PRA) were compared with the effects of the substances after 4 days of treatment. Both compounds caused a dose-dependent decrease in blood pressure but in contrast to urapidil the effect of dihydralazine was accompanied by large increases in heart rate and PRA. Dose-dependent tolerance to dihydralazine but not to urapidil was observed after treatment over only 4 days. However, basal blood pressure was significantly lowered after 4 days of treatment with urapidil at the high dosage and no further reduction was achieved on the fifth day. The importance of persistent counterregulation in the development of tolerance to the antihypertensive effect of dihydralazine is discussed.

Dynamic responses to intravenous urapidil and dihydralazine in normal subjects.

Hemodynamic responses after urapidil were compared with those after dihydralazine in placebo-controlled, double-blind studies after cumulative intravenous doses. We recorded heart rate, blood pressure, systolic time intervals corrected for heart rate (electromechanical systole and preejection period), electrical impedance cardiography [(dZ/dt)/RZ index and mean electrical thorax impedance], and M-mode echocardiogram (end-systolic and -diastolic diameters, end-systolic wall stress, fractional shortening, and cardiac output). Both drugs induced dose-dependent reductions in total peripheral resistance, which resulted in reduction in left ventricular end-systolic wall stress and increases in heart rate (limited at +10 bpm with urapidil), fractional shortening, cardiac output, and the (dZ/dt)/RZ index. With each drug, diastolic blood pressure fell by 5 mm Hg, the corrected preejection period shortened (dihydralazine greater than urapidil), the corrected electromechanical systole did not change, and mean electrical thorax impedance rose with urapidil. The spectrum of effects indicates that both drugs reduce left ventricular afterload, thereby increasing left ventricular pump performance. Urapidil also exerts some preload reduction.

Compared myocardial and vascular effects of captopril and dihydralazine during hypertension development in spontaneously hypertensive rats.

When administered to young spontaneously hypertensive rats (SHRs), dihydralazine (25 mg kg-1, daily) and captopril (100 mg kg-1, daily) prevent with the same efficacy genetic hypertension development (GHD). Dihydralazine treatment increased vascular mesenteric compliance, as shown by a significant decrease in the stiffness of the vessels (-27%), and induced slight reductions in contractility (-12%) and in wall to lumen (W/L) ratio (-15%). After treatment withdrawal, all these parameters returned to control values within 7 weeks, as did blood pressure. Captopril treatment also strongly increased the mesenteric vessels compliance, vessel stiffness being decreased by 16%, and reduced their contractility (-15%) and their W/L ratio (-30%). These effects as well as those exerted on blood pressure persisted up to 7 weeks after treatment ceased although there was a slight trend to a progressive reduction in the intensity of both phenomena. These experiments show that captopril but not dihydralazine has a long-lasting effect in opposing the functional and morphological vascular alterations occurring during GHD in SHRs and this phenomenon probably contributes to a large extent to the sustained preventive effects of the drug against GHD.

Comparative evaluation of the in vitro effects of hydralazine and hydralazine acetonide on arterial smooth muscle.

1. Dose-response relationships to K+ were determined in isolated strips of rabbit aorta. 2. K+ contractures were induced by 30 mM K+ in paired strips from individual animals. The effects of hydralazine and hydralazine acetone hydrazone (hydralazine acetonide) on these contractures were studied. 3. Hydralazine and hydralazine acetonide both produced dose-dependent decreases of K+-induced tone. Threshold concentrations for hydralazine were 11.89 +/- 4.5 X 10(-5) M (mean +/- s.d.) and for hydralazine actonide 9.7 +/- 4.6 X 10(-5) M (0.5 less than P less than 0.4). 4. The magnitude of the effect of hydralazine acetonide was greater than that of hydralazine at all concentrations above threshold, as reflected in a significant difference (P less than 0.05) in the slopes of dose-response curves to the two treatments. The vasodilator effects of hydralazine and the acetonide were terminated by washout of the bath. 5. The differences in effect were not due to instability of hydralazine under in vitro conditions. 6. It is concluded that hydralazine acetonide has intrinsic activity on vascular smooth muscle which differs significantly from that of the parent compound and that this may contribute to the hypotensive effects which follow administration of the parent compound.

Baroreflex response and vasodilating drugs in essential hypertension.

Blood pressure, heart rate, and arterial diameter of the brachial artery were studied in patients with sustained essential hypertension before and after administration of three vasodilating drugs: dihydralazine, diltiazem, and dinitrate isosorbide (ISDN). The diameter of the brachial artery was measured using a pulsed Doppler device, enabling the angle between the ultrasound beam and the vessel axis to be evaluated with a precision inferior to 2 percent. The three drugs had similar effects in decreasing the blood pressure and the forearm vascular resistance. Dihydralazine reduced the arterial diameter (p less than 0.001) and increased heart rate. Diltiazem and ISDN increased markedly the arterial diameter (p less than 0.001) but did not change heart rate. Dihydralazine decreased the tangential tension of the arterial wall, while diltiazem and ISDN did not. The study provided evidence that, with vasodilating drugs, the changes in the caliber of peripheral large arteries, which are a determinant of wall arterial tension, can influence the baroreflex-mediated tachycardia caused by use of the drugs.

Effects of dihydralazine infusion on the fetoplacental blood flow and maternal prostanoids.

The hemodynamic effects of intravenously infused dihydralazine (incremental doses up to 125 micrograms per minute during 60 minutes) were studied in ten women with acute or superimposed severe preeclampsia. The intervillous and umbilical vein blood flow were measured before and during dihydralazine infusion with 133Xenon method and with a combination of real-time and Doppler ultrasonic equipment, respectively. Maternal blood pressure decreased and pulse rate increased during the infusion. Dihydralazine did not change the intervillous blood flow but it increased the blood flow in umbilical vein. No effect on the 6-ketoprostaglandin F1 alpha in maternal plasma and urine or thromboxane B2 in maternal serum was observed. The results indicate that dihydralazine affects the placental and fetal circulations differently.

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.

Effect of dihydralazine on the fetus in the treatment of maternal hypertension.

The effect on the fetus of lowering the maternal blood pressure with intravenous dihydralazine was studied in 33 patients with diastolic blood pressure of 110 mmHg or more. Nineteen patients showed fetal heart rate (FHR) decelerations coinciding with a fall in blood pressure. Thirteen of the 19 patients had growth-retarded fetuses, while only 1 of 14 patients who showed no FHR changes had a growth-retarded fetus (P greater than .005). Continuous FHR monitoring during the administration of dihydralazine helped identify the compromised fetus if it had been unrecognized at other observations.

Dihydralazine and catecholamine-synthesizing enzymes in spontaneous hypertension.

Dihydralazine treatment which lowered blood pressure in young rats from the Lyon Hypertensive Strain (LHS), did not change phenylethanolamine-N-methyltransferase (PNMT) activity, but decreased tyrosine hydroxylase and dopamine-beta-hydroxylase activities in the C2 medullary region. These data suggest that the increase in PNMT activity, previously described for this strain, is not a consequence of the developing hypertension and that hypotensive treatment could inactivate some catecholaminergic neurons of the medulla oblongata.

Similar increase in circulating renin after equihypotensive doses of nitroprusside, dihydralazine or isradipine in conscious rabbits.

Calcium entry blockers but not nitroprusside sodium (NPS) or dihydralazine are known to enhance renin release in vitro. Isradipine (PN200-110), NPS or dihydralazine were infused at 15 min intervals into groups of 6 conscious, chronically instrumented rabbits each, at 3 doses carefully matched to elicit comparable falls in blood pressure. Plasma renin activity increased similarly with all treatments. In intact animals, the magnitude of the pressure decrease appears to be more important for renin release than the mechanism of vasodilation and a typical calcium entry blocker was indistinguishable from other vasodilators.

Effect of dihydralazine on the renal kallikrein-kinin system of the rat.

Dihydralazine (0.1 mg/kg), injected intravenously into male Sprague-Dawley rats, caused a decrease in mean arterial blood pressure and an increase in renal plasma flow, while urine volume remained unchanged. Dihydralazine had no effect on kallikrein excretion in the urine and on kallikrein activity in the renal cortex. No correlation was found between renal kallikrein and either renal plasma flow or mean arterial blood pressure. The excretion of kinins in the urine rose markedly after the administration of dihydralazine; no correlation between urinary kinins and urinary or renal kallikrein was observed. Dihydralazine had no influence on the kininogen content of blood-free renal cortex. The enzymatic activity of kininase II in renal cortex was not impaired by dihydralazine. It is suggested that the increased formation of kinins within the kidney could be involved in the vasodilating and blood pressure lowering effect of dihydralazine.

Effects of hydralazines on canine muscarinic ganglion transmission.

The effects of hydralazine, dihydralazine and 4-propyl-1-hydrazinophthalazine (4-propylhydralazine) were investigated on reflex pressor responses to increased intracranial fluid pressure (IIP) during occlusion of the abdominal aorta and the vena cava (MVO). It has been shown that application of MVO, caudal to the renal arteries, produces two independent vascular zones in the animal (Steinberg and Hilton, 1966a). Increasing intracranial fluid pressure during MVO elicits a reflex pressor response which consists of two components. One component is blocked by the nicotinic ganglionic blocking agent, chlorisondamine, and the other component is blocked by small doses of atropine. It was found that hydralazine and dihydralazine were effective in blocking residual pressor responses following partial blockade of reflex pressor responses to IIP with chlorisondamine. 4-Propylhydralazine, which is chemically similar to hydralazine and dihydralazine, was less active in inhibiting the residual pressor responses. It is suggested that hydralazine may act in part by interfering with muscarinic ganglionic transmission.

Morphological changes observed after stimulation or inhibition of adrenic system in rats chronically treated with hydrazinophtalazines.

Morphological studies of the heart muscle were carried out in healthy rats as well as treated with hydralazine or binazine after administration of adrenaline, noradrenaline, isoprenaline, regitine or propranolol. Hydrazinophtalazines (25 mg/kg body wt, daily) were given during 7 months. After discontinuation of this treatment the drugs were administered during 15 days. Evident changes (e.g. inflammatory infiltrations, focal necrosis and fibrosis) were found after administration of noradrenaline, adrenaline or isoprenaline. No differences were found in the reactivity to catecholamines in healthy and hydrazinophtalazine-treated rats.