HMGB1-induced activation of ER stress contributes to pulmonary artery hypertension in vitro and in vivo.

BACKGROUND: HMGB1 and ER stress have been considered to participate in the progression of pulmonary artery hypertension (PAH). However, the molecular mechanism underlying HMGB1 and ER stress in PAH remains unclear. This study aims to explore whether HMGB1 induces pulmonary artery smooth muscle cells (PASMCs) functions and pulmonary artery remodeling through ER stress activation. METHODS: Primary cultured PASMCs and monocrotaline (MCT)-induced PAH rats were applied in this study. Cell proliferation and migration were determined by CCK-8, EdU and transwell assay. Western blotting was conducted to detect the protein levels of protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor-4 (ATF4), seven in absentia homolog 2 (SIAH2) and homeodomain interacting protein kinase 2 (HIPK2). Hemodynamic measurements, immunohistochemistry staining, hematoxylin and eosin staining were used to evaluate the development of PAH. The ultrastructure of ER was observed by transmission electron microscopy. RESULTS: In primary cultured PASMCs, HMGB1 reduced HIPK2 expression through upregulation of ER stress-related proteins (PERK and ATF4) and subsequently increased SIAH2 expression, which ultimately led to PASMC proliferation and migration. In MCT-induced PAH rats, interfering with HMGB1 by glycyrrhizin, suppression of ER stress by 4-phenylbutyric acid or targeting SIAH2 by vitamin K3 attenuated the development of PAH. Additionally, tetramethylpyrazine (TMP), as a component of traditional Chinese herbal medicine, reversed hemodynamic deterioration and vascular remodeling by targeting PERK/ATF4/SIAH2/HIPK2 axis. CONCLUSIONS: The present study provides a novel insight to understand the pathogenesis of PAH and suggests that targeting HMGB1/PERK/ATF4/SIAH2/HIPK2 cascade might have potential therapeutic value for the prevention and treatment of PAH.

Jiedu Quyu Decoction mitigates monocrotaline-induced right-sided heart failure associated with pulmonary artery hypertension by inhibiting NLRP3 inflammasome in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Right-side heart failure could accelerate mortality in patients of pulmonary hypertension, Jiedu Quyu Decoction (JDQYF) was used to manage pulmonary hypertension, but its right-sided heart protective effect associated with pulmonary artery hypertension is still unclear. AIM OF THE STUDY: Here, we evaluated the therapeutic effect of JDQYF on monocrotaline-induced right-sided heart failure associated with pulmonary arterial hypertension in Sprague-Dawley (SD) rats and investigated the potential mechanism of action. MATERIALS AND METHODS: The main chemical components of JDQYF were detected and analyzed using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. The effects of JDQYF were investigated using a rat model of monocrotaline-induced right-sided heart failure associated with pulmonary arterial hypertension. We assessed the morphology of cardiac tissue using histopathology and the structure and function of the right heart using echocardiography. The biomarkers of heart failure, atrial natriuretic peptide and B-type natriuretic peptide, as well as serum pro-inflammatory markers, interleukin (IL)-1ß, and IL-18, were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the mRNA and protein expression levels of NLRP3 (NOD-, LRR-, and pyrin domain-containing 3), capase-1, IL-1ß, and IL-18 in the right heart tissue were examined by real-time quantitative reverse transcription PCR and western blotting. RESULTS: JDQYF improved ventricular function, alleviated pathological lesions in the right cardiac tissue, reduced the expression levels of biomarkers of heart failure and serum pro-inflammatory factors (IL-1ß and IL-18), and downregulated the mRNA and protein expression levels of NLRP3, caspase-1, IL-1ß, and IL-18 in the right cardiac tissue. CONCLUSIONS: JDQYF possesses cardioprotective effect against right heart failure induced by pulmonary arterial hypertension, possibly owing to reduction of cardiac inflammation through the inhibition of NLRP3 inflammasome activation.

A modified Fangji Huangqi decoction ameliorates pulmonary artery hypertension via phosphatidylinositide 3-kinases/protein kinase B-mediated regulation of proliferation and apoptosis of smooth muscle cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulmonary artery hypertension (PAH) is a progressive and fatal lung disease of multifactorial etiology, which arouses an enhanced interest in PAH disease therapy. Modified Fangji Huangqi decoction (MFJHQ), a traditional Chinese medicine (TCM) formula, has a crucial role in the treatment of PAH. However, the pharmacological roles and mechanisms of MFJHQ on PAH remain unknown. AIM OF THE STUDY: To investigate the effects and potential mechanism of MFJHQ on pulmonary vascular remodeling in PAH. MATERIAL AND METHODS: Ultra-performance liquid chromatography (UPLC) was employed to quantitate the principal components in MFJHQ. Rats were treated with MFJHQ by gavage for final 2 weeks in monocrotaline (MCT)-induced PAH rats. RNA-sequencing and network pharmacology analysis were performed to explore the potential mechanism. The primary rat pulmonary artery smooth muscle cells (PASMCs) were utilized to evaluate the regulatory effect of MFJHQ in vitro. RESULTS: Seven active components from MFJHQ were quantitated by UPLC. In rats with MCT-induced PAH, MFJHQ treatment significantly improved hemodynamic parameters, right ventricular hypertrophy index, lung function, and attenuated pulmonary vascular remodeling. Mechanistically, we further confirmed that MFJHQ inhibits MCT-induced phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt) pathway predicated by network pharmacology and RNA-sequencing analysis to reduce the proliferation of pulmonary arteries and promote pulmonary artery apoptosis in lung tissues. Additionally, MFJHQ hindered the proliferation and migration, and accelerated apoptosis in PDGF-BB-induced PASMCs in vitro, which can be enhanced by the presence of the PI3K inhibitor LY294002. CONCLUSIONS: Our results indicated that MFJHQ inhibited MCT-induced pulmonary vascular remodeling by decreasing proliferation and migration of PASMCs and promoting PASMC apoptosis through PI3K/Akt pathway, which provides a novel treatment option for PAH with multi-targeting mechanisms inspired by TCM theory.

Differential effect of basal vitamin D status in monocrotaline induced pulmonary arterial hypertension in normal and vitamin D deficient rats: Possible involvement of eNOS/TGF-ß/α-SMA signaling pathways.

Vitamin D deficiency is common and linked to poor prognosis in pulmonary arterial hypertension (PAH). We investigated the differential effect of basal vitamin D levels in monocrotaline (MCT) induced PAH in normal and vitamin D deficient (VDD) rats. Rats were fed a VDD diet and exposed to filtered fluorescent light to deplete vitamin D. Normal rats were pretreated with vitamin D 100 IU/d and treated with vitamin D 100 and 200 IU/d, while VDD rats received vitamin D 100 IU/d. Vitamin D receptor (VDR) silencing was done in human umbilical vein endothelial cells (HUVECs) using VDR siRNA. Calcitriol (50 nM/mL) was added to human pulmonary artery smooth muscle cells (HPASMCs) and HUVECs before and after the exposure to TGF-ß (10 ng/mL). Vitamin D 100 IU/d pretreatment in normal rats up-regulated the expression of eNOS and inhibited endothelial to mesenchymal transition significantly and maximally. Vitamin D 100 IU/d treatment in VDD rats was comparable to vitamin D 200 IU/d treated normal rats. These effects were significantly attenuated by L-NAME (20 mg/kg), a potent eNOS inhibitor. Exposure to TGF- ß significantly reduced the expression of eNOS and increased the mesenchymal marker expression in normal and VDR-silenced HUVECs and HPASMCs, which were averted by treatment and maximally inhibited by pretreatment with calcitriol (50 nM). To conclude, this study provided novel evidence suggesting the beneficial role of higher basal vitamin D levels, which are inversely linked with PAH severity.

Attenuating effect of magnesium on pulmonary arterial calcification in rodent models of pulmonary hypertension.

OBJECTIVE: Vascular calcification has been considered as a potential therapeutic target in pulmonary hypertension. Mg2+ has a protective role against calcification. This study aimed to investigate whether Mg2+ could alleviate pulmonary hypertension by reducing medial calcification of pulmonary arteries. METHODS: Monocrotaline (MCT)-induced and chronic hypoxia-induced pulmonary hypertension rats were given an oral administration of 10% MgSO4 (10 ml/kg per day). Additionally, we administered Mg2+ in calcified pulmonary artery smooth muscle cells (PASMCs) after incubating with ß-glycerophosphate (ß-GP, 10 mmol/l). RESULTS: In vivo, MCT-induced and chronic hypoxia-induced pulmonary hypertension indexes, including right ventricular systolic pressure, right ventricular mass index, and arterial wall thickness, as well as Alizarin Red S (ARS) staining-visualized calcium deposition, high calcium levels, and osteochondrogenic differentiation in pulmonary arteries, were mitigated by dietary Mg2+ intake. In vitro, ß-GP-induced calcium-rich deposits stained by ARS, calcium content, as well as the detrimental effects of calcification to proliferation, migration, and resistance to apoptosis of PASMCs were alleviated by high Mg2+ but exacerbated by low Mg2+. Expression levels of mRNA and protein of ß-GP-induced osteochondrogenic markers, RUNX Family Transcription Factor 2, and Msh Homeobox 2 were decreased by high Mg2+ but increased by low Mg2+; however, Mg2+ did not affect ß-GP-induced expression of SRY-Box Transcription Factor 9. Moreover, mRNA expression and protein levels of ß-GP-reduced calcification inhibitor, Matrix GLA protein was increased by high Mg2+ but decreased by low Mg2+. CONCLUSION: Mg2+ supplement is a powerful strategy to treat pulmonary hypertension by mitigating pulmonary arterial calcification as the calcification triggered physiological and pathological changes to PASMCs.

Dapagliflozin reduces the vulnerability of rats with pulmonary arterial hypertension-induced right heart failure to ventricular arrhythmia by restoring calcium handling.

BACKGROUND: Malignant ventricular arrhythmia (VA) is a major contributor to sudden cardiac death (SCD) in patients with pulmonary arterial hypertension (PAH)-induced right heart failure (RHF). Recently, dapagliflozin (DAPA), a sodium/glucose cotransporter-2 inhibitor (SGLT2i), has been found to exhibit cardioprotective effects in patients with left ventricular systolic dysfunction. In this study, we examined the effects of DAPA on VA vulnerability in a rat model of PAH-induced RHF. METHODS: Rats randomly received monocrotaline (MCT, 60 mg/kg) or vehicle via a single intraperitoneal injection. A day later, MCT-injected rats were randomly treated with placebo, low-dose DAPA (1 mg/kg/day), or high-dose (3 mg/kg/day) DAPA orally for 35 days. Echocardiographic analysis, haemodynamic experiments, and histological assessments were subsequently performed to confirm the presence of PAH-induced RHF. Right ventricle (RV) expression of calcium (Ca2+) handling proteins were detected via Western blotting. RV expression of connexin 43 (Cx43) was determined via immunohistochemical staining. An optical mapping study was performed to assess the electrophysiological characteristics in isolated hearts. Cellular Ca2+ imaging from RV cardiomyocytes (RVCMs) was recorded using Fura-2 AM or Fluo-4 AM. RESULTS: High-dose DAPA treatment attenuated RV structural remodelling, improved RV function, alleviated Cx43 remodelling, increased the conduction velocity, restored the expression of key Ca2+ handling proteins, increased the threshold for Ca2+ and action potential duration (APD) alternans, decreased susceptibility to spatially discordant APD alternans and spontaneous Ca2+ events, promoted cellular Ca2+ handling, and reduced VA vulnerability in PAH-induced RHF rats. Low-dose DAPA treatment also showed antiarrhythmic effects in hearts with PAH-induced RHF, although with a lower level of efficacy. CONCLUSION: DAPA administration reduced VA vulnerability in rats with PAH-induced RHF by improving RVCM Ca2+ handling.

Mineralocorticoid Receptor Antagonism by Finerenone Attenuates Established Pulmonary Hypertension in Rats.

BACKGROUND: We studied the ability of the nonsteroidal MR (mineralocorticoid receptor) antagonist finerenone to attenuate vascular remodeling and pulmonary hypertension using two complementary preclinical models (the monocrotaline and sugen/hypoxia rat models) of severe pulmonary hypertension. METHODS: We first examined the distribution pattern of MR in the lungs of patients with pulmonary arterial hypertension (PAH) and in monocrotaline and sugen/hypoxia rat lungs. Subsequent studies were performed to explore the effect of MR inhibition on proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. To validate the functional importance of MR activation in the pulmonary vascular remodeling characteristic of pulmonary hypertension, mice overexpressing human MR (hMR+) were studied, and curative treatments with finerenone (1 mg/kg per day by gavage), started 2 weeks after monocrotaline injection or 5 weeks after Sugen injection were realized. RESULTS: We demonstrated that MR is overexpressed in experimental and human PAH and that its inhibition following small interfering RNA-mediated MR silencing or finerenone treatment attenuates proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. In addition, we obtained evidence that hMR+ mice display increased right ventricular systolic pressure, right ventricular hypertrophy, and remodeling of pulmonary arterioles. Consistent with these observations, curative treatments with finerenone partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and vascular remodeling. Finally, we found that continued finerenone treatment decreases inflammatory cell infiltration and vascular cell proliferation in monocrotaline and sugen/hypoxia rat lungs. CONCLUSIONS: Finerenone treatment appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.

Srolo Bzhtang reduces inflammation and vascular remodeling via suppression of the MAPK/NF-κB signaling pathway in rats with pulmonary arterial hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Srolo Bzhtang (SBT), which consists of Solms-laubachia eurycarpa, Bergenia purpurascens, Glycyrrhiza uralensis, and lac secreted by Laccifer lacca Kerr (Lacciferidae Cockerell), is a well-known traditional Tibetan medicinal formula and was documented to cure "lung-heat" syndrome by eliminating "chiba" in the ancient Tibetan medical work Four Medical Tantras (Rgyud bzhi). Clinically, it is a therapy for pulmonary inflammatory disorders, such as pneumonia, chronic bronchitis, and chronic obstructive pulmonary disease. However, whether and how SBT participates in pulmonary arterial hypertension (PAH) is still unclear. AIM OF THE STUDY: We aimed to determine the role of SBT in attenuating pulmonary arterial pressure and vascular remodeling caused by monocrotaline (MCT) and hypoxia. To elucidate the potential mechanism underlying SBT-mediated PAH, we investigated the changes in inflammatory cytokines and mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) signaling pathway. MATERIALS AND METHODS: MCT- and hypoxia-induced PAH rat models were used. After administering SBT for four weeks, the rats were tested for hemodynamic indicators, hematological changes, pulmonary arterial morphological changes, and the levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in serum and lung tissues. Protein expression of the MAPK/NF-κB signaling pathway was determined using western blotting. RESULTS: SBT reduced pulmonary arterial pressure, vascular remodeling, and the levels of inflammatory cytokines induced by MCT and hypoxia in rats. Furthermore, SBT significantly suppressed the MAPK/NF-κB signaling pathway. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that SBT alleviates MCT- and hypoxia-induced PAH in rats, which is related to its anti-inflammatory actions involving inhibition of the MAPK/NF-κB signaling pathway.

Preparation of molecularly imprinted foam for selective extraction of toxic monocrotaline from herbs.

Monocrotaline (MCT), derived from most flowering plants, showed significant hepatotoxicity and carcinogenicity. Humans are easily exposed to MCT by eating traditional herbs or contaminated foods, posing a huge threat to human health. In order to selectively and conveniently separate and enrich MCT from these complex samples, we fabricated three-dimensional porous and homogeneous molecularly imprinted polymer foams (MIPFs) by using considerably inexpensive polyurethane foam (PU) as the carrier. The morphology, stability, adsorption properties and extraction parameters of MIPFs were investigated. The results indicated that an imprinted layer was coated on the surface of the carrier; the stability of MIPFs was excellent; In addition to hydrogen bonding and spatial complementarity, the electrostatic interactions were crucial for the recognition between MCT and MIPFs; MIPFs exhibited high adsorption capacity of 22.78 mg g-1, fast mass transfer rate, and satisfactory selectivity for MCT. Subsequently, MIPFs were applied as the solid phase extraction (SPE) absorbents and combined with high performance liquid chromatography (HPLC) to enrich and detect MCT in herbal plants. The results showed that MCT could be efficiently enriched, and the impurities could be dramatically reduced. MIPFs hold great potential for selective separation and detection of MCT in complex matrices, such as traditional Chinese medicine samples and food samples.

Shufeiya Recipe Improves Monocrotaline-Induced Pulmonary Hypertension in Rats by Regulating SIRT3/FOXO3a and Its Downstream Signaling Pathways.

Pulmonary hypertension (PH) is a chronic and progressive disease caused by obstructions and functional changes of small pulmonary arteries. Current treatment options of PH are costly with patients needing long-term taking medicine. The traditional Chinese medicine (TCM) compound "Shufeiya Recipe" was used to intervene in monocrotaline- (MCT-) induced pulmonary hypertension in rats. The rats were randomly divided into the control group, model group, positive drug (Sildenafil) group, and Shufeiya Recipe low-, moderate-, and high-dose groups. The improvement effect of the Shufeiya Recipe on the mean pulmonary artery pressure (mPAP) was assessed in PH rats, and pathological staining was used to observe the pathological changes of lung tissue. The impact of the Shufeiya Recipe on oxidative stress damage in rats with pulmonary hypertension and the regulation of SIRT3/FOXO3a and its downstream signaling pathways were determined. The results showed that Shufeiya Recipe could significantly downregulate mPAP and improve lung histopathological changes; downregulate serum levels of reactive oxygen species (ROS); upregulate the concentrations of COX-1 and COX-2 and the activity of Mn-SOD; inhibit oxidative response damage; promote the protein expression of SIRT3, FOXO3a, p-PI3K, p-AKT, and p-eNOS; increase the level of expression of NO, sGC, cGMP, and PKG; and downregulate the level of protein expression of Ras, p-MEK1/2, p-ERK1/2 and c-fos. These results indicate that Shufeiya Recipe can improve MCT-induced pulmonary hypertension in rats by regulating SIRT3/FOXO3a and its downstream PI3K/AKT/eNOS and Ras/ERK signaling pathways.

Effects of Crocin on CCL2/CCR2 Inflammatory Pathway in Monocrotaline-Induced Pulmonary Arterial Hypertension Rats.

Pulmonary arterial hypertension (PAH) is a malignant cardiopulmonary disease, in which pulmonary arterial remodeling is regarded as the prominent pathological feature. So far, the mechanism of PAH is still unclear, so its treatment remains a challenge. However, inflammation plays an important part in the occurrence and progression of PAH. It is well known that crocin has anti-inflammatory properties, so we investigated whether crocin could be a potential drug for the treatment of PAH rat models. Rats injected subcutaneously with monocrotaline (MCT) were treated with crocin via a gastric tube daily for four weeks. The results showed that crocin treatment significantly reduced the right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) in the PAH rat models. Moreover, crocin treatment reduced the proliferation of pulmonary arteriole smooth muscle cells (PASMCs). In addition, crocin treatment not only relieved inflammatory cell infiltration and collagen fiber hyperplasia in the lung and right ventricle, but also decreased the expression of the CCL2/CCR2 inflammatory pathway in the lung of PAH rat models. Furthermore, crocin treatment reduced the inflammatory cytokines and oxidative stress responses. In summary, crocin may play a protective role in MCT-induced PAH rats by alleviating inflammatory response, improving pulmonary arterial remodeling, and preventing PAH. Therefore, crocin as a new treatment for PAH may be quite worthy of consideration.

Pyrrolizidine alkaloids cause cell cycle and DNA damage repair defects as analyzed by transcriptomics in cytochrome P450 3A4-overexpressing HepG2 clone 9 cells.

Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs.

Role of oxidative stress versus lipids in monocrotaline-induced pulmonary hypertension and right heart failure.

Pulmonary hypertension (PH) is a global health issue with a prevalence of 10% in ages >65 years. Right heart failure (RHF) is the main cause of death in PH. We have previously shown that monocrotaline (MCT)-induced PH and RHF are due to an increase in oxidative stress. In this study, probucol (PROB), a strong antioxidant with a lipid-lowering property, versus lovastatin (LOV), a strong lipid-lowering drug with some antioxidant effects, were evaluated for their effects on the MCT-induced RHF. Rats were treated (I.P.) with PROB (10 mg/kg ×12) or LOV (4 mg/kg ×12), daily 6 days before and 6 days after a single MCT injection (60 mg/kg). Serial echocardiography was performed and at 4-week post-MCT, lung wet-to-dry weight, hemodynamics, RV glutathione peroxidase (GSHPx), superoxide dismutase (SOD), catalase, lipid peroxidation, and myocardial as well as plasma lipids were examined. MCT increased RV systolic and diastolic pressures, wall thickness, RV end diastolic diameter, mortality, and decreased ejection fraction as well as pulmonary artery acceleration time. These changes were mitigated by PROB while LOV had no effect. Furthermore, PROB prevented lipid peroxidation, lowered lipids, and increased GSHPx and SOD in RV myocardium. LOV did decrease the lipids but had no effect on antioxidants and lipid peroxidation. A reduction in oxidative stress and not the lipid-lowering effect of PROB may explain the prevention of MCT-induced PH, RHF, and mortality. Thus targeting of oxidative stress as an adjuvant therapy is suggested.

Preventive effects of nitrate-rich beetroot juice supplementation on monocrotaline-induced pulmonary hypertension in rats.

Beetroot (Beta vulgaris L.) has a high level of nitrate; therefore, its dietary intake could increase nitric oxide (NO) level in the body, possibly preventing the development of pulmonary hypertension (PH). In this study, we examined the effects of beetroot juice (BJ) supplementation on PH and the contribution of nitrate to such effects using a rat model of monocrotaline (MCT, 60 mg/kg s.c.)-induced PH. Rats were injected subcutaneously with saline or 60 mg/kg MCT and were sacrificed 28 days after the injection. In some rats injected with MCT, BJ was supplemented from the day of MCT injection to the day of sacrifice. First, MCT-induced right ventricular systolic pressure elevation, pulmonary arterial medial thickening and muscularization, and right ventricular hypertrophy were suppressed by supplementation with low-dose BJ (nitrate: 1.3 mmol/L) but not high-dose BJ (nitrate: 4.3 mmol/L). Of the plasma nitrite, nitrate, and their sum (NOx) levels, only the nitrate levels were found to be increased by the high-dose BJ supplementation. Second, in order to clarify the possible involvement of nitrate in the preventive effects of BJ on PH symptoms, the effects of nitrate-rich BJ (nitrate: 0.9 mmol/L) supplementation were compared with those of the nitrate-depleted BJ. While the former exerted preventive effects on PH symptoms, such effects were not observed in rats supplemented with nitrate-depleted BJ. Neither supplementation with nitrate-rich nor nitrate-depleted BJ affected plasma nitrite, nitrate, and NOx levels. These findings suggest that a suitable amount of BJ ingestion, which does not affect systemic NO levels, can prevent the development of PH in a nitrate-dependent manner. Therefore, BJ could be highly useful as a therapy in patients with PH.

Preclinical Investigation of Trifluoperazine as a Novel Therapeutic Agent for the Treatment of Pulmonary Arterial Hypertension.

Trifluoperazine (TFP), an antipsychotic drug approved by the Food and Drug Administration, has been show to exhibit anti-cancer effects. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive obliteration of small pulmonary arteries (PAs) due to exaggerated proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs). However, the therapeutic potential of TFP for correcting the cancer-like phenotype of PAH-PASMCs and improving PAH in animal models remains unknown. PASMCs isolated from PAH patients were exposed to different concentrations of TFP before assessments of cell proliferation and apoptosis. The in vivo therapeutic potential of TFP was tested in two preclinical models with established PAH, namely the monocrotaline and sugen/hypoxia-induced rat models. Assessments of hemodynamics by right heart catheterization and histopathology were conducted. TFP showed strong anti-survival and anti-proliferative effects on cultured PAH-PASMCs. Exposure to TFP was associated with downregulation of AKT activity and nuclear translocation of forkhead box protein O3 (FOXO3). In both preclinical models, TFP significantly lowered the right ventricular systolic pressure and total pulmonary resistance and improved cardiac function. Consistently, TFP reduced the medial wall thickness of distal PAs. Overall, our data indicate that TFP could have beneficial effects in PAH and support the view that seeking new uses for old drugs may represent a fruitful approach.

Safflower injection inhibits pulmonary arterial remodeling in a monocrotaline-induced pulmonary arterial hypertension rat model.

Pulmonary arterial hypertension (PAH) is a group of diseases with an increase of pulmonary artery pressure (PAP) and pulmonary vascular resistance. Here, the effects of safflower injection, a preparation of Chinese herbs, was investigated in a monocrotaline (MCT)-induced PAH rat model. PAP, carotid artery pressure (CAP), and the right ventricular hypertrophy index (RVHI) increased in the PAH group, while safflower injection was able to inhibit this increase to similar levels as observed in the normal group. The arteriole wall of the lungs and cardiac muscle were thickened and edema was observed in the PAH group, while these pathologies were improved in the herb-treated group in a dose-dependent manner. MCT treatment induced proliferation of pulmonary artery smooth muscle cells (PASMCs), which was inhibited by safflower injection in a dose-dependent manner. Our experimental results demonstrated that safflower injection can regulate pulmonary arterial remodeling through affecting the expression of connective tissue growth factor, transforming growth factor-ß, integrin, collagen or fibronectin, which subsequently affected the thicknesses of the arteriole walls of the lungs and cardiac muscle, and thereby benefits the control of PAH. This means safflower injection improved the abnormalities in PAP, CAP and RVHI, and pulmonary arterial remodeling through regulation of remodeling factors.

Therapeutic Potential of Regorafenib-A Multikinase Inhibitor in Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by a progressive elevation of mean arterial pressure followed by right ventricular failure and death. Previous studies have indicated that numerous inhibitors of receptor tyrosine kinase signaling could be either beneficial or detrimental for the treatment of PH. Here we investigated the therapeutic potential of the multi-kinase inhibitor regorafenib (BAY 73-4506) for the treatment of PH. A peptide-based kinase activity assay was performed using the PamStation®12 platform. The 5-bromo-2'-deoxyuridine proliferation and transwell migration assays were utilized in pulmonary arterial smooth muscle cells (PASMCs). Regorafenib was administered to monocrotaline- and hypoxia-induced PH in rats and mice, respectively. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. The kinase activity assay revealed upregulation of twenty-nine kinases in PASMCs from patients with idiopathic PAH (IPAH), of which fifteen were established as potential targets of regorafenib. Regorafenib showed strong anti-proliferative and anti-migratory effects in IPAH-PASMCs compared to the control PASMCs. Both experimental models indicated improved cardiac function and reduced pulmonary vascular remodeling upon regorafenib treatment. In lungs from monocrotaline (MCT) rats, regorafenib reduced the phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Overall, our data indicated that regorafenib plays a beneficial role in experimental PH.

Magnesium Supplementation Attenuates Pulmonary Hypertension via Regulation of Magnesium Transporters.

Pulmonary hypertension (PH) is characterized by profound vascular remodeling and altered Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Magnesium ion (Mg2+), a natural Ca2+ antagonist and a cofactor for numerous enzymes, is crucial for regulating diverse cellular functions, but its roles in PH remains unclear. Here, we examined the roles of Mg2+ and its transporters in PH development. Chronic hypoxia and monocrotaline induced significant PH in adult male rats. It was associated with a reduction of [Mg2+]i in PASMCs, a significant increase in gene expressions of Cnnm2, Hip14, Hip14l, Magt1, Mmgt1, Mrs2, Nipa1, Nipa2, Slc41a1, Slc41a2 and Trpm7; upregulation of SLC41A1, SLC41A2, CNNM2, and TRPM7 proteins; and downregulation of SLC41A3 mRNA and protein. Mg2+ supplement attenuated pulmonary arterial pressure, right heart hypertrophy, and medial wall thickening of pulmonary arteries, and reversed the changes in the expression of Mg2+ transporters. Incubation of PASMCs with a high concentration of Mg2+ markedly inhibited PASMC proliferation and migration, and increased apoptosis, whereas a low level of Mg2+ produced the opposite effects. siRNA targeting Slc41a1/2, Cnnm2, and Trpm7 attenuated PASMC proliferation and migration, but promoted apoptosis; and Slc41a3 overexpression also caused similar effects. Moreover, siRNA targeting Slc41a1 or high [Mg2+] incubation inhibited hypoxia-induced upregulation and nuclear translocation of NFATc3 in PASMCs. The results, for the first time, provide the supportive evidence that Mg2+ transporters participate in the development of PH by modulating PASMC proliferation, migration, and apoptosis; and Mg2+ supplementation attenuates PH through regulation of Mg2+ transporters involving the NFATc3 signaling pathway.

Preventive treatment with ginsenoside Rb1 ameliorates monocrotaline-induced pulmonary arterial hypertension in rats and involves store-operated calcium entry inhibition.

CONTEXT: Ginsenoside Rb1, the main active ingredient of ginseng, exhibits ex vivo depression of store-operated calcium entry (SOCE) and related vasoconstriction in pulmonary arteries derived from pulmonary hypertension (PH) rats. However, the in vivo effects of ginsenoside Rb1 on PH remain unclear. OBJECTIVE: This study explored the possibility of using ginsenoside Rb1 as an in vivo preventive medication for type I PH, i.e., pulmonary arterial hypertension (PAH), and potential mechanisms involving SOCE. MATERIALS AND METHODS: Male Sprague-Dawley rats (170-180 g) were randomly divided into Control, MCT, and MCT + Rb1 groups (n = 20). Control rats received only saline injection. Rats in the MCT + Rb1 and MCT groups were intraperitoneally administered single doses of 50 mg/kg monocrotaline (MCT) combined with 30 mg/kg/day ginsenoside Rb1 or equivalent volumes of saline for 21 consecutive days. Subsequently, comprehensive parameters related to SOCE, vascular tone, histological changes and hemodynamics were measured. RESULTS: Ginsenoside Rb1 reduced MCT-induced STIM1, TRPC1, and TRPC4 expression by 35.00, 31.96, and 32.24%, respectively, at the protein level. SOCE-related calcium entry and pulmonary artery contraction decreased by 162.6 nM and 71.72%. The mean pulmonary artery pressure, right ventricle systolic pressure, and right ventricular mass index decreased by 19.5 mmHg, 21.6 mmHg, and 39.50%. The wall thickness/radius ratios decreased by 14.67 and 17.65%, and the lumen area/total area ratios increased by 18.55 and 15.60% in intrapulmonary vessels with 51-100 and 101-150 µm o.d. CONCLUSION: Ginsenoside Rb1, a promising candidate for PH prevention, inhibited SOCE and related pulmonary vasoconstriction, and relieved MCT-induced PAH in rats.

Decreased Expression of Canstatin in Rat Model of Monocrotaline-Induced Pulmonary Arterial Hypertension: Protective Effect of Canstatin on Right Ventricular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease which causes right ventricular (RV) failure. Canstatin, a C-terminal fragment of type IV collagen α2 chain, is expressed in various rat organs. However, the expression level of canstatin in plasma and organs during PAH is still unclear. We aimed to clarify it and further investigated the protective effects of canstatin in a rat model of monocrotaline-induced PAH. Cardiac functions were assessed by echocardiography. Expression levels of canstatin in plasma and organs were evaluated by enzyme-linked immunosorbent assay and Western blotting, respectively. PAH was evaluated by catheterization. RV remodeling was evaluated by histological analyses. Real-time polymerase chain reaction was performed to evaluate RV remodeling-related genes. The plasma concentration of canstatin in PAH rats was decreased, which was correlated with a reduction in acceleration time/ejection time ratio and an increase in RV weight/body weight ratio. The protein expression of canstatin in RV, lung and kidney was decreased in PAH rats. While recombinant canstatin had no effect on PAH, it significantly improved RV remodeling, including hypertrophy and fibrosis, and prevented the increase in RV remodeling-related genes. We demonstrated that plasma canstatin is decreased in PAH rats and that administration of canstatin exerts cardioprotective effects.