GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and often death. Here we report that deficiency of transcription factor GATA6 is a shared pathological feature of PA endothelial (PAEC) and smooth muscle cells (PASMC) in human PAH and experimental PH, which is responsible for maintenance of hyper-proliferative cellular phenotypes, pulmonary vascular remodeling and pulmonary hypertension. We further show that GATA6 acts as a transcription factor and direct positive regulator of anti-oxidant enzymes, and its deficiency in PAH/PH pulmonary vascular cells induces oxidative stress and mitochondrial dysfunction. We demonstrate that GATA6 is regulated by the BMP10/BMP receptors axis and its loss in PAECs and PASMC in PAH supports BMPR deficiency. In addition, we have established that GATA6-deficient PAEC, acting in a paracrine manner, increase proliferation and induce other pathological changes in PASMC, supporting the importance of GATA6 in pulmonary vascular cell communication. Treatment with dimethyl fumarate resolved oxidative stress and BMPR deficiency, reversed hemodynamic changes caused by endothelial Gata6 loss in mice, and inhibited proliferation and induced apoptosis in human PAH PASMC, strongly suggesting that targeting GATA6 deficiency may provide a therapeutic advance for patients with PAH.

Cross-talk between TSC2 and the extracellular matrix controls pulmonary vascular proliferation and pulmonary hypertension.

Increased proliferation and survival of cells in small pulmonary arteries (PAs) drive pulmonary arterial hypertension (PAH). Because cell growth mediated by the mTOR-containing mTORC1 complex is inhibited by tuberous sclerosis complex 2 (TSC2), we investigated the role of this GTPase-activating protein in PAH pathology. TSC2 abundance was decreased in remodeled small PAs and PA vascular smooth muscle cells (PAVSMCs) from patients with PAH or from rodent pulmonary hypertension (PH) models, as well as PAVSMCs maintained on substrates that reproduced pathology-induced stiffness. Accordingly, mice with smooth muscle-specific reduction in TSC2 developed PH. At the molecular level, decreased TSC2 abundance led to stiffness-induced PAVSMC proliferation, increased abundance of the mechanosensitive transcriptional coactivators YAP/TAZ, and enhanced mTOR kinase activity. Moreover, extracellular matrix (ECM) produced by TSC2-deficient PAVSMCs stimulated the proliferation of nondiseased PA adventitial fibroblasts and PAVSMCs through fibronectin and its receptor, the α5ß1 integrin. Reconstituting TSC2 in PAVSMCs from patients with PAH through overexpression or treatment with the SIRT1 activator SRT2104 decreased YAP/TAZ abundance, mTOR activity, and ECM production, as well as inhibited proliferation and induced apoptosis. In two rodent models of PH, SRT2104 treatment restored TSC2 abundance, attenuated pulmonary vascular remodeling, and ameliorated PH. Thus, TSC2 in PAVSMCs integrates ECM composition and stiffness with pro-proliferative and survival signaling, and restoring TSC2 abundance could be an attractive therapeutic option to treat PH.

Akt-Dependent Glycolysis-Driven Lipogenesis Supports Proliferation and Survival of Human Pulmonary Arterial Smooth Muscle Cells in Pulmonary Hypertension.

Hyper-proliferation of pulmonary arterial vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Lipogenesis is linked to numerous proliferative diseases, but its role in PAVSMC proliferation in PAH remains to be elucidated. We found that early-passage human PAH PAVSMC had significant up-regulation of key fatty acids synthesis enzymes ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN), and increased unstimulated proliferation compared to control human PAVSMC. Treatment with an allosteric ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) significantly decreased proliferation and induced apoptosis of human PAH PAVSMC. Intracellular lipid content and proliferation of PAH PAVSMC were not reduced by incubation in lipid-depleted media but suppressed by a non-metabolizable analog of glucose 2-Deoxy-D-glucose (2-DG) and partially restored by addition of pyruvate. Protein kinase Akt was upregulated in human PAH PAVSMC in a sirtuin 7 (SIRT7)- and c-Jun N-terminal kinase (JNK)-dependent manner. Pharmacological inhibition of Akt down-regulated ACLY and ACC, significantly reduced intracellular lipid content, inhibited proliferation and induced apoptosis of human PAH PAVSMC. Taken together, these data demonstrate that human PAH PAVSMC have up-regulated lipogenesis, which is supported in an Akt- and glycolysis-dependent manner and is required for increased proliferation and survival. Our data suggest that there is a mechanistic link between glycolysis, lipogenesis, and the proliferation of human PAH PAVSMC and call for further studies to determine the potential attractiveness of a SIRT7/JNK-Akt-lipogenesis axis as a target pathway to inhibit PAVSMC hyper-proliferation in PAH.

Noncanonical HIPPO/MST Signaling via BUB3 and FOXO Drives Pulmonary Vascular Cell Growth and Survival.

RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.

English Feature Recognition Based on GA-BP Neural Network Algorithm and Data Mining.

With the development of society and the promotion of science and technology, English, as the largest universal language in the world, is used by more and more people. In the life around us, there is information in English all the time. However, because the process of manual recognition of English letters is very labor-intensive and inefficient, the demand for computer recognition of English letters is increasing. This paper studies the influence of the parameters of BP neural network and genetic algorithm on the whole network, including the input, output, and number of hidden layer nodes. Finally, it improves and determines the settings and values of the relevant parameters. On this basis, it shows the rationality of the selected parameters through experiments. The results show that only GA-BP neural network and feature data mining algorithm can complete feature extraction and become the main function of feature classification at the same time. After enough initial data sample analysis training, the GA-BP neural network was found to have good data fault tolerance and feature recognition. The experimental results show that the genetic algorithm can find the best weights and thresholds and the weights and thresholds are given to the BP neural network. After training, the recognition of handwritten letters can be realized. Finally, the convergence of the two algorithms is compared through experiments, which shows that the overall performance of the BP neural network algorithm is improved after genetic algorithm optimization. It can be seen that the genetic algorithm has a good effect in improving the BP neural network and this method has a broad prospect in English feature recognition.

Wilson Disease: Intersecting DNA Methylation and Histone Acetylation Regulation of Gene Expression in a Mouse Model of Hepatic Copper Accumulation.

BACKGROUND & AIMS: The pathogenesis of Wilson disease (WD) involves hepatic and brain copper accumulation resulting from pathogenic variants affecting the ATP7B gene and downstream epigenetic and metabolic mechanisms. Prior methylome investigations in human WD liver and blood and in the Jackson Laboratory (Bar Harbor, ME) C3He-Atp7btx-j/J (tx-j) WD mouse model revealed an epigenetic signature of WD, including changes in histone deacetylase (HDAC) 5. We tested the hypothesis that histone acetylation is altered with respect to copper overload and aberrant DNA methylation in WD. METHODS: We investigated class IIa HDAC4 and HDAC5 and H3K9/H3K27 histone acetylation in tx-j mouse livers compared with C3HeB/FeJ (C3H) control in response to 3 treatments: 60% kcal fat diet, D-penicillamine (copper chelator), and choline (methyl group donor). Experiments with copper-loaded hepatoma G2 cells were conducted to validate in vivo studies. RESULTS: In 9-week tx-j mice, HDAC5 levels increased significantly after 8 days of a 60% kcal fat diet compared with chow. In 24-week tx-j mice, HDAC4/5 levels were reduced 5- to 10-fold compared with C3H, likely through mechanisms involving HDAC phosphorylation. HDAC4/5 levels were affected by disease progression and accompanied by increased acetylation. D-penicillamine and choline partially restored HDAC4/5 and H3K9ac/H3K27ac to C3H levels. Integrated RNA and chromatin immunoprecipitation sequencing analyses revealed genes regulating energy metabolism and cellular stress/development, which, in turn, were regulated by histone acetylation in tx-j mice compared with C3H mice, with Pparα and Pparγ among the most relevant targets. CONCLUSIONS: These results suggest dietary modulation of class IIa HDAC4/5, and subsequent H3K9/H3K27 acetylation/deacetylation can regulate gene expression in key metabolic pathways in the pathogenesis of WD.

Caveolar peroxynitrite formation impairs endothelial TRPV4 channels and elevates pulmonary arterial pressure in pulmonary hypertension.

Recent studies have focused on the contribution of capillary endothelial TRPV4 channels to pulmonary pathologies, including lung edema and lung injury. However, in pulmonary hypertension (PH), small pulmonary arteries are the focus of the pathology, and endothelial TRPV4 channels in this crucial anatomy remain unexplored in PH. Here, we provide evidence that TRPV4 channels in endothelial cell caveolae maintain a low pulmonary arterial pressure under normal conditions. Moreover, the activity of caveolar TRPV4 channels is impaired in pulmonary arteries from mouse models of PH and PH patients. In PH, up-regulation of iNOS and NOX1 enzymes at endothelial cell caveolae results in the formation of the oxidant molecule peroxynitrite. Peroxynitrite, in turn, targets the structural protein caveolin-1 to reduce the activity of TRPV4 channels. These results suggest that endothelial caveolin-1-TRPV4 channel signaling lowers pulmonary arterial pressure, and impairment of endothelial caveolin-1-TRPV4 channel signaling contributes to elevated pulmonary arterial pressure in PH. Thus, inhibiting NOX1 or iNOS activity, or lowering endothelial peroxynitrite levels, may represent strategies for restoring vasodilation and pulmonary arterial pressure in PH.

The Effects of Healthy Aging on Right Ventricular Structure and Biomechanical Properties: A Pilot Study.

Healthy aging has been associated with alterations in pulmonary vascular and right ventricular (RV) hemodynamics, potentially leading to RV remodeling. Despite the current evidence suggesting an association between aging and alterations in RV function and higher prevalence of pulmonary hypertension in the elderly, limited data exist on age-related differences in RV structure and biomechanics. In this work, we report our preliminary findings on the effects of healthy aging on RV structure, function, and biomechanical properties. Hemodynamic measurements, biaxial mechanical testing, constitutive modeling, and quantitative transmural histological analysis were employed to study two groups of male Sprague-Dawley rats: control (11 weeks) and aging (80 weeks). Aging was associated with increases in RV peak pressures (+17%, p = 0.017), RV contractility (+52%, p = 0.004), and RV wall thickness (+38%, p = 0.001). Longitudinal realignment of RV collagen (16.4°, p = 0.013) and myofibers (14.6°, p = 0.017) were observed with aging, accompanied by transmural cardiomyocyte loss and fibrosis. Aging led to increased RV myofiber stiffness (+141%, p = 0.003), in addition to a bimodal alteration in the biaxial biomechanical properties of the RV free wall, resulting in increased tissue-level stiffness in the low-strain region, while progressing into decreased stiffness at higher strains. Our results demonstrate that healthy aging may modulate RV remodeling via increased peak pressures, cardiomyocyte loss, fibrosis, fiber reorientation, and altered mechanical properties in male Sprague-Dawley rats. Similarities were observed between aging-induced remodeling patterns and those of RV remodeling in pressure overload. These findings may help our understanding of age-related changes in the cardiovascular fitness and response to disease.

Dechlorination and demethylation of ochratoxin A enhance blocking activity of PXR activation, suppress PXR expression and reduce cytotoxicity.

The pregnane X receptor (PXR) has been established to induce chemoresistance and metabolic diseases. Ochratoxin A (OTA), a mycotoxin, decreases the expression of PXR protein in human primary hepatocytes. OTA is chlorinated and has a methylated lactone ring. Both structures are associated with OTA toxicity. The study was to test the hypothesis that structural modifications differentially impact PXR blocking activity over cytotoxicity. To test this hypothesis, OTA-M and OTA-Cl/M were synthesized. OTA-M lacked the methyl group of the lactone-ring, whereas OTA-Cl/M had neither the methyl group nor the chlorine atom. The blocking activity of PXR activation was determined in a stable cell line, harboring both PXR (coding sequence) and its luciferase element reporter. OTA-Cl/M showed the highest blocking activity, followed by OTA-M and OTA. OTA-Cl/M was 60 times as potent as the common PXR blocker ketoconazole based on calculated IC50 values. OTA-Cl/M decreased by 90 % the expression of PXR protein and was the least cytotoxic among the tested compounds. Molecular docking identified that OTA and its derivatives interacted with different sets of residues in PXR, providing a molecular basis for selectivity. Excessive activation of PXR has been implicated in chemoresistance and metabolic diseases. Downregulation of PXR protein expression likely delivers an effective mechanism against structurally diverse PXR agonists.

Differential effects of integrin-linked kinase inhibitor Cpd22 on severe pulmonary hypertension in male and female rats.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease with no cure. Inhibition of integrin-linked kinase (ILK) reverses experimental pulmonary hypertension (PH) in male mice, but its effect on severe experimental PH in either male or female animals is unknown. We examined effects of ILK inhibitor Cpd22 on rats with SU5416/hypoxia-induced PH; treatment was performed at six to eight weeks after PH initiation. Five weeks after PH initiation, male and female rats developed similar levels of PH. Eight weeks after PH induction, vehicle-treated male rats had more severe PH than females. Cpd22-treated males, but not females, showed complete suppression of phospho-Akt in small pulmonary arteries (PAs), significantly lower PA medial thickness and percentage of fully occluded arteries, decreased systolic right ventricle (RV) pressure, PA pressure, RV hypertrophy, RV end-diastolic pressure, and improved RV contractility index compared to vehicle-treated group. Cpd22 suppressed proliferation of human male and female PAH pulmonary artery vascular smooth muscle cell (PAVSMC). 17ß-estradiol had no effect as a single agent but significantly attenuated Cpd22-dependent inhibition of proliferation in female, but not male, PAH PAVSMC. Taken together, these data demonstrate that male rats develop more severe PH than females but respond better to Cpd22 treatment by reducing pulmonary vascular remodeling, PH, and RV hypertrophy and improving RV functional outcomes. 17ß-estradiol diminishes anti-proliferative effect of Cpd22 in female, but not male, human PAH PAVSMC. These findings suggest potential attractiveness of ILK inhibition to reduce established PH in males and suggest that the combination with estrogen-lowering drugs could be considered to maximize anti-proliferative and anti-remodeling effects of ILK inhibitors in females.

Taurine Enhances the Protective Actions of Fish Oil Against D-Galactosamine-Induced Metabolic Changes and Hepatic Lipid Accumulation and Injury in the Rat.

This study has evaluated the effects of a supplementation with taurine (TAU) on the actions of fish oil (FO) against the hypoglycemia, hypoproteinemia, and hepatic accumulation of lipids and liver damage caused by D-galactosamine (GAL) in the rat. To this end, male Sprague-Dawley rats (200-225 g), in groups of 6, were orally treated with physiological saline (2.5 mL, control group), FO (60 mg/kg), TAU (2.4 mmol/kg) or FO-TAU for three consecutive days and before a single oral dose of GAL (400 mg/kg) given on day 3. In parallel, rats receiving only GAL on day 3 or N-acetylcysteine (NAC, 2.4 mmol/kg) for 3 days before GAL served as controls. On day 4 blood samples were collected by cardiac puncture and used to either measure glucose (GLC) or to obtain plasma fractions. Immediately thereafter, the livers were excised, made into a homogenate in phosphate buffered saline pH 7.4, and centrifuged to obtain clear supernatant. Plasma samples were assayed for their total protein (TP), triglycerides (TG), cholesterol (CHOL), phospholipids (PLP), free fatty acids (FFA) and total bilirubin (TB) and direct bilirubin (DB) contents, and for the activities of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP). The liver homogenates were used to measure TG, CHOL, PLP and total lipids (TL) contents. Without exceptions, GAL was found to markedly affect (p < 0.001) all of the experimental parameters examined, with increases occurring in all instances except for the values of the plasma GLC, TP and PLP which were decreased. A pretreatment with either FO or TAU led to significant attenuation of the effects of GAL and which, in most cases, were of similar magnitude. On the other hand, a combined pretreatment with FO plus TAU usually resulted in a greater protection than with either agent alone (p ≤ 0.05). NAC, serving as a reference treatment, was, in most instances, equipotent with FO alone and. in addition, was the only agent that significantly attenuated the increases in both liver weight and liver weight to body weight ratio caused by GAL.

Refined assessment of heavy metal-associated health risk due to the consumption of traditional animal medicines in humans.

Little is known about the extent of heavy metal accumulation in traditional Chinese medicines (TCMs). In this study, the levels of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) in traditional animal medicines were monitored using inductively coupled plasma mass spectroscopy (ICP-MS). Additionally, for the first time, a heavy metal risk assessment strategy was used to evaluate the potential risks of traditional animal medicines by calculating estimated daily intake (EDI), target hazard quotient (THQ), and cancer risk (CR). To obtain a refined risk assessment, the frequency of exposure to traditional animal medicines was determined from questionnaire data, and the safe factor for TCM was applied. Based on the standard levels for leech, it was found that earthworm, hive, scorpion, and leech accumulated high levels of heavy metals. The combined THQ (cTHQ) values indicated that ingestion of most traditional animal medicines would not pose a risk to the health of either male or female human beings. However, it was indicated that attention should be paid to the potential risk associated with cicada slough, earthworm, scorpion, turtle shells, and hive. Among heavy metals, As and Hg contributed to a major extent to the risk to human health. The CR assessment for Pb and As indicated that, with the exception of earthworm, the cancer risk was less than the acceptable lifetime risk for both males and females. Owing to the higher body weight, both THQ and CR were generally lower for males than for females.

Inhibitory Antibodies against Activin A and TGF-ß Reduce Self-Supported, but Not Soluble Factors-Induced Growth of Human Pulmonary Arterial Vascular Smooth Muscle Cells in Pulmonary Arterial Hypertension.

Increased growth and proliferation of distal pulmonary artery vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary arterial hypertension (PAH). Transforming Growth Factor-ß (TGF-ß) superfamily plays a critical role in PAH, but relative impacts of self-secreted Activin A, Gremlin1, and TGF-ß on PAH PAVSMC growth and proliferation are not studied. Here we report that hyper-proliferative human PAH PAVSMC have elevated secretion of TGF-ß1 and, to a lesser extent, Activin A, but not Gremlin 1, and significantly reduced Ser465/467-Smad2 and Ser423/425-Smad3 phosphorylation compared to controls. Media, conditioned by PAH PAVSMC, markedly increased Ser465/467-Smad2, Ser423/425-Smad3, and Ser463/465-Smad1/5 phosphorylation, up-regulated Akt, ERK1/2, and p38 MAPK, and induced significant proliferation of non-diseased PAVSMC. Inhibitory anti-Activin A antibody reduced PAH PAVSMC growth without affecting canonical (Smads) or non-canonical (Akt, ERK1/2, p38 MAPK) effectors. Inhibitory anti-TGF-ß antibody significantly reduced P-Smad3, P-ERK1/2 and proliferation of PAH PAVSMC, while anti-Gremlin 1 had no anti-proliferative effect. PDGF-BB diminished inhibitory effects of anti-Activin A and anti-TGF-ß antibodies. None of the antibodies affected growth and proliferation of non-diseased PAVSMC induced by PAH PAVSMC-secreted factors. Together, these data demonstrate that human PAH PAVSMC have secretory, proliferative phenotype that could be targeted by anti-Activin A and anti-TGF-ß antibodies; potential cross-talk with PDGF-BB should be considered while developing therapeutic interventions.

Preparation and characterization of a lovastatin-loaded protein-free nanostructured lipid carrier resembling high-density lipoprotein and evaluation of its targeting to foam cells.

This study was designed to investigate whether a non-protein nanostructured lipid carrier (NLC) resembling high-density lipoprotein (HDL) could deliver a hydrophobic anti-atherogenic drug, lovastatin, to foam cells. Lovastatin-loaded NLC (LT-NLC) was prepared by a nanoprecipitation/solvent diffusion method. The LT-NLC-apoprotein (LT-NLC-apo) was prepared by incubating LT-NLC with native HDL. The physicochemical parameters of LT-NLC were characterized in terms of particle size, zeta potential, morphology, entrapment efficiency, and crystallization behavior. Targeting behavior and mechanism were demonstrated by the incubation of LT-NLC-apo with a RAW 264.7 macrophage-derived foam cell model in the presence or absence of very-low-density lipoprotein (VLDL) and lipase. The results showed that LT-NLC was solid spherical or oval in shape with an average diameter of 13.8 ± 2.2 nm, zeta potential of -29.3 ± 0.2 mV and entrapment efficiency of 96.2 ± 1.3%. Phagocytosis studies showed that uptake of LT-NLC-apo by macrophages was significantly lower than LT-NLC (p < 0.01), suggesting that LT-NLC-apo could possibly escape recognition from macrophages in vivo. The uptake was increased twofold when LT-NLC-apo was incubated with transfected foam cells containing VLDL and lipase. These results indicated that non-protein NLC resembling HDL could be a useful tool to deliver lipophilic anti-atherogenic drugs to foam cells, and that uptake could be enhanced by the VLDL receptor pathway.