Transcriptomic profiling highlights cell proliferation in the progression of experimental pulmonary hypertension in rats.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary vascular remolding and occlusion, leading to the elevated pulmonary arterial pressures, right ventricular hypertrophy, and eventual heart failure if left untreated. Understanding the molecular mechanisms underlying the development and progression of pulmonary hypertension (PH) is crucial for devising efficient therapeutic approaches for the disease. Lung homogenates were collected weekly and underwent RNA-sequencing in the monocrotaline (MCT)-induced PH rat model to explore genes associated with PH progression. Statistical analyses revealed 1038, 1244, and 3125 significantly altered genes (P < 0.05, abs (log2fold change) > log21.5) between control and MCT-exposed rats during the first, second, and third week, respectively. Pathway enrichment analyses revealed involvement of cell cycle and innate immune system for the upregulated genes, GPCR and VEGF signaling for the downregulated genes. Furthermore, qRT-PCR validated upregulation of representative genes associated with cell cycle including Cdc25c (cell division cycle 25C), Cdc45, Top2a (topoisomerase IIα), Ccna2 (cyclin A2) and Ccnb1 (cyclin B1). Western blot and immunofluorescence analysis confirmed increases in PCNA, Ccna2, Top2a, along with other proliferation markers in the lung tissue of MCT-treated rats. In summary, RNA sequencing data highlights the significance of cell proliferation in progression of rodent PH.

Proteomic and Phosphoproteomic Analysis of Right Ventricular Hypertrophy in the Pulmonary Hypertension Rat Model.

Pulmonary arterial hypertension (PAH) is a progressive disease that affects both the lungs and heart. Right ventricle (RV) hypertrophy is a primary pathological feature of PAH; however, its underlying molecular mechanisms remain insufficiently studied. In this study, we employed tandem mass tag (TMT)-based quantitative proteomics for the integrative analysis of the proteome and phosphoproteome of the RV derived from monocrotaline-induced PAH model rats. Compared with control samples, 564 significantly upregulated proteins, 616 downregulated proteins, 622 downregulated phosphopeptides, and 683 upregulated phosphopeptides were identified (P < 0.05, abs (log2 (fold change)) > log2 1.2) in the MCT samples. The quantitative real-time polymerase chain reaction (qRT-PCR) validated the expression levels of top 20 significantly altered proteins, including Nppa (natriuretic peptides A), latent TGF-ß binding protein 2 (Ltbp2), periostin, connective tissue growth factor 2 (Ccn2), Ncam1 (neural cell adhesion molecule), quinone reductase 2 (Nqo2), and tropomodulin 4 (Tmod4). Western blotting confirmed the upregulation of Ncam1 and downregulation of Nqo2 and Tmod4 in both MCT-induced and hypoxia-induced PH rat models. Pathway enrichment analyses indicated that the altered proteins are associated with pathways, such as vesicle-mediated transport, actin cytoskeleton organization, TCA cycle, and respiratory electron transport. These significantly changed phosphoproteins were enriched in pathways such as diabetic cardiomyopathy, hypertrophic cardiomyopathy, glycolysis/gluconeogenesis, and cardiac muscle contraction. In summary, this study provides an initial analysis of the RV proteome and phosphoproteome in the progression of PAH, highlighting several RV dysfunction-associated proteins and pathways.

Supraclavicular brown adipocytes originate from Tbx1+ myoprogenitors.

Brown adipose tissue (BAT) dissipates energy as heat, contributing to temperature control, energy expenditure, and systemic homeostasis. In adult humans, BAT mainly exists in supraclavicular areas and its prevalence is associated with cardiometabolic health. However, the developmental origin of supraclavicular BAT remains unknown. Here, using genetic cell marking in mice, we demonstrate that supraclavicular brown adipocytes do not develop from the Pax3+/Myf5+ epaxial dermomyotome that gives rise to interscapular BAT (iBAT). Instead, the Tbx1+ lineage that specifies the pharyngeal mesoderm marks the majority of supraclavicular brown adipocytes. Tbx1Cre-mediated ablation of peroxisome proliferator-activated receptor gamma (PPARγ) or PR/SET Domain 16 (PRDM16), components of the transcriptional complex for brown fat determination, leads to supraclavicular BAT paucity or dysfunction, thus rendering mice more sensitive to cold exposure. Moreover, human deep neck BAT expresses higher levels of the TBX1 gene than subcutaneous neck white adipocytes. Taken together, our observations reveal location-specific developmental origins of BAT depots and call attention to Tbx1+ lineage cells when investigating human relevant supraclavicular BAT.

Quantitative Proteomic and Phosphoproteomic Profiling of Lung Tissues from Pulmonary Arterial Hypertension Rat Model.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by elevated pulmonary vascular resistance and increased pressure in the distal pulmonary arteries. Systematic analysis of the proteins and pathways involved in the progression of PAH is crucial for understanding the underlying molecular mechanism. In this study, we performed tandem mass tags (TMT)-based relative quantitative proteomic profiling of lung tissues from rats treated with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 6759 proteins were quantified, among which 2660 proteins exhibited significant changes (p-value < 0.05, fold change < 0.83 or >1.2). Notably, these changes included several known PAH-related proteins, such as Retnla (resistin-like alpha) and arginase-1. Furthermore, the expression of potential PAH-related proteins, including Aurora kinase B and Cyclin-A2, was verified via Western blot analysis. In addition, we performed quantitative phosphoproteomic analysis on the lungs from MCT-induced PAH rats and identified 1412 upregulated phosphopeptides and 390 downregulated phosphopeptides. Pathway enrichment analysis revealed significant involvement of pathways such as complement and coagulation cascades and the signaling pathway of vascular smooth muscle contraction. Overall, this comprehensive analysis of proteins and phosphoproteins involved in the development and progression of PAH in lung tissues provides valuable insights for the development of potential diagnostic and treatment targets for PAH.

NF-κB-dependent repression of Sox18 transcription factor requires the epigenetic regulators histone deacetylases 1 and 2 in acute lung injury.

In acute lung injury (ALI), the NF-κB-mediated downregulation of Sox18 gene expression leads to the disruption of the pulmonary endothelial barrier. Previous studies have suggested that the action of NF-κB as a transcriptional repressor also requires the action of class I histone deacetylases (HDACs). Thus, the purpose of this study was to investigate and further delineate the mechanism of Sox18 repression during lipopolysaccharide (LPS) induced ALI. Using selective inhibitors and specific siRNA-driven depletion of HDACs 1-3 in human lung microvascular endothelial cells (HLMVEC) we were able to demonstrate a critical role for HDACs 1 and 2 in the LPS-mediated repression of Sox18 gene expression and the loss of endothelial monolayer integrity. Moreover, our data demonstrate that HDAC1 associates with a transcription-repressive complex within the NF-κB-binding site of Sox18 promoter. Further, we were able to show that the selective inhibitor of HDAC1, tacedinaline, significantly reduced the endothelial permeability and injury associated with LPS challenge in the mouse lung. Taken together, our data demonstrate, for the first time, that transcription repressors HDACs 1 and 2 are involved in pathological mechanism of ALI and can be considered as therapeutic targets.

Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension.

Rationale: Enhanced proliferation and distal migration of human pulmonary arterial smooth muscle cells (hPASMCs) both contribute to the progressive increases in pulmonary vascular remodeling and resistance in pulmonary arterial hypertension (PAH). Our previous studies revealed that Rictor deletion, to disrupt mTOR Complex 2 (mTORC2), over longer periods result in a paradoxical rise in platelet-derived growth factor receptor (PDGFR) expression in PASMCs. Thus, the purpose of this study was to evaluate the role of combination therapy targeting both mTOR signaling with PDGFR inhibition to attenuate the development and progression of PAH. Methods and Results: Immunoblotting analyses revealed that short-term exposure to rapamycin (6h) significantly reduced phosphorylation of p70S6K (mTORC1-specific) in hPASMCs but had no effect on the phosphorylation of AKT (p-AKT S473, considered mTORC2-specific). In contrast, longer rapamycin exposure (>24 h), resulted in differential AKT (T308) and AKT (S473) phosphorylation with increases in phosphorylation of AKT at T308 and decreased phosphorylation at S473. Phosphorylation of both PDGFRα and PDGFRß was increased in hPASMCs after treatment with rapamycin for 48 and 72 h. Based on co-immunoprecipitation studies, longer exposure to rapamycin (24-72 h) significantly inhibited the binding of mTOR to Rictor, mechanistically suggesting mTORC2 inhibition by rapamycin. Combined exposure of rapamycin with the PDGFR inhibitor, imatinib significantly reduced the proliferation and migration of hPASMCs compared to either agent alone. Pre-clinical studies validated increased therapeutic efficacy of rapamycin combined with imatinib in attenuating PAH over either drug alone. Specifically, combination therapy further attenuated the development of monocrotaline (MCT)- or Hypoxia/Sugen-induced pulmonary hypertension (PH) in rats as demonstrated by further reductions in the Fulton index, right ventricular systolic pressure (RVSP), pulmonary vascular wall thickness and vessel muscularization, and decreased proliferating cell nuclear antigen (PCNA) staining in PASMCs. Conclusion: Prolonged rapamycin treatment activates PDGFR signaling, in part, via mTORC2 inhibition. Combination therapy with rapamycin and imatinib may be a more effective strategy for the treatment of PAH.

Molecular Characterization and Elucidation of Pathways to Identify Novel Therapeutic Targets in Pulmonary Arterial Hypertension.

Background: Pulmonary arterial hypertension (PAH) is a life-threatening chronic cardiopulmonary disease. However, there are limited studies reflecting the available biomarkers from separate gene expression profiles in PAH. This study explored two microarray datasets by an integrative analysis to estimate the molecular signatures in PAH. Methods: Two microarray datasets (GSE53408 and GSE113439) were exploited to compare lung tissue transcriptomes of patients and controls with PAH and to estimate differentially expressed genes (DEGs). According to common DEGs of datasets, gene and protein overrepresentation analyses, protein-protein interactions (PPIs), DEG-transcription factor (TF) interactions, DEG-microRNA (miRNA) interactions, drug-target protein interactions, and protein subcellular localizations were conducted in this study. Results: We obtained 38 common DEGs for these two datasets. Integration of the genome transcriptome datasets with biomolecular interactions revealed hub genes (HSP90AA1, ANGPT2, HSPD1, HSPH1, TTN, SPP1, SMC4, EEA1, and DKC1), TFs (FOXC1, FOXL1, GATA2, YY1, and SRF), and miRNAs (hsa-mir-17-5p, hsa-mir-26b-5p, hsa-mir-122-5p, hsa-mir-20a-5p, and hsa-mir-106b-5p). Protein-drug interactions indicated that two compounds, namely, nedocromil and SNX-5422, affect the identification of PAH candidate biomolecules. Moreover, the molecular signatures were mostly localized in the extracellular and nuclear areas. Conclusions: In conclusion, several lung tissue-derived molecular signatures, highlighted in this study, might serve as novel evidence for elucidating the essential mechanisms of PAH. The potential drugs associated with these molecules could thus contribute to the development of diagnostic and therapeutic strategies to ameliorate PAH.

Salusin-ß Promotes Vascular Calcification via Nicotinamide Adenine Dinucleotide Phosphate/Reactive Oxygen Species-Mediated Klotho Downregulation.

Aims: Vascular calcification (VC) is a hallmark feature of cardiovascular disease and a significant risk factor for morbidity and mortality. Salusin-ß exerts cardiovascular regulating effects in hypertension, atherosclerosis, and diabetes. The present study was designed to examine the roles of salusin-ß in the progression of VC and its downstream signaling mechanisms. Results: Salusin-ß expression in both the aortas of VC rats induced by vitamin D3 and nicotine and vascular smooth muscle cells (VSMCs) incubated with calcifying media was increased. Salusin-ß knockdown remarkably reduced VC, whereas overexpression of salusin-ß exacerbated VC both in vitro and in vivo. Overexpression of salusin-ß promoted the VSMC osteochondrogenic transition, decreased Klotho protein levels, enhanced Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the translocation of p47phox to the membrane, increased the expression of nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase subunits and the production of reactive oxygen species (ROS) with or without calcifying media; however, salusin-ß deficiency played the opposite roles. The calcification and downregulated Klotho protein levels induced by salusin-ß were restored by ROS scavenger N-acetyl-l-cysteine, diphenyleneiodonium chloride [an inhibitor of flavin-containing enzyme, including NAD(P)H oxidase], or gene knockdown of NAD(P)H oxidase (NOX)-2, p22phox, or p47phox but were not affected by NOX-1 and NOX-4 knockdown. Klotho knockdown attenuated the protective effect of salusin-ß deficiency on VSMC calcification. By contrast, exogenous Klotho ameliorated the development of VC and ROS generation induced by salusin-ß overexpression. Innovation: Salusin-ß is a critical modulator in VC. Conclusion: Salusin-ß regulates VC through activation of NAD(P)H/ROS-mediated Klotho downregulation, suggesting that salusin-ß may be a novel target for treatment of VC.

Echocardiographic assessment of right ventricular function in experimental pulmonary hypertension.

Echocardiography, a non-invasive and cost-effective method for monitoring cardiac function, is commonly used for evaluation and pre-clinical diagnostics of pulmonary hypertension (PH). Previous echocardiographic studies in experimental models of PH are fragmentary in terms of the evaluation of right ventricle (RV) function. In this study, three rodent models of PH: a mouse model of hypoxia-induced PH, a rat model of hypoxia+Sugen induced PH and a rat model of monocrotaline-induced PH, were employed to measure RV fractional area change (RVFAC), RV free wall thickness (RVFWT), pulmonary acceleration time (PAT), pulmonary ejection time (PET), and tricuspid annular plane systolic excursion (TAPSE). We found that, in these models, RVFWT significantly increased, but RVFAC, PAT, or PAT/PET ratios and TAPSE values significantly decreased. Accurate and complete TAPSE patterns were demonstrated in the three rodent models of PH. The RV echocardiography data matched the corresponding invasive hemodynamic and heart histologic data in each model. This serves as a reference study for real-time and non-invasive evaluation of RV function in rodent models of PH using echocardiography.

Neuroprotective Effects of Jitai Tablet, a Traditional Chinese Medicine, on the MPTP-Induced Acute Model of Parkinson's Disease: Involvement of the Dopamine System.

Jitai tablet (JTT) is a traditional Chinese medicine used to treat neuropsychiatric disorders. We previously demonstrated that JTT treatment led to increased level of dopamine transporter (DAT) in the striatum, thus indicating that JTT might have therapeutic potential for Parkinson's disease (PD), which is characterized by dysregulated dopamine (DA) transmission and decreased striatal DAT expression. The aim of this study was to investigate the neuroprotective effect of JTT on MPTP-induced PD mice. Using locomotor activity test and rotarod test, we evaluated the effects of JTT (0.50, 0.15, or 0.05 g/kg) on MPTP-induced behavioral impairments. Tyrosine hydroxylase TH-positive neurons in the substantia nigra and DAT and dopamine D2 receptor (D2R) levels in the striatum were detected by immunohistochemical staining and/or autoradiography. Levels of DA and its metabolites were determined by HPLC. In MPTP-treated mice, behavioral impairments were alleviated by JTT treatment. Moreover, JTT protected against impairment of TH-positive neurons and attenuated the MPTP-induced decreases in DAT and D2R. Finally, high dose of JTT (0.50 g/kg) inhibited the MPTP-induced increase in DA metabolism rate. Taken together, results from our present study provide evidence that JTT offers neuroprotective effects against the neurotoxicity of MPTP and thus might be a potential treatment for PD.

Preliminary evaluation of the interactions of Panax ginseng and Salvia miltiorrhiza Bunge with 5-fluorouracil on pharmacokinetics in rats and pharmacodynamics in human cells.

An increasing number of cancer patients are using herbs in combination with conventional chemotherapeutic treatment. It is therefore important to study the potential consequences of the interactions between herbs and anticancer drugs. The effects of extracts from Panax ginseng (PGS) and Salvia miltiorrhiza Bunge (SMB) on the pharmacokinetics of 5-fluorouracil (5-FU) were performed in vivo and detected by high performance liquid chromatography (HPLC), while, an ATP assay was used to study the pharmacodynamic interactions in vitro. The results of the pharmacokinetic experiments showed a significant increase in the elimination half-life (t1/2(k e )) of 5-FU in the PGS-pretreated group and in the area under the curve (AUC) in the SMB-pretreated group compared with the control group. However, after SMB pretreatment, weight loss was observed in rats. The results of pharmacodynamic experiments showed that neither PGS nor SMB, when used alone, directly inhibited cancer cell growth at 0.1-100 µg/ml. Moreover, PGS had a synergistic cytotoxic effect with 5-FU on human gastric cancer cells but not on normal gastric cells. The results imply that when combined with 5-FU, PGS may be a better candidate for further study. This study might provide insights for the selection of herbal-chemotherapy agent interactions.

Anticancer activity of an extract from needles and twigs of Taxus cuspidata and its synergistic effect as a cocktail with 5-fluorouracil.

BACKGROUND: Botanical medicines are increasingly combined with chemotherapeutics as anticancer drug cocktails. This study aimed to assess the chemotherapeutic potential of an extract of Taxus cuspidata (TC) needles and twigs produced by artificial cuttage and its co-effects as a cocktail with 5-fluorouracil (5-FU). METHODS: Components of TC extract were identified by HPLC fingerprinting. Cytotoxicity analysis was performed by MTT assay or ATP assay. Apoptosis studies were analyzed by H & E, PI, TUNEL staining, as well as Annexin V/PI assay. Cell cycle analysis was performed by flow cytometry. 5-FU concentrations in rat plasma were determined by HPLC and the pharmacokinetic parameters were estimated using 3p87 software. Synergistic efficacy was subjected to median effect analysis with the mutually nonexclusive model using Calcusyn1 software. The significance of differences between values was estimated by using a one-way ANOVA. RESULTS: TC extract reached inhibition rates of 70-90% in different human cancer cell lines (HL-60, BGC-823, KB, Bel-7402, and HeLa) but only 5-7% in normal mouse T/B lymphocytes, demonstrating the broad-spectrum anticancer activity and low toxicity to normal cells of TC extract in vitro. TC extract inhibited cancer cell growth by inducing apoptosis and G(2)/M cell cycle arrest. Most interestingly, TC extract and 5-FU, combined as a cocktail, synergistically inhibited the growth of cancer cells in vitro, with Combination Index values (CI) ranging from 0.90 to 0.26 at different effect levels from IC50 to IC90 in MCF-7 cells, CI ranging from 0.93 to 0.13 for IC40 to IC90 in PC-3M-1E8 cells, and CI < 1 in A549 cells. In addition, the cocktail had lower cytotoxicity in normal human cell (HEL) than 5-FU used alone. Furthermore, TC extract did not affect the pharmacokinetics of 5-FU in rats. CONCLUSIONS: The combinational use of the TC extract with 5-FU displays strong cytotoxic synergy in cancer cells and low cytotoxicity in normal cells. These findings suggest that this cocktail may have a potential role in cancer treatment.

Effect of green tea on pharmacokinetics of 5-fluorouracil in rats and pharmacodynamics in human cell lines in vitro.

Tea drinking is widely practiced in the world and has recently increased among cancer patients. However, the effects of concurrent consumption of tea on the bioavailability and the net therapeutic potential of co-administered chemical drugs are not clear. In this study, the effects of green tea on the pharmacokinetics of 5-fluorouracil (5-FU) in rats and the pharmacodynamics in human cell lines in vitro were studied. The pharmacokinetic experiment indicated that there was an approximately 151% increase in the maximum plasma concentration (C(max)) and an approximately 425% increase in the area under the plasma concentration curve (AUC) of 5-FU in the green tea-treated group compared with the control group. Green tea consumption increased the plasma concentration of 5-FU. In addition, the pharmacodynamics experiment showed that at the moderate dose level (equivalent to <6 cups daily in human), neither fresh green tea extract nor (-)-epigallocatechin-3-gallate (EGCG) showed significant additive effects on the cytotoxicity of 5-FU in human cell lines. The results showed that it is crucial to perform therapeutic drug monitoring (TDM) when the cancer patients have a habit of drinking green tea.