Quercetin prevents osteoarthritis progression possibly via regulation of local and systemic inflammatory cascades.

Due to the lack of effective treatments, osteoarthritis (OA) remains a challenge for clinicians. Quercetin, a bioflavonoid, has shown potent anti-inflammatory effects. However, its effect on preventing OA progression and the underlying mechanisms are still unclear. In this study, Sprague-Dawley male rats were divided into five groups: control group, OA group (monosodium iodoacetate intra-articular injection), and three quercetin-treated groups. Quercetin-treated groups were treated with intragastric quercetin once a day for 28 days. Gross observation and histopathological analysis showed cartilage degradation and matrix loss in the OA group. High-dose quercetin-group joints showed failure in OA progression. High-dose quercetin inhibited the OA-induced expression of MMP-3, MMP-13, ADAMTS4, and ADAMTS5 and promoted the OA-reduced expression of aggrecan and collagen II. Levels of most inflammatory cytokines and growth factors tested in synovial fluid and serum were upregulated in the OA group and these increases were reversed by high-dose quercetin. Similarly, subchondral trabecular bone was degraded in the OA group and this effect was reversed in the high-dose quercetin group. Our findings indicate that quercetin has a protective effect against OA development and progression possibly via maintaining the inflammatory cascade homeostasis. Therefore, quercetin could be a potential therapeutic agent to prevent OA progression in risk groups.

Chronic hypoxia promoted pulmonary arterial smooth muscle cells proliferation through upregulated calcium-sensing receptorcanonical transient receptor potential 1/6 pathway.

OBJECTIVES: Although both calcium-sensing receptor (CaSR) and canonical transient receptor potential (TRPC) proteins contribute to chronic hypoxia (CH)-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation, the relationship between CaSR and TRPC in hypoxic PASMCs proliferation remains poorly understood. The goal of this study was to identify that CH promotes PASMCs proliferation through CaSR-TRPC pathway. METHODS: Rat PASMCs were isolated and treated with CH. Cell proliferation was assessed by cell counting, CCK-8 assay, and EdU incorporation. CaSR and TRPC expressions were determined by qPCR and Western blotting. Store-operated Ca2+ entry (SOCE) was assessed by extracellular Ca2+ restoration. RESULTS: In PASMCs, CH enhanced the cell number, cell viability and DNA synthesis, which is accompanied by upregulated expression of CaSR, TRPC1 and TRPC6. Negative CaSR modulators (NPS2143, NPS2390) inhibited, whereas positive modulators (spermine, R568) enhanced, the CH-induced increases in cell number, cell viability and DNA synthesis in PASMCs. Knockdown of CaSR by siRNA inhibited the CH-induced upregulation of TRPC1 and TRPC6 and enhancement of SOCE and attenuated the CH-induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. However, neither siTRPC1 nor siTRPC6 had an effect on the CH-induced CaSR upregulation, although both significantly attenuated the CH-induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. CONCLUSION: These results demonstrate that upregulated CaSR-TRPC1/6 pathway mediating PASMCs proliferation is an important pathogenic mechanism under hypoxic conditions.

Phenotypically Silent Bone Morphogenetic Protein Receptor 2 Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing the Risk for Neointimal Transformation.

BACKGROUND: Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4, are candidates for the second hit. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. METHODS: Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2-mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes. RESULTS: Lung inflammation, induced by intratracheal delivery of 5-LO-expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2+/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2+/- rats gained endogenous 5-LO expression associated with elevated leukotriene B4 biosynthesis. Bmpr2-mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-ß) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-ß antagonism suggests that TGF-ß is critical for neointimal transformation. CONCLUSIONS: In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-ß signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.

PM2.5 promotes human bronchial smooth muscle cell migration via the sonic hedgehog signaling pathway.

BACKGROUND: The contribution of airway remodeling in chronic obstructive pulmonary disease (COPD) has been well documented, with airway smooth muscle cell proliferation and migration playing a role in the remodeling process. Here, we aimed to verify the effects of fine particulate matter (PM2.5) on human bronchial smooth muscle cell (HBSMC) migration and to explore the underlying signaling pathways. METHODS: HBSMC apoptosis, proliferation and migration were measured using flow cytometry, cell counting and transwell migration assays, respectively. The role of the hedgehog pathway in cell migration was assessed by western blotting to measure the expression of Sonic hedgehog (Shh), Gli1 and Snail. Furthermore, siRNA was used to knock down Gli1 or Snail expression. RESULTS: PM2.5 induced HBSMC apoptosis in a dose-dependent manner, although certain concentrations of PM2.5 did not induce HBSMC proliferation or apoptosis. Interestingly, cell migration was stimulated by PM2.5 doses far below those that induced apoptosis. Additional experiments revealed that these PM2.5 doses enhanced the expression of Shh, Gli1 and Snail in HBSMCs. Furthermore, PM2.5-induced cell migration and protein expression were enhanced by recombinant Shh and attenuated by cyclopamine. Similar results were obtained by knocking down Gli1 or Snail. CONCLUSIONS: These findings suggest that PM2.5, which may exert its effects through the Shh signaling pathway, is necessary for the migration of HBSMCs. These data define a novel role for PM2.5 in airway remodeling in COPD.

Nicotine-Induced Airway Smooth Muscle Cell Proliferation Involves TRPC6-Dependent Calcium Influx Via α7 nAChR.

BACKGROUND/AIMS: The proliferation of human bronchial smooth muscle cells (HBSMCs) is a key pathophysiological component of airway remodeling in chronic obstructive pulmonary disease (COPD) for which pharmacotherapy is limited, and only slight improvements in survival have been achieved in recent decades. Cigarette smoke is a well-recognized risk factor for COPD; however, the pathogenesis of cigarette smoke-induced COPD remains incompletely understood. This study aimed to investigate the mechanisms by which nicotine affects HBSMC proliferation. METHODS: Cell viability was assessed with a CCK-8 assay. Proliferation was measured by cell counting and EdU immunostaining. Fluorescence calcium imaging was performed to measure intracellular Ca2+ concentration ([Ca2+]i). RESULTS: The results showed that nicotine promotes HBSMC proliferation, which is accompanied by elevated store-operated calcium entry (SOCE), receptor-operated calcium entry (ROCE) and basal [Ca2+]i in HBSMCs. Moreover, we also confirmed that canonical transient receptor potential protein 6 (TRPC6) and α7 nicotinic acetylcholine receptor (α7 nAChR) are involved in nicotine-induced upregulation of cell proliferation. Furthermore, we verified that activation of the PI3K/Akt signaling pathway plays a pivotal role in nicotine-enhanced proliferation and calcium influx in HBSMCs. Inhibition of α7 nAChR significantly decreased Akt phosphorylation levels, and LY294002 inhibited the protein expression levels of TRPC6. CONCLUSION: Herein, these data provide compelling evidence that calcium entry via the α7 nAChR-PI3K/Akt-TRPC6 signaling pathway plays an important role in the physiological regulation of airway smooth muscle cell proliferation, representing an important target for augmenting airway remodeling.

Chronic Hypoxia Increases Intracellular Ca(2+) Concentration via Enhanced Ca(2+) Entry Through Receptor-Operated Ca(2+) Channels in Pulmonary Venous Smooth Muscle Cells.

BACKGROUND: Hypoxic pulmonary hypertension (HPH) is characterized by pulmonary vascular remodeling. Intracellular Ca(2+)concentration ([Ca(2+)]i) is an essential signal for myocyte proliferation. Whether chronic hypoxia (CH) affects the basal [Ca(2+)]I and Ca(2+)entry through store- and/or receptor-operated calcium channels (SOCC, ROCC), and whether canonical transient receptor potential (TRPC) proteins are involved in CH-induced Ca(2+)influx and proliferation in pulmonary venous smooth muscle cells (PVSMCs) is examined. METHODS AND RESULTS: Rats were exposed to CH. PVSMCs were isolated from distal pulmonary veins. In freshly isolated PVSMCs, CH increased the basal [Ca(2+)]i; removal of Ca(2+)or application of SKF-96365 reversed the elevated [Ca(2+)]i, whereas nifedipine had no effect. Receptor-operated Ca(2+)entry (ROCE) was expressed in PVSMCs. In freshly isolated PVSMCs from CH rats, ROCE was enhanced, whereas store-operated Ca(2+)entry had no alteration. Furthermore, real-time polymerase chain reaction and western blotting showed that mRNA and protein expression level of TRPC6, but neither TRPC1 nor TRPC3, in pulmonary venous smooth muscle (PV) from CH rats and PVSMCs exposed to CH was greater than in normal PV and PVSMCs. The knockdown of TRPC6 in hypoxic PVSMCs with siRNA inhibited the enhanced ROCE and attenuated CH-induced PVSMCs proliferation. CONCLUSIONS: The enhanced Ca(2+)entry through ROCC, due to upregulated TRPC6, is a novel pathogenic mechanism contributing to the increased basal [Ca(2+)]iin PVSMCs and excessive PVSMC proliferation during the development of HPH.

Nicotine elevated intracellular Ca²âº in rat airway smooth muscle cells via activating and up-regulating α7-nicotinic acetylcholine receptor.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling with airway smooth muscle (ASM) hypertrophy and hyperplasia. Since tobacco use is the key risk factor for the development of COPD and intracellular Ca(2+) concentration ([Ca(2+)]i) plays a major role in both cell proliferation and differentiation, we hypothesized that nicotinic acetylcholine receptor (nAChR) activation plays a role in the elevation of [Ca(2+)]i in airway smooth muscle cells (ASMCs). METHODS: We examined the expression of nAChR and characterized the functions of α7-nAChR in ASMCs. RESULTS: RT-PCR analysis showed that α2-7, ß2, and ß3-nAChR subunits are expressed in rat ASMCs, with α7 being one of the most abundantly expressed subtypes. Chronic nicotine exposure increased α7-nAChR mRNA and protein expression, and elevated resting [Ca(2+)]i in cultured rat ASMCs. Acute application of nicotine evoked a rapid increase in [Ca(2+)]i in a concentration-dependent manner, and the response was significantly enhanced in ASMCs cultured with 1 µM nicotine for 48 hours. Nicotine-induced Ca(2+) response was reversibly blocked by the α7-nAChR nicotinic antagonists, methyllycaconitine and α-bungarotoxin. Small interfering RNA suppression of α7-nAChR also substantially blunted the Ca(2+) responses induced by nicotine. CONCLUSION: These observations suggest that nicotine elevates [Ca(2+)]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling.that nicotine elevates [Ca(2+)]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling.

Development of a new method for the isolation and culture of pulmonary arterial endothelial cells from rat pulmonary arteries.

Pulmonary endothelial dysfunction plays an integral role in the pathogenesis and development of pulmonary hypertension. It is difficult and inconvenient to obtain pulmonary arterial endothelial cells (PAECs) from humans and large animals. Some methods for the isolation of PAECs from rats require complex equipment and expensive reagents. In this study, we describe a new method of obtaining cultures of PAECs isolated from rat pulmonary arteries with Chinese acupuncture needles. We acquired PAECs in 5 steps. These were: the isolation of pulmonary arteries, exposure of endothelium, enzymatic digestion, concentration of resuspended pellets and incubation. PAECs were characterized by morphological activity and by immunostaining for von Willebrand factor, CD31 and CD34, but not for α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Furthermore, transmission electron microscopy was carried out, confirming the presence of Weibel-Palade bodies that are characteristic ultrastructures of vascular endothelial cells. In conclusion, we established a simple and economical technique to isolate and culture PAECs from rat pulmonary arteries. These PAECs exhibit features consistent with vascular endothelial cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary arterial endothelium.

Development and systematic oxidative stress of a rat model of chronic bronchitis and emphysema induced by biomass smoke.

BACKGROUND: Epidemiological research and meta-analyses of published data have shown that biomass smoke (BS) is a risk factor for chronic obstructive pulmonary disease (COPD). However, the link between BS and COPD lacks experimental confirmation. OBJECTIVES: To verify whether BS can induce pathologic changes and systemic oxidative stress, which may be relevant to the development of emphysema and chronic bronchitis in rats. METHODS: Rats were exposed to BS, cigarette smoke (CS), or clean air (sham) for 14 weeks. During the exposure, the O2, SO2, and CO levels were monitored. Pathological changes in the lungs, systemic oxidative stress, and inflammation biomarkers, together with GSTM1 and GSTP1 mRNA expression in the lung were measured. The glutamate-cysteine ligase catalytic subunit (GCLC) protein expression in the lung was measured using immunohistochemistry and western blotting. RESULTS: The O2, CO, and SO2 levels were 20.31 ± 0.03%, 981.72 ± 64.76, and 2.59 ± 0.26 mg/m(3) for the BS group, respectively, while their levels in the CS group were 20.28 ± 0.15%, 745.56 ± 30.83, and 12.64 ± 0.591 mg/m(3) respectively. As with the rats exposed to CS, the BS rats showed an increased number of inflammatory cells in the bronchoalveolar lavage fluid, an increased pulmonary mean linear intercept and a decreased pulmonary mean alveolar number. Characteristics of chronic bronchitis and peribronchial fibrosis were also found in the BS-exposed rat lungs. Reduced body weight, systemic oxidative stress, and increased GCLC protein expression in the lungs were observed in the rats exposed to BS and CS. CONCLUSIONS: BS can cause emphysema and chronic bronchitis similar to that caused by CS, which is accompanied by systemic oxidative stress and inflammation.

Impaired exercise capacity in individuals with non-obstructive small airway dysfunction.

Background: Whether individuals with non-obstructive spirometry-defined small airway dysfunction (SAD) have impaired exercise capacity is unclear, particularly in never-smokers. This study clarifies the degree of impaired exercise capacity and its potential cause in individuals with non-obstructive SAD. Methods: This community-based, multiyear cross-sectional study analyzed data collected in Guangdong, China from 2012-2019 by the National Science and Technology Support Plan Program. Measurements of exercise capacity [peak work rate and peak oxygen uptake ( V ˙ O 2peak )] in participants with non-obstructive spirometry-defined SAD (n=157) were compared with those in controls (n=85) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) I patients (n=239). Subgroup analyses were performed by smoking status. Results: The risk of impaired exercise capacity was significantly higher in participants with non-obstructive SAD [ V ˙ O 2peak <84%predicted, adjusted odds ratio (aOR) =2.53; 95% confidence interval (CI): 1.42-4.52] than in controls but was not significantly different from that in GOLD I patients. Results were consistent within subgroups of smoking status (ever-smokers: non-obstructive SAD vs. controls, aOR =2.44; 95% CI: 1.08-5.51; never-smokers: non-obstructive SAD vs. controls, aOR =2.38, 95% CI: 1.02-5.58). Participants with non-obstructive SAD had a significantly lower peak work rate (ß=-10.5; 95% CI: -16.3 to -4.7) and V ˙ O 2peak (%predicted, ß=-4.0; 95% CI: -7.7 to -0.2) and tended to have higher ventilatory equivalents for carbon dioxide at the ventilatory threshold ( V ˙ E / V ˙ CO 2AT , ß=1.1; 95% CI: -0.1 to 2.3) when compared with controls. Both peak work rate and V ˙ O 2peak were negatively correlated with V ˙ E / V ˙ CO 2AT . Conclusions: Although not meeting the current criteria for chronic obstructive pulmonary disease, individuals with non-obstructive SAD have impaired exercise capacity that may be associated with ventilatory inefficiency regardless of smoking status.

Pseudomonas aeruginosa: A typical biofilm forming pathogen and an emerging but underestimated pathogen in food processing.

Pseudomonas aeruginosa (P. aeruginosa) is a notorious gram-negative pathogenic microorganism, because of several virulence factors, biofilm forming capability, as well as antimicrobial resistance. In addition, the appearance of antibiotic-resistant strains resulting from the misuse and overuse of antibiotics increases morbidity and mortality in immunocompromised patients. However, it has been underestimated as a foodborne pathogen in various food groups for instance water, milk, meat, fruits, and vegetables. Chemical preservatives that are commonly used to suppress the growth of food source microorganisms can cause problems with food safety. For these reasons, finding effective, healthy safer, and natural alternative antimicrobial agents used in food processing is extremely important. In this review, our ultimate goal is to cover recent advances in food safety related to P. aeruginosa including antimicrobial resistance, major virulence factors, and prevention measures. It is worth noting that food spoilage caused by P. aeruginosa should arouse wide concerns of consumers and food supervision department.

Intense pulsed light for inactivating planktonic and biofilm molds in food.

It has been reported that about a quarter of the world's agriculture products is unable to be consumed each year because of mold contamination, resulting in incalculable economic losses. Despite modern food technology and the various preservation techniques available, the problem of mold contamination of food is still not adequately controlled. In this study, we simulated the biofilm formed by Aspergillus niger and Penicillium glaucum in liquid and solid food in 96 well cell culture plates and polycarbonate membrane models, respectively, and investigated the fungicidal effect of IPL on planktonic and biofilm molds at three different capacitance parameters at room and refrigerator temperatures. The results show that IPL can achieve fungicidal rates of over 99% for planktonic molds and over 90% for biofilm molds, and that the smaller the capacitance, the more frequent the irradiation required to achieve the same fungicidal rate. In addition, temperature, A. niger or Penicillium glaucum have no effect on the fungicidal effect of IPL. We believe that IPL is a promising non-thermal physical sterilization technique for fungal inhibition on food surfaces.

Regulatory network controls microbial biofilm development, with Candida albicans as a representative: from adhesion to dispersal.

Microorganisms mainly exist in the form of biofilm in nature. Biofilm can contaminate food and drinking water system, as well as cause chronic wound infections, thereby posing a potential threat to public health safety. In the last two decades, researchers have made efforts to investigate the genetic contributors control different stages of biofilm development (adherence, initiation, maturation, and dispersal). As an opportunistic pathogen, C. albicans causes severe superficial or systemic infections with high morbidity and mortality under conditions of immune dysfunction. It has been reported that 80% of C. albicans infections are directly or indirectly associated with biofilm formation on host or abiotic surfaces including indwelling medical devices, resulting in high morbidity and mortality. Significantly, the outcome of C. albicans biofilm development includes enhanced invasion, exacerbated inflammatory responses and intrinsic resistance to antimicrobial chemotherapy. Thus, this review aimed at providing a comprehensive overview of the regulatory network controls microbial biofilm development, with C. albicans as a representative, served as reference for therapeutic targets.

Establishment and application of a rapid visual detection method for Listeria monocytogenes based on polymerase spiral reaction (PSR).

Listeria monocytogenes is a common foodborne pathogen that presents in various food products, posing important threat to public health. The aim of this study was to establish a rapid and sensitive method with visualization to detect L. monocytogenes based on polymerase spiral reaction (PSR). Primers targeting conserved hlyA gene sequence of L. monocytogenes were designed based on bioinformatics analyses on the current available L. monocytogenes genomes. The isothermal amplification PSR can be completed under constant temperature (65ᵒC) within 60 min with high specificity and sensitivity. Twenty-five reference strains were used to evaluate the specificity of the developed reaction. The results showed that the sensitive of the reaction for L. monocytogenes in purified genomic DNA and artificially contaminated food samples were 41 pg/µL and 103 CFU/mL, respectively. It was 100-fold more sensitive than conventional PCR. In conclusion, this novel PSR method is rapid, cost-efficient, timesaving, and applicable on artificially contaminated food samples, providing broad prospects into the detection of foodborne microbes with the promising on-site inspection.

Correction to: Platelet-derived growth factor-BB induces pulmonary venous smooth muscle cells proliferation by upregulating calcium sensing receptor under hypoxic conditions.

[This corrects the article DOI: 10.1007/s10616-021-00456-5.].

Quercetin modulates the gut microbiota as well as the metabolome in a rat model of osteoarthritis.

Although the mechanism of osteoarthritis (OA) has been widely studied and the use of quercetin for OA therapy is well documented, the relevant characteristics of the microbiome and metabolism remain unclear. This study reports changes in the gut microbiota and metabolism during quercetin therapy for OA in a rat model and provides an integrative analysis of the biomechanism. In this study, the rats were categorized into 3 different groups: the OA model, quercetin treatment, and control groups. The OA rats was conducted using a monoiodoacetate (MIA) injection protocol. The rats in the quercetin group received daily intragastric administration of quercetin from day 1 to day 28. Stool samples were collected, and DNA was extracted. We used an integrated approach that combined the sequencing of whole 16S rRNA, short-chain fatty acid (SCFA) measurements and metabolomics analysis by mass spectrometry (MS) to characterize the functional impact of quercetin on the gut microbiota and metabolism in a rat model of OA. The use of quercetin partially abrogated intestinal flora disorder and reversed fecal metabolite abnormalities. Compared with the control rats, the OA rats showed differences at both the class level (Clostridia, Bacteroidia, and Bacilli) and the genus level (Lactobacillus and unidentified Ruminococcaceae). Acetic acid, propionic acid and 24 metabolites were significantly altered among the three groups. However, the changes were significantly abrogated in quercetin-treated OA rats. Consequently, this study provided important evidence regarding perturbations of the gut microbiome and the function of these changes in a potential new mechanism of quercetin treatment.

Platelet-derived growth factor-BB induces pulmonary venous smooth muscle cells proliferation by upregulating calcium sensing receptor under hypoxic conditions.

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, which exists in both pulmonary arteries and pulmonary veins. Pulmonary vascular remodeling stems from excessive proliferation of pulmonary vascular myocytes. Platelet-derived growth factor-BB (PDGF-BB) is a vital vascular regulator whose level increases in PH human lungs. Although the mechanisms by which pulmonary arterial smooth muscle cells respond to PDGF-BB have been studied extensively, the effects of PDGF-BB on pulmonary venous smooth muscle cells (PVSMCs) remain unknown. We herein examined the involvement of calcium sensing receptor (CaSR) in PDGF-BB-induced PVSMCs proliferation under hypoxic conditions. In PVSMCs isolated from rat intrapulmonary veins, PDGF-BB increased the cell number and DNA synthesis under normoxic and hypoxic conditions, which was accompanied by upregulated CaSR expression. The influences of PDGF-BB on proliferation and CaSR expression in hypoxic PVSMCs were greater than that in normoxic PVSMCs. In hypoxic PVSMCs superfused with Ca2+-free solution, restoration of extracellular Ca2+ induced an increase of [Ca2+]i, which was significantly smaller than that in PDGF-BB-treated hypoxic PVSMCs. The positive CaSR modulator spermine enhanced, whereas the negative CaSR modulator NPS2143 attenuated, the extracellular Ca2+-induced [Ca2+]i increase in PDGF-BB-treated hypoxic PVSMCs. Furthermore, the spermine enhanced, whereas the NPS2143 inhibited, PDGF-BB-induced proliferation in hypoxic PVSMCs. Silencing CaSR with siRNA attenuated the extracellular Ca2+-induced [Ca2+]i increase in PDGF-BB-treated hypoxic PVSMCs and inhibited PDGF-BB-induced proliferation in hypoxic PVSMCs. In conclusion, these results demonstrated that CaSR mediating PDGF-BB-induced excessive PVSMCs proliferation is an important mechanism involved in the initiation and progression of PVSMCs proliferation under hypoxic conditions.

Changes in the gut microbiome and metabolome in a rat model of pulmonary arterial hypertension.

The gut microbiota is widely considered to be involved in several diseases, including atherosclerosis, obesity, chronic obstructive pulmonary disease (COPD) and pulmonary arterial hypertension (PAH). This study aimed to determine if changes in the gut microbiome and metabolome play a major role in the early pathogenesis of PAH. Male Wistar rats were injected with monocrotaline (MCT) (55 mg/kg) at day 1 and injected with calcium-sensing receptor (CaSR) antagonist NPS2143 (4.5 mg/kg/d) from days 1 to 21. Fecal samples were obtained. The gut microbiota and metabolome were analyzed by 16S rRNA gene sequencing and mass spectrometry-based analysis to investigate the effect of PAH in this rat model. MCT injection had a marked effect on the composition of the gut microbiota. This finding was further confirmed by metabolomic analysis with identification of several metabolites relevant to the gut microflora. However, NPS2143 partially abrogated this intestinal flora disorder and reversed fecal metabolite abnormalities. In conclusion, our study shows correlations between changes in the gut microbiome and the metabolome in PAH, which are affected by NPS2143.

Lung Features in Individuals with Biomass Smoke Exposure Characterized by CT Scan and Changes in Pulmonary Function.

BACKGROUND AND OBJECTIVE: To determine the effects of BSE (biomass smoke exposure) on pulmonary and non-pulmonary changes in patients with COPD compared with normal individuals. METHODS: Using a cohort, we recruited 16 healthy individuals with BSE (BSE normal), 19 patients with BSE+COPD, 13 healthy individuals with cigarette smoke exposure (CSE normal), 25 patients with CSE+COPD, and 25 healthy controls. Patients with GOLD stage I and II COPD were included. Baseline data (demographic data, BSE or CSE, lung function, and CT findings) and follow-up lung function data were collected. CT parameters of emphysema, pulmonary small vessels, airway remodeling, pectoralis muscles, and erector spinae muscle were measured. RESULTS: Individuals with BSE were mainly women (32/35, 91.43%). Compared with the CSE+COPD group, the BSE+COPD group demonstrated slower lung function decline, increased lower lung emphysema, narrower airway lumen dimensions and increased airway wall thickening in the moderate and small airways (all P<0.05). Compared with healthy controls, the CSE normal and BSE normal groups exhibited significant reductions in pulmonary small vessel area and obvious airway remodeling in small airways (P<0.05). Compared with the BSE normal group, the BSE+COPD group showed significantly more severe emphysema and airway remodeling, as well as reduced left pectoralis major muscle area (all P<0.05). CONCLUSION: Healthy individuals with BSE had reduced pulmonary small vessel area and evidence of airway remodeling; patients with BSE and COPD showed more severe emphysema, airway remodeling, and reductions in pectoralis major muscle area. CLINICAL TRIAL REGISTRATION: ChiCTR-OO-14004264.

Sildenafil inhibits chronically hypoxic upregulation of canonical transient receptor potential expression in rat pulmonary arterial smooth muscle.

In pulmonary arterial smooth muscle cells (PASMCs), Ca2+ influx through store-operated Ca2+ channels thought to be composed of canonical transient receptor potential (TRPC) proteins is an important determinant of intracellular free calcium concentration ([Ca2+](i)) and pulmonary vascular tone. Sildenafil, a type V phosphodiesterase inhibitor that increases cellular cGMP, is recently identified as a promising agent for treatment of pulmonary hypertension. We previously demonstrated that chronic hypoxia elevated basal [Ca2+](i) in PASMCs due in large part to enhanced store-operated Ca2+ entry (SOCE); moreover, ex vivo exposure to prolonged hypoxia (4% O2 for 60 h) upregulated TRPC1 and TRPC6 expression in PASMCs. We examined the effect of sildenafil on basal [Ca2+](i), SOCE, and the expression of TRPC in PASMCs under prolonged hypoxia exposure. We also examined the effect of sildenafil on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle (PA) from rats that developed chronically hypoxic pulmonary hypertension (CHPH). Compared with vehicle control, treatment with sildenafil (300 nM) inhibited prolonged hypoxia induced increases of 1) basal [Ca2+](i), 2) SOCE, and 3) mRNA and protein expression of TRPC in PASMCs. Moreover, sildenafil (50 mg . kg(-1) . day(-1)) inhibited mRNA and protein expression of TRPC1 and TRPC6 in PA from chronically hypoxic (10% O2 for 21 days) rats, which was associated with decreased right ventricular pressure and right ventricular hypertrophy. Furthermore, we found, in PASMCs exposed to prolonged hypoxia, that knockdown of TRPC1 or TRPC6 by their specific small interference RNA attenuated the hypoxic increases of SOCE and basal [Ca2+]i, suggesting a cause and effect link between increases of TRPC1 and TRPC6 expression and the hypoxic increases of SOCE and basal [Ca2+]i. These results suggest that sildenafil may alter basal [Ca2+](i) in PASMCs by decreasing SOCE through downregulation of TRPC1 and TRPC6 expression, thereby contributing to decreased vascular tone of pulmonary arteries during the development of CHPH.