Comprehensive Analysis of Fibroblast Activation Protein Expression in Interstitial Lung Diseases.

Rationale: Sustained activation of lung fibroblasts and the resulting oversynthesis of the extracellular matrix are detrimental events for patients with interstitial lung diseases (ILDs). Lung biopsy is a primary evaluation technique for the fibrotic status of ILDs, and is also a major risk factor for triggering acute deterioration. Fibroblast activation protein (FAP) is a long-known surface biomarker of activated fibroblasts, but its expression pattern and diagnostic implications in ILDs are poorly defined. Objectives: The present study aims to comprehensively investigate whether the expression intensity of FAP could be used as a potential readout to estimate or measure the amounts of activated fibroblasts in ILD lungs quantitatively. Methods: FAP expression in human primary lung fibroblasts as well as in clinical lung specimens was first tested using multiple experimental methods, including real-time quantitative PCR (qPCR), Western blot, immunofluorescence staining, deep learning measurement of whole slide immunohistochemistry, as well as single-cell sequencing. In addition, FAP-targeted positron emission tomography/computed tomography imaging PET/CT was applied to various types of patients with ILD, and the correlation between the uptake of FAP tracer and pulmonary function parameters was analyzed. Measurements and Main Results: Here, it was revealed, for the first time, FAP expression was upregulated significantly in the early phase of lung fibroblast activation event in response to a low dose of profibrotic cytokine. Single-cell sequencing data further indicate that nearly all FAP-positive cells in ILD lungs were collagen-producing fibroblasts. Immunohistochemical analysis validated that FAP expression level was closely correlated with the abundance of fibroblastic foci on human lung biopsy sections from patients with ILDs. We found that the total standard uptake value (SUV) of FAP inhibitor (FAPI) PET (SUVtotal) was significantly related to lung function decline in patients with ILD. Conclusions: Our results strongly support that in vitro and in vivo detection of FAP can assess the profibrotic activity of ILDs, which may aid in early diagnosis and the selection of an appropriate therapeutic window.

Deficiency of HtrA3 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via TGF-ß1/Smad Signaling Pathway.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by excessive extracellular matrix deposition. No effective treatments are currently available for IPF. High-temperature requirement A3 (HtrA3) suppresses tumor development by antagonizing transforming growth factor ß (TGF-ß) signaling; however, little is known about the role of HtrA3 in IPF. This study investigated the role of HtrA3 in IPF and underlying mechanisms. METHODS: Lung tissues were collected from patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis, and HtrA3 expression was measured in tissue samples. Then, HtrA3 gene knockout mice were treated with BLM to induce pulmonary fibrosis and explore the effects and underlying mechanism of HtrA3 on pulmonary fibrosis. RESULTS: HtrA3 was up-regulated in the lung tissues of patients with IPF and the pulmonary fibrotic mouse model compared to corresponding control groups. HtrA3 knockout decreased pulmonary fibrosis-related protein expression, alleviated the symptoms of pulmonary fibrosis, and inhibited epithelial-mesenchymal transition (EMT) in BLM-induced lung tissue compared with BLM-induced wild-type mice. The TGF-ß1/Smad signaling pathway was activated in fibrotic lung tissue, whereas HtrA3 knockout inhibited this pathway. CONCLUSION: The expression level of HtrA3 is increased in fibrotic lungs. HtrA3 knockout alleviates the symptoms of pulmonary fibrosis probably via the TGF-ß1/Smad signaling pathway. Therefore, HtrA3 inhibition is a potential therapeutic target for pulmonary fibrosis.

Periostin: A Potential Therapeutic Target For Pulmonary Hypertension?

RATIONALE: POSTN (Periostin) is an ECM (extracellular matrix) protein involved in tissue remodeling in response to injury and a contributing factor in tumorigenesis, suggesting that POSTN plays a role in the pathogenesis of pulmonary hypertension (PH). OBJECTIVE: We aimed to gain insight into the mechanistic contribution of POSTN in experimental mouse models of PH and correlate these findings with PH in humans. METHODS AND RESULTS: We used genetic epistasis approaches in human pulmonary artery endothelial cells (hPAECs), human pulmonary artery smooth muscle cells, and experimental mouse models of PH (Sugen 5416/hypoxia or chronic hypoxia) to discern the role of POSTN and its relationship to HIF (hypoxia-inducible factor)-1α signaling. We found that POSTN expression was correlated with the extent of PH in mouse models and in humans. Decreasing POSTN improved hemodynamic and cardiac responses in PH mice, blunted the release of growth factors and HIF-1α, and reversed the downregulated BMPR (bone morphogenetic protein receptor)-2 expression in hPAECs from patients with PH, whereas increasing POSTIN had the opposite effects and induced a hyperproliferative and promigratory phenotype in both hPAECs and human pulmonary artery smooth muscle cells. Overexpression of POSTN-induced activation of HIFs and increased the production of ET (endothelin)-1 and VEGF (vascular endothelial growth factor) in hPAECs. SiRNA-mediated knockdown of HIF-1α abolished the proangiogenic effect of POSTN. Blockade of TrkB (tyrosine kinase receptor B) attenuated the effect of POSTN on HIF-1α expression, while inhibition of HIF-1α reduced the expression of POSTN and TrkB. These results suggest that hPAECs produce POSTN via a HIF-1α-dependent mechanism. CONCLUSIONS: Our study reveals that POSTN expression is increased in human and animal models of PH and fosters PH development via a positive feedback loop between HIF-1α and POSTN during hypoxia. We propose that manipulating POSTIN expression may be an efficacious therapeutic target in the treatment of PH. Our results also suggest that POSTN may serve as a biomarker to estimate the severity of PH.

TGR5 receptor activation attenuates diabetic retinopathy through suppression of RhoA/ROCK signaling.

Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus. Abnormal energy metabolism in microvascular endothelium is involved in the progression of diabetic retinopathy. Bile Acid G-Protein-Coupled Membrane Receptor (TGR5) has emerged as a novel regulator of metabolic disorders. However, the role of TGR5 in diabetes mellitus-induced microvascular dysfunction in retinas is largely unknown. Herein, enzyme-linked immunosorbent assay was used for analyzing bile acid (BA) profiles in diabetic rat retinas and retinal microvascular endothelial cells (RMECs) cultured in high glucose medium. The effects of TGR5 agonist on streptozotocin (STZ)-induced diabetic retinopathy were evaluated by HE staining, TUNEL staining, retinal trypsin digestion, and vascular permeability assay. A pharmacological inhibitor of RhoA was used to study the role of TGR5 on the regulation of Rho/Rho-associated coiled-coil containing protein kinase (ROCK) and western blot, immunofluorescence and siRNA silencing were performed to study the related signaling pathways. Here we show that bile acids were downregulated during DR progression in the diabetic rat retinas and RMECs cultured in high glucose medium. The TGR5 agonist obviously ameliorated diabetes-induced retinal microvascular dysfunction in vivo, and inhibited the effect of TNF-α on endothelial cell proliferation, migration, and permeability in vitro. In contrast, knockdown of TGR5 by siRNA aggravated TNF-α-induced actin polymerization and endothelial permeability. Mechanistically, the effects of TGR5 on the improvement of endothelial function was due to its regulatory role on the ROCK signaling pathway. An inhibitor of RhoA significantly reversed the loss of tight junction protein under TNF-α stimulation. Taken together, our findings suggest that insufficient BA signaling plays an important pathogenic role in the development of DR. Upregulation or activation of TGR5 may inhibit RhoA/ROCK-dependent actin remodeling and represent an important therapeutic intervention for DR.

Upregulation of MicroRNA 711 Mediates HIV-1 Vpr Promotion of Kaposi's Sarcoma-Associated Herpesvirus Latency and Induction of Pro-proliferation and Pro-survival Cytokines by Targeting the Notch/NF-κB-Signaling Axis.

Coinfection with HIV-1 and Kaposi's sarcoma-associated herpesvirus (KSHV) often leads to AIDS-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The interaction between HIV and KSHV plays a pivotal role in the progression of these malignancies. We have previously demonstrated that, by upregulating miR-942-5p, HIV-1 viral protein R (Vpr) inhibits KSHV lytic replication by targeting IκBα to activate the NF-κB signaling (Q. Yan, C. Shen, J. Qin, W. Li, M. Hu, H. Lu, D. Qin, J. Zhu, S. J. Gao, C. Lu, J Virol 90:8739-8753, 2016). Here, we show that Vpr inactivates Notch signaling, resulting in inhibition of KSHV lytic replication and induction of pro-proliferative and -survival cytokines, including interleukin-2 (IL-2), TIMP-1, IGF-1, and NT-4. Mechanistically, Vpr upregulates miR-711, which directly targets the Notch1 3' untranslated region. Suppression of miR-711 relieved Notch1 and reduced Vpr inhibition of KSHV lytic replication and Vpr induction of pro-proliferation and -survival cytokines, while overexpression of miR-711 exhibited the opposite effect. Finally, overexpression of Notch1 reduced Vpr induction of NF-κB activity by promoting IκBα promoter activity. Our novel findings reveal that by upregulating miR-711 to target Notch1, Vpr silences Notch signaling to activate the NF-κB pathway by reducing IκBα expression, leading to inhibition of KSHV lytic replication and induction of pro-proliferation and -survival cytokines. Therefore, the miR-711/Notch/NF-κB axis is important in the pathogenesis of AIDS-related malignancies and could be an attractive therapeutic target.IMPORTANCE HIV-1 infection significantly increases the risk of KS and PEL in KSHV-infected individuals. Our previous study has shown that HIV-1 Vpr regulates the KSHV life cycle by targeting IκBα to activate NF-κB signaling through upregulating cellular miR-942-5p. In this study, we have further found that Vpr inactivates Notch signaling to promote KSHV latency and production of pro-proliferation and -survival cytokines. Another Vpr-upregulated cellular microRNA, miR-711, participates in this process by directly targeting Notch1. As a result, Notch1 upregulation of the IκBα promoter activity is attenuated, resulting in reduced levels of IκBα transcript and protein. Overall, these results illustrate an alternative mechanism of HIV-1 Vpr regulation of KSHV latency and aberrant cytokines through the miR-711/Notch/NF-κB axis. Our novel findings further demonstrate the role of an HIV-1-secreted regulatory protein in the KSHV life cycle and KSHV-related malignancies.

Azithromycin inhibits muscarinic 2 receptor-activated and voltage-activated Ca2+ permeant ion channels and Ca2+ sensitization, relaxing airway smooth muscle contraction.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

CCL5 deficiency rescues pulmonary vascular dysfunction, and reverses pulmonary hypertension via caveolin-1-dependent BMPR2 activation.

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with PAH impairs pulmonary arterial endothelial cells (PAECs) function. This can adversely affect PAEC survival and promote PASMCs proliferation. We hypothesized that interventions to normalize the expression of genes that are targets of the BMPR2 signaling could restore PAECs function and prevent or reverse PAH. Here we characterized for the first time, in human PAECs, chemokine (C-C motif) ligand 5 (CCL5/RANTES) deficiency restore BMP-mediated PAECs function. In the cell culture experiments, we found that CCL5 deficiency increased apoptosis and tube formation of PAECs, but suppressed proliferation and migration of PASMCs. Silencing CCL5 expression in PAH PAECs restored bone morphogenetic protein (BMP) signaling responses and promoted phosphorylation of SMADs and transcription of ID genes. Moreover, CCL5 deficiency inhibited angiogenesis by increasing pSMAD-dependent and-independent BMPR2 signaling. This was linked mechanistically to enhanced interaction of BMPR2 with caveolin-1 via CCL5 deficiency-mediated stabilization of endothelial surface caveolin-1. Consistent with these functions, deletion of CCL5 significantly attenuated development of Sugen5416/hypoxia-induced PAH by restoring BMPR2 signaling in mice. Taken together, our findings suggest that CCL5 deficiency could reverse obliterative changes in pulmonary arteries via caveolin-1-dependent amplification of BMPR2 signaling. Our results shed light on better understanding of the disease pathobiology and provide a possible novel target for the treatment of PAH.

The SH3BGR/STAT3 Pathway Regulates Cell Migration and Angiogenesis Induced by a Gammaherpesvirus MicroRNA.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically associated with KS, a highly disseminated angiogenic tumor of hyperproliferative spindle endothelial cells. KSHV encodes 25 mature microRNAs but their roles in KSHV-induced tumor dissemination and angiogenesis remain unknown. Here, we investigated KSHV-encoded miR-K12-6-3p (miR-K6-3p) promotion of endothelial cell migration and angiogenesis, which are the underlying mechanisms of tumor dissemination and angiogenesis. We found that ectopic expression of miR-K6-3p promoted endothelial cell migration and angiogenesis. Mass spectrometry, bioinformatics and luciferase reporter analyses revealed that miR-K6-3p directly targeted sequence in the 3' untranslated region (UTR) of SH3 domain binding glutamate-rich protein (SH3BGR). Overexpression of SH3BGR reversed miR-K6-3p induction of cell migration and angiogenesis. Mechanistically, miR-K6-3p downregulated SH3BGR, hence relieved STAT3 from SH3BGR direct binding and inhibition, which was required for miR-K6-3p maximum activation of STAT3 and induction of cell migration and angiogenesis. Finally, deletion of miR-K6 from the KSHV genome abrogated its effect on the SH3BGR/STAT3 pathway, and KSHV-induced migration and angiogenesis. Our results illustrated that, by inhibiting SH3BGR, miR-K6-3p enhances cell migration and angiogenesis by activating the STAT3 pathway, and thus contributes to the dissemination and angiogenesis of KSHV-induced malignancies.

HIV-1 Vpr Inhibits Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by Inducing MicroRNA miR-942-5p and Activating NF-κB Signaling.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) infection is required for the development of several AIDS-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The high incidence of AIDS-KS has been ascribed to the interaction of KSHV and HIV-1. We have previously shown that HIV-1-secreted proteins Tat and Nef regulate the KSHV life cycle and synergize with KSHV oncogenes to promote angiogenesis and tumorigenesis. Here, we examined the regulation of KSHV latency by HIV-1 viral protein R (Vpr). We found that soluble Vpr inhibits the expression of KSHV lytic transcripts and proteins, as well as viral particle production by activating NF-κB signaling following internalization into PEL cells. By analyzing the expression profiles of microRNAs combined with target search by bioinformatics and luciferase reporter analyses, we identified a Vpr-upregulated cellular microRNA (miRNA), miR-942-5p, that directly targeted IκBα. Suppression of miR-942-5p relieved the expression of IκBα and reduced Vpr inhibition of KSHV lytic replication, while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV lytic replication. Our findings collectively illustrate that, by activating NF-κB signaling through upregulating a cellular miRNA to target IκBα, internalized HIV-1 Vpr inhibits KSHV lytic replication. These results have demonstrated an essential role of Vpr in the life cycle of KSHV. IMPORTANCE: Coinfection by HIV-1 promotes the aggressive growth of Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). In this study, we have shown that soluble HIV-1 Vpr inhibits KSHV lytic replication by activating NF-κB signaling following internalization into PEL cells. Mechanistic studies revealed that a cellular microRNA upregulated by Vpr, miR-942-5p, directly targeted IκBα. Suppression of miR-942-5p relieved IκBα expression and reduced Vpr inhibition of KSHV replication, while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV replication. These results indicate that by activating NF-κB signaling through upregulating a cellular miRNA to target IκBα, internalized Vpr inhibits KSHV lytic replication. This work illustrates a molecular mechanism by which HIV-1-secreted regulatory protein Vpr regulates KSHV latency and the pathogenesis of AIDS-related malignancies.

Inhibition of Kaposi's sarcoma-associated herpesvirus lytic replication by HIV-1 Nef and cellular microRNA hsa-miR-1258.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several AIDS-related malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. The interaction of human immunodeficiency virus type 1 (HIV-1) and KSHV has a central role in promoting the aggressive manifestations of AIDS-KS. We have previously shown that negative factor (Nef), a secreted HIV-1 protein, synergizes with KSHV viral interleukin-6 (vIL-6) to promote angiogenesis and tumorigenesis by activating the AKT pathway (X. Zhu, et al., Oncogene, 22 April 2013, http://dx.doi.org/10.1038/onc.2013.136). Here, we further demonstrated the role of soluble and ectopic Nef in the regulation of KSHV latency. We found that both soluble Nef protein and ectopic expression of Nef by transfection suppressed the expression of KSHV viral lytic mRNA transcripts and proteins and the production of infectious viral particles. MicroRNA (miRNA) microarray analysis identified a number of Nef-regulated miRNAs. Bioinformatics and luciferase reporter analyses showed that one of the Nef-upregulated miRNAs, cellular miRNA 1258 (hsa-miR-1258), directly targeted a seed sequence in the 3' untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls KSHV reactivation from latency. Ectopic expression of hsa-miR-1258 impaired RTA synthesis and enhanced Nef-mediated inhibition of KSHV replication, whereas repression of hsa-miR-1258 has the opposite effect. Mutation of the seed sequence in the RTA 3'UTR abolished downregulation of RTA by hsa-miR-1258. Collectively, these novel findings demonstrate that, by regulating cellular miRNA, Nef may inhibit KSHV replication to promote viral latency and contribute to the pathogenesis of AIDS-related malignancies. IMPORTANCE: This study found that Nef, a secreted HIV-1 protein, suppressed KSHV lytic replication to promote KSHV latency. Mechanistic studies indicated that a Nef-upregulated cellular miRNA, hsa-miR-1258, inhibits KSHV replication by directly targeting a seed sequence in the KSHV RTA 3'UTR. These results illustrate that, in addition to viral miRNAs, cellular miRNAs also play an important role in regulating the life cycle of KSHV. Overall, this is the first study to report the involvement of Nef in KSHV latency, implying its likely important role in the pathogenesis of AIDS-related malignancies.

Andrographolide Attenuates Established Pulmonary Hypertension via Rescue of Vascular Remodeling.

Pulmonary hypertension (PH) is characterized by vascular remodeling caused by marked proliferation of pulmonary artery smooth muscle cells (PASMCs). Andrographolide (ANDRO) is a potent anti-inflammatory agent which possesses antioxidant, and has anticarcinogenic activity. The present study examined potential therapeutic effects of ANDRO on PH in both chronic hypoxia and Sugen5416/hypoxia mouse PH models. Effects of ANDRO were also studied in cultured human PASMCs isolated from either healthy donors or PH patients. In vivo, ANDRO decreased distal pulmonary arteries (PAs) remodeling, mean PA pressure and right ventricular hypertrophy in chronic hypoxia- and Sugen/hypoxia-induced PH in mice. ANDRO reduced cell viability, proliferation and migration, but increased cell apoptosis in the PASMCs isolated from PH patients. ANDRO also reversed the dysfunctional bone morphogenetic protein receptor type-2 (BMPR2) signaling, suppressed [Ca2+]i elevation, reactive oxygen species (ROS) generation, and the upregulated expression of IL-6 and IL-8, ET-1 and VEGF in PASMCs from PH patients. Moreover, ANDRO significantly attenuated the activation of TLR4/NF-κB, ERK- and JNK-MAPK signaling pathways and reversed the inhibition of p38-MAPK in PASMCs of PH patients. Further, ANDRO blocked hypoxia-triggered ROS generation by suppressing NADPH oxidase (NOX) activation and augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression both in vitro and in vivo. Conventional pulmonary vasodilators have limited efficacy for the treatment of severe PH. We demonstrated that ANDRO may reverse pulmonary vascular remodeling through modulation of NOX/Nrf2-mediated oxidative stress and NF-κB-mediated inflammation. Our findings suggest that ANDRO may have therapeutic value in the treatment of PH.

DR-region of Na+/K+-ATPase is a target to ameliorate hepatic insulin resistance in obese diabetic mice.

Reduced hepatic Na+/K+-ATPase (NKA) activity and NKAα1 expression are engaged in the pathologies of metabolism diseases. The present study was designed to investigate the potential roles of NKAα1 in hepatic gluconeogenesis and glycogenesis in both hepatocytes and obese diabetic mice. Methods: Insulin resistance was mimicked by glucosamine (GlcN) in either human hepatocellular carcinoma (HepG2) cells or primary mouse primary hepatocytes. Obese diabetic mice were induced by high-fat diet (HFD) feeding for 12 weeks. Results: We found that both NKA activity and NKAα1 protein level were downregulated in GlcN-treated hepatocytes and in the livers of obese diabetic mice. Pharmacological inhibition of NKA with ouabain worsened, while activation of NKAα1 with an antibody against an extracellular DR region of NKAα1 subunit (DR-Ab) prevented GlcN-induced increase in gluconeogenesis and decrease in glycogenesis. Likewise, the above results were also corroborated by the opposite effects of genetic knockout/overexpression of NKAα1 on both gluconeogenesis and glycogenesis. In obese diabetic mice, hepatic activation or overexpression of NKAα1 stimulated the PI3K/Akt pathway to suppress hyperglycemia and improve insulin resistance. More importantly, loss of NKA activities in NKAα1+/- mice was associated with more susceptibility to insulin resistance following HFD feeding. Conclusions: Our findings suggest that NKAα1 is a physiological regulator of glucose homoeostasis and its DR-region is a novel target to treat hepatic insulin resistance.

Polygonum aviculare L. extract and quercetin attenuate contraction in airway smooth muscle.

Because of the serious side effects of the currently used bronchodilators, new compounds with similar functions must be developed. We screened several herbs and found that Polygonum aviculare L. contains ingredients that inhibit the precontraction of mouse and human airway smooth muscle (ASM). High K+-induced precontraction in ASM was completely inhibited by nifedipine, a selective blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). However, nifedipine only partially reduced the precontraction induced by acetylcholine chloride (ACH). Additionally, the ACH-induced precontraction was partly reduced by pyrazole-3 (Pyr3), a selective blocker of TRPC3 and stromal interaction molecule (STIM)/Orai channels. These channel-mediated currents were inhibited by the compounds present in P. aviculare extracts, suggesting that this inhibition was mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. Moreover, these channel-mediated currents were inhibited by quercetin, which is present in P. aviculare extracts. Furthermore, quercetin inhibited ACH-induced precontraction in ASM. Overall, our data indicate that the ethyl acetate fraction of P. aviculare and quercetin can inhibit Ca2+-permeant LVDCCs, TRPC3 and STIM/Orai channels, which inhibits the precontraction of ASM. These findings suggest that P. aviculare could be used to develop new bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease.

Semen cassiae Extract Inhibits Contraction of Airway Smooth Muscle.

ß2-adrenoceptor agonists are commonly used as bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), however, they induce severe side effects. Therefore, developing new bronchodilators is essential. Herbal plants were extracted and the extracts' effect on airway smooth muscle (ASM) precontraction was assessed. The ethyl alcohol extract of semen cassiae (EESC) was extracted from Semen cassia. The effects of EESC on the ACh- and 80 mM K+-induced sustained precontraction in mouse and human ASM were evaluated. Ca2+ permeant ion channel currents and intracellular Ca2+ concentration were measured. HPLC analysis was employed to determine which compound was responsible for the EESC-induced relaxation. The EESC reversibly inhibited the ACh- and 80 mM K+-induced precontraction. The sustained precontraction depends on Ca2+ influx, and it was mediated by voltage-dependent L-type Ca2+ channels (LVDCCs), store-operated channels (SOCs), TRPC3/STIM/Orai channels. These channels were inhibited by aurantio-obtusin, one component of EESC. When aurantio-obtusin removed, EESC's action disappeared. In addition, aurantio-obtusin inhibited the precontraction of mouse and human ASM and intracellular Ca2+ increases. These results indicate that Semen cassia-contained aurantio-obtusin inhibits sustained precontraction of ASM via inhibiting Ca2+-permeant ion channels, thereby, which could be used to develop new bronchodilators.

Distinct Effects of Ca2+ Sparks on Cerebral Artery and Airway Smooth Muscle Cell Tone in Mice and Humans.

The effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone, as well as the underlying mechanisms, are not clear. In this investigation, we elucidated the underlying mechanisms of the distinct effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone. In CASMCs, owing to the functional loss of Ca2+-activated Cl- (Clca) channels, Ca2+ sparks activated large-conductance Ca2+-activated K+ channels (BKs), resulting in a decreases in tone against a spontaneous depolarization-caused high tone in the resting state. In ASMCs, Ca2+ sparks induced relaxation through BKs and contraction via Clca channels. However, the integrated result was contraction because Ca2+ sparks activated BKs prior to Clca channels and Clca channels-induced depolarization was larger than BKs-caused hyperpolarization. However, the effects of Ca2+ sparks on both cell types were determined by L-type voltage-dependent Ca2+ channels (LVDCCs). In addition, compared with ASMCs, CASMCs had great and higher amplitude Ca2+ sparks, a higher density of BKs, and higher Ca2+ and voltage sensitivity of BKs. These differences enhanced the ability of Ca2+ sparks to decrease CASMC and to increase ASMC tone. The higher Ca2+ and voltage sensitivity of BKs in CASMCs than ASMCs were determined by the ß1 subunits. Moreover, Ca2+ sparks showed the similar effects on human CASMC and ASMC tone. In conclusions, Ca2+ sparks decrease CASMC tone and increase ASMC tone, mediated by BKs and Clca channels, respectively, and finally determined by LVDCCs.

Nur77 downregulation triggers pulmonary artery smooth muscle cell proliferation and migration in mice with hypoxic pulmonary hypertension via the Axin2-ß-catenin signaling pathway.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by remodeling of the pulmonary vasculature, including marked proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Members of the nuclear receptor 4A (NR4A) subfamily are involved in a variety of biological events, such as cell apoptosis, proliferation, inflammation, and metabolism. Activation of Nur77 (an orphan nuclear receptor that belongs to NR4A subfamily) has recently been reported to be as a beneficial agent in the treatment of cardiovascular and metabolic diseases. In the present study, we investigated the effects of NR4A on human PASMCs function in vitro and determined the underlying mechanisms. We found a robust expression of NR4A receptors in lung tissues of PAH patients and hypoxic mice but a highly significant downregulation within pulmonary arteries (PAs) as assessed by quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. In vitro, NR4A receptors were found significantly decreased in PASMCs derived from PAH patients. To explore the pathological effects of decreased Nur77 in PASMCs, PASMCs were transduced with siRNA against Nur77. The siRNA-mediated knockdown of Nur77 significantly augmented PASMCs proliferation and migration. In contrast, Nur77 overexpression prevented PASMCs from proliferation and migration. Mechanistically, overexpression of Axis inhibition protein 2 (Axin2) or inhibition of ß-catenin signaling was shown to be responsible for Nur77 knockdown-induced proliferation of PASMCs. Following hypoxia-induced angiogenesis of the pulmonary artery in C57BL/6 mice, small-molecule Nur77 agonists-Octaketide Cytosporone B (Csn-B) can significantly decreased thickness of vascular wall and markedly attenuated the development of chronic hypoxia-induced PAH in vivo. Therefore, reconstitution of Nur77 levels represents a promising therapeutic option to prevent vascular remodeling processes.

A KSHV microRNA enhances viral latency and induces angiogenesis by targeting GRK2 to activate the CXCR2/AKT pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Most tumor cells in these malignancies are latently infected by KSHV. Thus, viral latency is critical for the development of tumor and induction of tumor-associated angiogenesis. KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced angiogenesis remains unknown. We have recently shown that miR-K12-3 (miR-K3) promoted cell migration and invasion by targeting GRK2/CXCR2/AKT signaling (PLoS Pathog, 2015;11(9):e1005171). Here, we further demonstrated a role of miR-K3 and its induced signal pathway in KSHV latency and KSHV-induced angiogenesis. We found that overexpression of miR-K3 not only promoted viral latency by inhibiting viral lytic replication, but also induced angiogenesis. Further, knockdown of GRK2 inhibited KSHV replication and enhanced KSHV-induced angiogenesis by enhancing the CXCR2/AKT signals. As a result, blockage of CXCR2 or AKT increased KSHV replication and decreased angiogenesis induced by PEL cells in vivo. Finally, deletion of miR-K3 from viral genome reduced KSHV-induced angiogenesis and increased KSHV replication. These findings indicate that the miR-K3/GRK2/CXCR2/AKT axis plays an essential role in KSHV-induced angiogenesis and promotes KSHV latency, and thus may be a potential therapeutic target of KSHV-associated malignancies.

Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.

Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293 T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.