CypA Inhibitors Research Articles

Thai traditional medicines reduce CD147 levels in lung cells: Potential therapeutic candidates for cancers, inflammations, and COVID-19

Ethnopharmacological relevanceThe cluster of differentiation 147 (CD147) is identified as the signaling protein relevant importantly in various cancers, inflammations, and coronavirus disease 2019 (COVID-19) via interacting with extracellular cyclophilin A (CypA). The reduction of CD147 levels inhibits the progression of CD147-associated diseases. Thai traditional medicines (TTMs): Keaw-hom (KH), Um-ma-ruek-ka-wa-tee (UM), Chan-ta-lee-la (CT), and Ha-rak (HR) have been used as anti-pyretic and anti-respiratory syndromes caused from various conditions including cancers, inflammations, and infections. Thus, these medicines would play a crucial role in the reduction of CD147 levels. Aim of the studyThis article aimed to investigate the effects of KH, UM, CT, and HR for reducing the CD147 levels through in vitro study. Additionally, in silico study was employed to screen the active compounds reflexing the reduction of CD147 levels. Materials and methodsThe immunofluorescent technique was used to evaluate the reduction of CD147 levels in human lung epithelial cells (BEAS-2B) stimulated with CypA for eight extracts of KH, UM, CT, and HR obtained from water decoction (D) and 70% ethanol maceration (M) including, KHD, UMD, CTD, HRD, KHM, UMM, CTM, and HRM. ResultsUM extracts showed the most efficiency for reduction of CD147 levels in the cytoplasm and perinuclear of BEAS-2B cells stimulated with CypA. Phenolic compounds composing polyphenols, polyphenol sugars, and flavonoids were identified as the major chemical components of UMD and UMM. Further, molecular docking calculations identified polyphenol sugars as CypA inhibitors. ConclusionsUMD and UMM are potential for reduction of CD147 levels which provide a useful information for further development of UM as potential therapeutic candidates for CD147-associated diseases such as cancers, inflammations, and COVID-19.

Inhibition of Cyclophilin A-Metalloproteinase-9 Pathway Alleviates the Development of Neuropathic Pain by Promoting Repair of the Blood-Spinal Cord Barrier.

Dysfunction of the blood-spinal cord barrier (BSCB) contributes to the occurrence and development of neuropathic pain (NP). Previous studies revealed that the activation of cyclophilin A (CypA)-metalloproteinase-9 (MMP9) signaling pathway can disrupt the integrity of the blood-brain barrier (BBB) and aggravate neuroinflammatory responses. However, the roles of CypA-MMP9 signaling pathway on BSCB in NP have not been studied. This study aimed to investigate the effect of CypA on the structure and function of the BSCB and pain behaviors in mice with NP. We first created the mouse chronic constriction injury (CCI) model, and they were then intraperitoneally injected with the CypA inhibitor cyclosporine A (CsA) or vehicle. Pain behaviors, the structure and function of the BSCB, the involvement of the CypA-MMP9 signaling pathway, microglia activation, and expression levels of proinflammatory factors in mice were examined. CCI mice presented mechanical allodynia and thermal hyperalgesia, impaired permeability of the BSCB, downregulated tight junction proteins, activated CypA-MMP9 signaling pathway, microglia activation, and upregulated proinflammatory factors, which were significantly alleviated by inhibition of CypA. Collectively, the CypA-MMP9 signaling pathway is responsible for CCI-induced NP in mice by impairing the structure and function of the BSCB, and activating microglia and inflammatory responses.

Natural Cyclophilin A Inhibitors Suppress the Growth of Cancer Stem Cells in Non-Small Cell Lung Cancer by Disrupting Crosstalk between CypA/CD147 and EGFR.

Non-small cell lung cancer (NSCLC) is a fatal malignant tumor with a high mortality rate. Cancer stem cells (CSCs) play pivotal roles in tumor initiation and progression, treatment resistance, and NSCLC recurrence. Therefore, the development of novel therapeutic targets and anticancer drugs that effectively block CSC growth may improve treatment outcomes in patients with NSCLC. In this study, we evaluated, for the first time, the effects of natural cyclophilin A (CypA) inhibitors, including 23-demethyl 8,13-deoxynargenicin (C9) and cyclosporin A (CsA), on the growth of NSCLC CSCs. C9 and CsA more sensitively inhibited the proliferation of epidermal growth factor receptor (EGFR)-mutant NSCLC CSCs than EGFR wild-type NSCLC CSCs. Both compounds suppressed the self-renewal ability of NSCLC CSCs and NSCLC-CSC-derived tumor growth in vivo. Furthermore, C9 and CsA inhibited NSCLC CSC growth by activating the intrinsic apoptotic pathway. Notably, C9 and CsA reduced the expression levels of major CSC markers, including integrin α6, CD133, CD44, ALDH1A1, Nanog, Oct4, and Sox2, through dual downregulation of the CypA/CD147 axis and EGFR activity in NSCLC CSCs. Our results also show that the EGFR tyrosine kinase inhibitor afatinib inactivated EGFR and decreased the expression levels of CypA and CD147 in NSCLC CSCs, suggesting close crosstalk between the CypA/CD147 and EGFR pathways in regulating NSCLC CSC growth. In addition, combined treatment with afatinib and C9 or CsA more potently inhibited the growth of EGFR-mutant NSCLC CSCs than single-compound treatments. These findings suggest that the natural CypA inhibitors C9 and CsA are potential anticancer agents that suppress the growth of EGFR-mutant NSCLC CSCs, either as monotherapy or in combination with afatinib, by interfering with the crosstalk between CypA/CD147 and EGFR.

Cyclophilin A Inhibitors Suppress Proliferation and Induce Apoptosis of MKN45 Gastric Cancer Stem-like Cells by Regulating CypA/CD147-Mediated Signaling Pathway

Gastric cancer stem cells (GCSCs) are a subgroup of gastric cancer (GC) cells with high self-renewal and multi-lineage differentiation abilities that lead to tumor initiation, metastasis, drug resistance, and tumor relapse. Therefore, the eradication of GCSCs can contribute to the effective treatment of advanced or metastatic GC. In our previous study, compound 9 (C9), a novel derivative of nargenicin A1, was identified as a potential natural anticancer agent that specifically targeted cyclophilin A (CypA). However, its therapeutic effect and molecular mechanisms of action on GCSC growth have not been assessed. In this study, we investigated the effects of natural CypA inhibitors, including C9 and cyclosporin A (CsA), on the growth of MKN45-derived GCSCs. Compound 9 and CsA effectively suppressed cell proliferation by inducing cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade in MKN45 GCSCs. In addition, C9 and CsA potently inhibited tumor growth in the MKN45 GCSC-grafted chick embryo chorioallantoic membrane (CAM) model. Furthermore, the two compounds significantly decreased the protein expression of key GCSC markers including CD133, CD44, integrin α6, Sox2, Oct4, and Nanog. Notably, the anticancer activities of C9 and CsA in MKN45 GCSCs were associated with the regulation of CypA/CD147-mediated AKT and mitogen-activated protein kinase (MAPK) signaling pathways. Collectively, our findings suggest that the natural CypA inhibitors C9 and CsA could be novel anticancer agents used to combat GCSCs by targeting the CypA/CD147 axis.

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

Reactive oxygen species contribute to delirium-like behavior by activating CypA/MMP9 signaling and inducing blood-brain barrier impairment in aged mice following anesthesia and surgery.

Postoperative delirium (POD) is common in the elderly and is associated with poor clinical outcomes. Reactive oxygen species (ROS) and blood-brain barrier (BBB) damage have been implicated in the development of POD, but the association between these two factors and the potential mechanism is not clear. Cyclophilin A (CypA) is a specifically chemotactic leukocyte factor that can be secreted in response to ROS, which activates matrix metalloproteinase 9 (MMP9) and mediates BBB breakdown. We, therefore, hypothesized that ROS may contribute to anesthesia/surgery-induced BBB damage and delirium-like behavior via the CypA/MMP9 pathway. To test these hypotheses, 16-month-old mice were subjected to laparotomy under 3% sevoflurane anesthesia (anesthesia/surgery) for 3 h. ROS scavenger (N-acetyl-cysteine) and CypA inhibitor (Cyclosporin A) were used 0.5 h before anesthesia/surgery. A battery of behavior tests (buried food test, open field test, and Y maze test) was employed to evaluate behavioral changes at 24 h before and after surgery in the mice. Levels of tight junction proteins, CypA, MMP9, postsynaptic density protein (PSD)-95, and synaptophysin in the prefrontal cortex were assessed by western blotting. The amounts of ROS and IgG in the cortex of mice were observed by fluorescent staining. The concentration of S100β in the serum was detected by ELISA. ROS scavenger prevented the reduction in TJ proteins and restored the permeability of BBB as well as reduced the levels of CypA/MMP9, and further alleviated delirium-like behavior induced by anesthesia/surgery. Furthermore, the CypA inhibitor abolished the increased levels of CypA/MMP, which reversed BBB damage and ameliorated delirium-like behavior caused by ROS accumulation. Our findings demonstrated that ROS may participate in regulating BBB permeability in aged mice with POD via the CypA/MMP9 pathway, suggesting that CypA may be a potential molecular target for preventing POD.

Porphyromonas gingivalis lipopolysaccharide and gingival fibroblast augment MMP-9 expression of monocytic U937 cells through cyclophilin A.

Intercellular cross-talking was suggested in matrix metalloproteinase (MMP)-9 expression with unknown mechanisms. Studies showed cyclophilin A (CypA) playing an important role in regulating MMP-9 expression in varied diseases. The aim of the study was to examine the CyPA on the MMP-9 augmentation in monocytic U937 cells after Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) treatment and human gingival fibroblast (hGF) co-culture. In independent culture or co-culture of hGF and U937 cell, quantitative real-time polymerase chain reaction (qPCR) and zymography were selected to examine the mRNA and protein activity of MMP-9, respectively. The CyPA expression was determined by qPCR. LPS could enhance MMP-9 mRNA expression and enzyme activity in U937 cell. However, the enhancements were not observed in hGF. Similarly, LPS enhanced CyPA mRNA in U937, but not in hGF. After co-cultured with hGF, however, MMP-9 and CyPA in U937 increased regardless of the presence/absence of LPS. In U937 cells, the extra-supplied CyPA increased MMP-9 mRNA and enzyme activity, whereas the CyPA inhibitor, cyclosporine A, suppressed the LPS- and co-culture-enhanced MMP-9. Moreover, the inhibitors for MAP kinase, including PD98059 (ERK) and SP600125 (JNK), suppressed the CyPA-enhanced MMP-9 in U937. Through the CyPA pathway, the LPS and the hGF could augment the MMP-9 expression in the U937 cells.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

Cyclophilin A: a key player for etiological agent infection.

Cyclophilin A (CypA), a key member of the immunophilin family, is the most abundantly expressed isozyme of the 18 known human cyclophilins. Besides acting as an intracellular receptor for cyclosporine A, CypA plays a vital role in microorganismal infections, cardiovascular diseases, liver diseases, kidney diseases, neurodegeneration, cancer, rheumatoid arthritis, periodontitis, sepsis, asthma, and aging. This review focuses on the pivotal roles of CypA in the infection of etiological agents, which manifests mainly in promoting or inhibiting viral replication based on the host cell type and viral species. CypA can interact with viral proteins and thus regulate the replication cycle of the virus. CypA is involved in pathogenic bacterial infections by regulating the formation of host actin skeleton or membrane translocation of bacterial toxins, or mediated the adhesion of Mycoplasma genitalium during the infection processes by acting as a cellular receptor of M. genitalium. CypA also plays a critical role in infection or the life cycle of certain parasites or host immune regulation. Moreover, we summarized the current understanding of CypA inhibitors acting as host-targeting antiviral agents, thus opening an avenue for the treatment of multiple viral infections due to their broad antiviral effects and ability to effectively prevent drug resistance. Therefore, the antiviral effect of CypA has the potential to promote CypA inhibitors as host-targeting drugs to CypA-involved etiological agent infections and human diseases.Key points• CypA is involved in the replication and infection of several viruses, pathogenic bacteria, mycoplasma, and parasites.• CypA inhibitors are in a strong position to inhibit the infection of viruses, bacterial, and mycoplasma.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

SummaryProlactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Deficiency of CD147 Attenuated Non-alcoholic Steatohepatitis Progression in an NLRP3-Dependent Manner.

Cluster of differentiation 147 (CD147) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD147 overexpression has been reported to facilitate the development of hepatocellular carcinoma (HCC) and influence immunologic disorders. Although increased expression of CD147 was reported in non-alcoholic steatohepatitis (NASH), functions of CD147 in NASH have not been evaluated. Firstly, we confirmed that CD147 expression was increased in the liver tissues from methionine-choline-deficient (MCD) diet-induced NASH model mice and NASH patients. Mice with hepatocyte-specific CD147 deletion exhibited attenuated NASH phenotypes, including reduced steatosis, liver injury, hepatocyte apoptosis and inflammatory cytokines IL-1β/IL-18 secretion. Following the administration of the MCD diet, NLRP3 expression was increased gradually along with CD147 expression. Furthermore, CD147 deletion inhibited the NF-κB/NLRP3 signaling pathway in both MCD diet-induced mice and primary hepatocytes. Finally, CypA inhibitor TMN355 attenuated liver steatosis and injury and inhibited NF-κB/NLRP3 signaling pathway. Therefore, our results suggest that CD147 played a vital role in NASH pathogenesis by regulating the inflammatory response, and CypA/CD147 could be attractive therapeutic targets for NASH treatment.

SAR study of bisamides as cyclophilin a inhibitors for the development of host-targeting therapy for hepatitis C virus infection

The therapy of chronic hepatitis C virus infections has significantly improved with the development of direct-acting antivirals (DAAs), which contain NS3/4A protease, NS5A, and NS5B polymerase inhibitors. However, mutations in specific residues in these viral target genes are associated with resistance to the DAAs. Especially inhibitors of NS3/4A protease and NS5A, such as grazoprevir and velpatasvir, have a low barrier to resistant mutations. As a result, the mutations influence the virological outcomes after DAA treatment. CypA inhibitors, as host-targeted agents, act on host factors to inhibit HCV replication, exhibiting a high resistance barrier and pan-genotype activities against HCV. Therefore, they can be developed into alternative, more effective anti-HCV agents. However, CypA inhibitors are natural products and analogs. Based on previous studies, bisamide derivatives were designed and synthesized to develop a novel class of CypA inhibitors. Bisamide derivative 7c is a promising compound with potent anti-HCV activity at subtoxic concentrations. Surface plasmon resonance experiments revealed that 7c directly binds to CypA. All these studies indicated that the derivative 7c is a potent CypA inhibitor, which can be used as a host-targeted agent in combination with other antiviral agents for anti-HCV treatment.

Cyclophilin A as a Novel Mediator in Ventilator‐Induced Lung Injury

RationaleCyclophilin A (CypA) is a ubiquitously distributed cytoplasmic protein. While focus has previously been on its intracellular roles, extracellular CypA and its receptor CD147 have emerged as a novel signalling axis in various inflammatory diseases. We explore the role of the CypA:CD147 axis in ventilator‐induced lung injury (VILI) and hypothesise that CypA is secreted into the alveolar space following epithelial insult where it promotes inflammation during acute lung injury (ALI).MethodsC57BL/6 mice received low‐stretch (LS; tidal volume: 8 mL/kg) or high‐stretch (HS: 40 mL/kg) ventilation for up to three hours (n=6–8/group). For inhibition experiments, mice received a 50 μL intratracheal dose of either a non‐permeable extracellular‐specific CypA inhibitor, MM‐284 (0.67 mM), or vehicle control, DMSO (10%), immediately prior to HS ventilation. Bronchoalveolar lavage fluid (BALF) CypA and soluble receptor for advanced glycation endproducts (sRAGE) were assessed by ELISA; cell activation status (surface ICAM‐1 expression) and intracellular CypA (iCypA) by flow cytometry; and matrix metalloproteinase‐2 (MMP‐2) by gelatin zymography.ResultsDuring VILI, there is a significant early upregulation of BALF CypA at two hours (LS: 663±268 vs HS: 2,586±1,054 pg/mL; p<0.05), preceding signs of physiological deterioration, with further increase at three hours. In contrast, plasma CypA levels showed no significant changes across three hours regardless of ventilation strategy and were consistently below BALF levels. iCypA decreased in EpCAM‐/T1α+ and EpCAM+/T1α‐epithelial cells but not alveolar macrophages. MM‐284 mediated inhibition of extracellular CypA led to protection from the development of VILI (vehicle: 50% vs MM‐284: 100% survival; p<0.05), attenuated lung wet:dry weight ratio (7.0±1.2 vs 5.0±0.2; p<0.001), BALF protein (3.7±1.4 vs 1.3±0.2 mg/mL; p<0.001), BALF sRAGE (40.3±11.2 vs 8.3±3.8 ng/mL; p<0.0001), and deteriorations in elastance and oxygenation (interaction p<0.01). Surprisingly, CypA inhibition appeared to have no impact on leukocyte recruitment into lung tissue but was associated with a reduction in stretch‐induced activation of alveolar macrophages (135.6±18.4 vs 21.8±45.4 ICAM‐1 MFI; p<0.001) and both EpCAM‐/T1α+ (129.1±28.7 vs 91.9±6.1 ICAM‐1 MFI; p<0.05) and EpCAM+/T1α‐(99.8±12.1 vs 76.5±15.2 ICAM‐1 MFI; p<0.05) epithelial cells. Further, CypA inhibition also led to a decrease in active MMP‐2 in BALF (0.9±0.5 vs 0.4±0.2 relative band intensity; p<0.05).ConclusionsWe have demonstrated the involvement of a novel mediator, CypA, in VILI. The early upregulation in BALF and corresponding decrease of iCypA in epithelial cells suggest that CypA is produced locally following epithelial injury. Inhibition of extracellular CypA promoted an impressive attenuation of VILI, particularly parameters related to alveolar‐capillary barrier integrity (e.g. BALF sRAGE, oedema formation, oxygenation, and MMP activity). Together, our findings support the CypA:CD147 axis as a novel mediator of epithelial cell dysfunction during the pathogenesis of VILI.Support or Funding InformationBritish Journal of Anaesthesia/Royal College of Anaesthetists

Molecular design, synthesis, and biological evaluation of bisamide derivatives as cyclophilin A inhibitors for HCV treatment

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. Direct-acting antivirals (DAAs), including inhibitors of nonstructural proteins (NS3/4A protease, NS5A, and NS5B polymerase), represent key components of anti-HCV treatment. However, some DAAs are associated with increased drug resistance and undesired side effects. Previous reports have shown that bisamides could be a novel class of cyclophilin A (CypA) inhibitors for treating HCV as a member of combinational therapies. To fully elucidate structure-activity relationships of bisamide derivatives and find a better hit compound with diverse binding modes, 16 biamides were designed with the help of docking program. They were then synthesized using one-pot four-component Ugi reaction. 7e with selectivity index of more than 18.9 (50% effective concentration of 5.3 μM, but no cytotoxicity at 100 μM) and unique binding mode that could be dived into gatekeeper pocket was selected as a new hit compound. Surface plasmon resonance experiments revealed that 7e is able to bind to CypA with a KD of 3.66 μM. Taken together, these results suggest that 7e as a CypA inhibitor could be used as an alternative anti-HCV agent in combinational therapy in the future.

Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication

The well-known immunosuppressive drug cyclosporin A inhibits replication of various viruses including coronaviruses by binding to cellular cyclophilins thus inactivating their cis-trans peptidyl-prolyl isomerase function. Viral nucleocapsid proteins are inevitable for genome encapsidation and replication. Here we demonstrate the interaction between the N protein of HCoV-229E and cyclophilin A, not cyclophilin B. Cyclophilin inhibitors abolish this interaction. Upon infection, cyclophilin A stays evenly distributed throughout the cell, whereas cyclophilin B concentrates at ER-bleb-like structures. We further show the inhibitory potential of non-immunosuppressive CsA derivatives Alisporivir, NIM811, compound 3 on HCoV-229E-GFP and -Luciferase replication in human Huh-7.5 hepatoma cells at 18 and 48 h time points post infection with EC50 s at low micromolar ranges. Thus, non-immunosuppressive CsA derivatives effectively inhibit HCoV-229E replication suggesting them as possible candidates for the treatment of HCoV infection. The interruption of interaction between CypA and N protein by CsA and its derivatives suggest a mechanism how CypA inhibitors suppress viral replication.

Cyclophilin A contributes to aortopathy induced by postnatal loss of smooth muscle TGFBR1.

Recent recognition that TGF-β signaling disruption is involved in the development of aortic aneurysms has led to renewed investigations into the role of TGF-β biology in the aortic wall. We previously found that the type I receptor of TGF-β (TGFBR2) receptor contributes to formation of ascending aortic aneurysms and dissections (AADs) induced by smooth muscle cell (SMC)-specific, postnatal deletion of Tgfbr1 (Tgfbr1iko). Here, we aimed to decipher the mechanistic signaling pathway underlying the pathogenic effects of TGFBR2 in this context. Gene expression profiling demonstrated that Tgfbr1iko triggers an acute inflammatory response in developing AADs, and Tgfbr1iko SMCs express an inflammatory phenotype in culture. Comparative proteomics profiling and mass spectrometry revealed that Tgfbr1iko SMCs respond to TGF-β1 stimulation via robust up-regulation of cyclophilin A (CypA). This up-regulation is abrogated by inhibition of TGFBR2 kinase activity, small interfering RNA silencing of Tgfbr2 expression, or inhibition of SMAD3 activation. In mice, Tgfbr1iko rapidly promotes CypA production in SMCs of developing AADs, whereas treatment with a CypA inhibitor attenuates aortic dilation by 56% (P = 0.003) and ameliorates aneurysmal degeneration (P = 0.016). These protective effects are associated with reduced aneurysm-promoting inflammation. Collectively, these results suggest a novel mechanism, wherein loss of type I receptor of TGF-β triggers promiscuous, proinflammatory TGFBR2 signaling in SMCs, thereby promoting AAD formation.-Zhou, G., Liao, M., Wang, F., Qi, X., Yang, P., Berceli, S. A., Sharma, A. K., Upchurch, G. R., Jr., Jiang, Z. Cyclophilin A contributes to aortopathy induced by postnatal loss of smooth muscle TGFBR1.

MiRNA143 Downregulated the Expression of Cyclophilin A to Inhibit the Progression of Atherosclerosis

To investigate the influence of microRNA (miR)-143 on atherosclerosis and its working mechanism via cyclophilin A. Human acute monocytic leukemia cells (human acute monocytic leukemia cells, human THP-1 monocytes) were treated with ox-LDL 80 mg/L for 48 h to establish a foam cell model. The modeling was evaluated by oil red O staining. The expressions levels of miRNA-143 and CyPA were detected by q-PCR. RNA extracted was confirmed by SiO2 nano magnetic bead. Dual-luciferase reporter gene assay was used to verify the targeted regulatory relationship between miRNA-143 and CyPA. Next, the cells were transfected with miR-143 mimics and treated with a CyPA inhibitor (cyclophilin A). The expression levels of CyPA and downstream matrix metalloproteinases-9 (MMP-9) in the downstream were detected by q-PCR and western blot, respectively. No or few lipid droplets were observed in the normal THP-1 cells. After treatment with ox-LDL for 48 h, a large amount of red-stained lipid was observed in the cytoplasm, indicating that the THP-1 derived foam cell models were successfully constructed. q-PCR results indicated that miRNA-143 was significantly downregulated in the THP-1-derived foam cell model, whereas CyPA was upregulated (P < 0.05); the dual luciferase reporter gene assay was performed for miRNA-143 and 3'-UTR of CyPA, and the fluorescence intensity of the 3'-UTR vector group was significantly lower than that of the empty vector group (P < 0.05). Fluorescence intensity detection of mutations at the target site showed that it had no effect was produced on the fluorescence intensity. This implied a negative regulatory effect conferred of miRNA-143 on CyPA; As compared to the model group, transfection with miRNA-143 mimics led to a significant downregulation of CyPA mRNA (P < 0.05), which was accompanied by significant downregulation of both MMP-9 mRNA and protein (P < 0.05). MMP-9 was also significantly downregulated upon the addition of the CyPA inhibitor cyclosporine A (P < 0.05).

Positive Correlation between Activated CypA/CD147 Signaling and MMP-9 Expression in Mice Inflammatory Periapical Lesion.

Aim Cyclophilin A (CypA)/CD147 signaling plays critical roles in the regulation of inflammation and bone metabolism. This study aimed to investigate the participation of CypA/CD147 in mice periapical lesions progression and its relationship with bone resorption. Methodology Periapical lesions were induced by pulp exposure in the first lower molars of 40 C57BL/6J mice. The mice were sacrificed on days 0, 7, 14, 21, 28, 35, 42, and 49. Mandibles were harvested for X-ray imaging, microcomputed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. Western blot was employed to further detect the related molecular signaling pathways in LPS-stimulated RAW 264.7 cells treated with CypA inhibitor. Results The volume and area of the periapical lesions increased from day 0 to day 35 and remained comparably stable until day 49. Immunohistochemistry demonstrated that the CypA expression levels also increased from day 0 to day 35 and decreased until day 49, similar to CD147 expression (R2 = 0.4423, P < 0.05), osteoclast number (R2 = 0.5101, P < 0.01), and the expression of osteoclastogenesis-related matrix metalloproteinase 9 (MMP-9) (R2 = 0.4715, P < 0.05). Serial sections further confirmed the colocalization of CypA and CD147 on osteoclasts with immunohistochemistry. And the distribution of CypA-positive or CD147-positive cells was positively correlated with the dynamics of MMP-9-positive cells by using immunofluorescence analysis. Furthermore, CD147 and MMP-9 expression in RAW 264.7 cells were both downregulated with CypA inhibitor treatment (P < 0.05). Conclusions The present study reveals the positive correlation of CypA/CD147 signaling and osteoclast-related MMP-9 expression in mice inflammatory periapical lesions progression. Therefore, intervention of CypA/CD147 signaling could probably provide a potential therapeutic target for attenuating inflammatory bone resorption.

Overall Structural Model of NS5A Protein from Hepatitis C Virus and Modulation by Mutations Confering Resistance of Virus Replication to Cyclosporin A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a RNA-binding phosphoprotein composed of a N-terminal membrane anchor (AH), a structured domain 1 (D1), and two intrinsically disordered domains (D2 and D3). The knowledge of the functional architecture of this multifunctional protein remains limited. We report here that NS5A-D1D2D3 produced in a wheat germ cell-free system is obtained under a highly phosphorylated state. Its NMR analysis revealed that these phosphorylations do not change the disordered nature of D2 and D3 domains but increase the number of conformers due to partial phosphorylations. By combining NMR and small angle X-ray scattering, we performed a comparative structural characterization of unphosphorylated recombinant D2 domains of JFH1 (genotype 2a) and the Con1 (genotype 1b) strains produced in Escherichia coli. These analyses highlighted a higher intrinsic folding of the latter, revealing the variability of intrinsic conformations in HCV genotypes. We also investigated the effect of D2 mutations conferring resistance of HCV replication to cyclophilin A (CypA) inhibitors on the structure of the recombinant D2 Con1 mutants and their binding to CypA. Although resistance mutations D320E and R318W could induce some local and/or global folding perturbation, which could thus affect the kinetics of conformer interconversions, they do not significantly affect the kinetics of CypA/D2 interaction measured by surface plasmon resonance (SPR). The combination of all our data led us to build a model of the overall structure of NS5A, which provides a useful template for further investigations of the structural and functional features of this enigmatic protein.

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells

BackgroundRetroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC.ResultsIn this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation.ConclusionsWe showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.