Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-ß1-Induced OCT4/Nanog Pathway.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-ß1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-ß1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-ß1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-ß1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-ß1-induced OCT4/Nanog-dependent pathway.

Functional and structural phenotyping of cardiomyocytes in the 3D organization of embryoid bodies exposed to arsenic trioxide.

Chronic exposure to environmental pollutants threatens human health. Arsenic, a world-wide diffused toxicant, is associated to cardiac pathology in the adult and to congenital heart defects in the foetus. Poorly known are its effects on perinatal cardiomyocytes. Here, bioinformatic image-analysis tools were coupled with cellular and molecular analyses to obtain functional and structural quantitative metrics of the impairment induced by 0.1, 0.5 or 1.0 µM arsenic trioxide exposure on the perinatal-like cardiomyocyte component of mouse embryoid bodies, within their 3D complex cell organization. With this approach, we quantified alterations to the (a) beating activity; (b) sarcomere organization (texture, edge, repetitiveness, height and width of the Z bands); (c) cardiomyocyte size and shape; (d) volume occupied by cardiomyocytes within the EBs. Sarcomere organization and cell morphology impairment are paralleled by differential expression of sarcomeric α-actin and Tropomyosin proteins and of acta2, myh6 and myh7 genes. Also, significant increase of Cx40, Cx43 and Cx45 connexin genes and of Cx43 protein expression profiles is paralleled by large Cx43 immunofluorescence signals. These results provide new insights into the role of arsenic in impairing cytoskeletal components of perinatal-like cardiomyocytes which, in turn, affect cell size, shape and beating capacity.

Mumio (Shilajit) as a potential chemotherapeutic for the urinary bladder cancer treatment.

Mumio (Shilajit) is a traditional medicinal drug known and used for hundreds of years. Bladder cancer is one of the most common cancer types and better treatments are needed. This study analysed the in vitro effect of Mumio on urinary bladder cancer cells (T24 and 5637) in comparison to normal uroepithelial cells (SV-HUC1). Cytotoxicity of Mumio was analysed in these cell lines via MTT and real-time cell growth assays as well via the assessment of the cytoskeleton, apoptosis, and cell cycle. Mumio affected the viability of both cell types in a time and concentration dependent manner. We observed a selectivity of Mumio against cancer cells. Cell cycle and apoptosis analysis showed that Mumio inhibited G0/G1 or S phase cell cycle, which in turn induced apoptosis. Our results showed that Mumio was significantly more cytotoxic to urinary bladder cancer cells than to normal cells. These results are promising and indicate Mumio as a great candidate for urinary bladder cancer treatment and further investigations should be performed.

Electroacupuncture improves repeated social defeat stress-elicited social avoidance and anxiety-like behaviors by reducing Lipocalin-2 in the hippocampus.

BACKGROUND: Post-traumatic stress disorder (PTSD) is a trauma-related disorder that is associated with pro-inflammatory activation and neurobiological impairments in the brain and leads to a series of affective-like behaviors. Electroacupuncture (EA) has been proposed as a clinically useful therapy for several brain diseases. However, the potential role of EA treatment in PTSD and its molecular and cellular mechanisms has rarely been investigated. METHODS: We used an established preclinical social defeat stress mouse model to study whether EA treatment modulates PTSD-like symptoms and understand its underlying mechanisms. To this end, male C57BL/6 mice were subjected to repeated social defeat stress (RSDS) for 6 consecutive days to induce symptoms of PTSD and treated with EA at Baihui (GV 20) and Dazhui (GV 14) acupoints. RESULTS: The stimulation of EA, but not needle insertion at Baihui (GV 20) and Dazhui (GV 14) acupoints effectively improved PTSD-like behaviors such as, social avoidance and anxiety-like behaviors. However, EA stimulation at the bilateral Tianzong (SI11) acupoints did not affect the PTSD-like behaviors obtained by RSDS. EA stimulation also markedly inhibited astrocyte activation in both the dorsal and ventral hippocampi of RSDS-treated mice. Using next-generation sequencing analysis, our results showed that EA stimulation attenuated RSDS-enhanced lipocalin 2 expression in the hippocampus. Importantly, using double-staining immunofluorescence, we observed that the increased lipocalin 2 expression in astrocytes by RSDS was also reduced by EA stimulation. In addition, intracerebroventricular injection of mouse recombinant lipocalin 2 protein in the lateral ventricles provoked social avoidance, anxiety-like behaviors, and the activation of astrocytes in the hippocampus. Interestingly, the overexpression of lipocalin 2 in the brain also altered the expression of stress-related genes, including monoamine oxidase A, monoamine oxidase B, mineralocorticoid receptor, and glucocorticoid receptor in the hippocampus. CONCLUSIONS: This study suggests that the treatment of EA at Baihui (GV 20) and Dazhui (GV 14) acupoints improves RSDS-induced social avoidance, anxiety-like behaviors, astrocyte activation, and lipocalin 2 expression. Furthermore, our findings also indicate that lipocalin 2 expression in the brain may be an important biomarker for the development of PTSD-related symptoms.

Rosiglitasone and ROCK Inhibitors Modulate Fibrogenetic Changes in TGF-ß2 Treated Human Conjunctival Fibroblasts (HconF) in Different Manners.

PURPOSE: The effects of Rho-associated coiled-coil containing protein kinase (ROCK) 1 and 2 inhibitor, ripasudil hydrochloride hydrate (Rip), ROCK2 inhibitor, KD025 or rosiglitazone (Rosi) on two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) treated by transforming growth factor (TGFß2) were studied. METHODS: Two-dimension and three-dimension cultured HconF were examined by transendothelial electrical resistance (TEER, 2D), size and stiffness (3D), and the expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, fibronectin (FN), and α-smooth muscle actin (αSMA) by quantitative PCR (2D, 3D) in the presence of Rip, KD025 or Rosi. RESULTS: TGFß2 caused a significant increase in (1) the TEER values (2D) which were greatly reduced by Rosi, (2) the stiffness of the 3D organoids which were substantially reduced by Rip or KD025, and (3) TGFß2 induced a significant up-regulation of all ECMs, except for COL6 (2D) or αSMA (3D), and down-regulation of COL6 (2D). Rosi caused a significant up-regulation of COL1, 4 and 6 (3D), and down-regulation of COL6 (2D) and αSMA (3D). Most of these TGFß2-induced expressions in the 2D and αSMA in the 3D were substantially inhibited by KD025, but COL4 and αSMA in 2D were further enhanced by Rip. CONCLUSION: The findings reported herein indicate that TGFß2 induces an increase in fibrogenetic changes on the plane and in the spatial space, and are inhibited by Rosi and ROCK inhibitors, respectively.

Ultrastructural aberrations, histological disruption and upregulation of the VEGF, CD34 and ASMA immunoexpression in the myocardium of anemic albino rats.

Iron deficiency anemia (IDA) is a global health problem affecting various body systems and tissues including the cardiovascular system. Several literatures described the associated physiological and clinical changes in the cardiovascular system and heart. However, the associated structural changes were poorly investigated. Therefore, the main aim of the present work was to elucidate whether IDA induces structural changes and alterations in the VEGF, CD34 and ASMA immunoexpression in the myocardium of albino rats. Thirty adult male albino rats were divided into two groups (fifteen rats each); control and anemic. Hematological data for all animals were assessed weekly and statistically analyzed. Three weeks later, animals were sacrificed, and heart specimens were obtained and processed for light and electron microscopy. All hematological parameters showed a statistically significant decrease in the anemic group. Structurally, the anemic group showed markedly degenerated, disrupted and disorganized cardiomyocytes in addition to markedly congested blood vessels, fibroblasts, collagen fibers deposition and perivascular cellular infiltration were noted. Also, positive immunostaining for VEGF, CD34 and ASMA was observed. Ultra-structurally, the myocardium of the anemic group showed disrupted and degenerated myofibrils with degenerated nuclei, perinuclear edema, widened interstitial spaces and marked collagen deposition. Mitochondria markedly increased with abnormal shapes. IDA induced myocardial injury that may propagate to regeneration through activated CD34 progenitor cells and increased VEGF or to degeneration and fibrosis through collagen fibers deposition and enhanced ASMA. So, early diagnosis and treatment of IDA is mandatory to avoid the associated myocardial structural changes.

Digital Imaging Analysis Reveals Reduced Alveolar α-Smooth Muscle Actin Expression in Severe Asthma.

Expansion of α-smooth muscle actin (α-SMA)-expressing airway smooth muscle of the large airways in asthma is well-studied. However, the contribution of α-SMA-expressing cells in the more distal alveolated parenchyma, including pericytes and myofibroblasts within the alveolar septum, to asthma pathophysiology remains relatively unexplored. The objective of this study was to evaluate α-SMA expression in the alveolated parenchyma of individuals with severe asthma (SA), compared with healthy controls or individuals with chronic obstructive pulmonary disease. Using quantitative digital image analysis and video-assisted thoracoscopic surgery lung biopsies, we show that alveolated parenchyma α-SMA expression is markedly reduced in SA in comparison to healthy controls (mean %positive pixels: 12% vs. 23%, P=0.005). Chronic obstructive pulmonary disease cases showed a similar, but trending, decrease in α-SMA positivity compared with controls (mean %positivity: 17% vs. 23%, P=0.107), which may suggest loss of α-SMA expression is a commonality of obstructive lung diseases. The SA group had similar staining for ETS-related gene protein, a specific endothelial marker, comparatively to controls (mean %positive nuclei: 34% vs. 42%, P=0.218), which suggests intact capillary endothelium and likely intact capillary-associated, α-SMA-positive pericytes. These findings suggest that the loss of α-SMA expression in SA may be because of changes in myofibroblast α-SMA expression or cell number. Further study is necessary to fully evaluate possible mechanisms and consequences of this phenomenon.

Pro-Resolving Factors Released by Macrophages After Efferocytosis Promote Mucosal Wound Healing in Inflammatory Bowel Disease.

Nonresolving inflammation is a critical driver of several chronic inflammatory diseases, including inflammatory bowel diseases (IBD). This unresolved inflammation may result from the persistence of an initiating stimulus or from the alteration of the resolution phase of inflammation. Elimination of apoptotic cells by macrophages (a process called efferocytosis) is a critical step in the resolution phase of inflammation. Efferocytosis participates in macrophage reprogramming and favors the release of numerous pro-resolving factors. These pro-resolving factors exert therapeutic effects in experimental autoimmune arthritis. Here, we propose to evaluate the efficacy of pro-resolving factors produced by macrophages after efferocytosis, a secretome called SuperMApo, in two IBD models, namely dextran sodium sulfate (DSS)-induced and T cell transfer-induced colitis. Reintroducing these pro-resolving factors was sufficient to decrease clinical, endoscopic and histological colitis scores in ongoing naive T cell-transfer-induced colitis and in DSS-induced colitis. Mouse primary fibroblasts isolated from the colon demonstrated enhanced healing properties in the presence of SuperMApo, as attested by their increased migratory, proliferative and contractive properties. This was confirmed by the use of human fibroblasts isolated from patients with IBD. Exposure of an intestinal epithelial cell (IEC) line to these pro-resolving factors increased their proliferative properties and IEC acquired the capacity to capture apoptotic cells. The improvement of wound healing properties induced by SuperMApo was confirmed in vivo in a biopsy forceps-wound colonic mucosa model. Further in vivo analysis in naive T cell transfer-induced colitis model demonstrated an improvement of intestinal barrier permeability after administration of SuperMApo, an intestinal cell proliferation and an increase of α-SMA expression by fibroblasts, as well as a reduction of the transcript coding for fibronectin (Fn1). Finally, we identified TGF-ß, IGF-I and VEGF among SuperMApo as necessary to favor mucosal healing and confirmed their role both in vitro (using neutralizing antibodies) and in vivo by depleting these factors from efferocytic macrophage secretome using antibody-coated microbeads. These growth factors only explained some of the beneficial effects induced by factors released by efferocytic macrophages. Overall, the administration of pro-resolving factors released by efferocytic macrophages limits intestinal inflammation and enhance tissue repair, which represents an innovative treatment of IBD.

Distinct effects of pharmacological inhibition of stromelysin1 on endothelial-to-mesenchymal transition and myofibroblast differentiation.

Endothelial-to-mesenchymal transition (EndMT) and fibroblast-to-myofibroblast (FibroMF) differentiation are frequently reported in organ fibrosis. Stromelysin1, a matrix metalloprotease-3 (MMP3) has been indicated in vascular pathologies and organ injuries that often lead to fibrosis. In the current study, we investigated the role of stromelysin1 in EndMT and FibroMF differentiation, which is currently unknown. In our results, whereas TGFß2 treatment of endothelial cells (ECs) induced EndMT associated with increased expression of stromelysin1 and mesenchymal markers such as α-smooth muscle actin (αSMA), N-cadherin, and activin linked kinase-5 (ALK5), inhibition of stromelysin1 blunted TGFß2-induced EndMT. In contrast, treatment of NIH-3T3 fibroblasts with TGFß1 promoted FibroMF differentiation accompanied by increased expression of αSMA, N-cadherin, and ALK5. Intriguingly, stromelysin1 inhibition in TGFß1-stimulated myofibroblasts further exacerbated fibroproliferation with increased FibroMF marker expression. Gene Expression Omnibus (GEO) data analysis indicated increased stromelysin1 expression associated with EndMT and decreased stromelysin1 expression in human pulmonary fibrosis fibroblasts. In conclusion, our study has identified that EndMT and FibroMF differentiation are reciprocally regulated by stromelysin1.

TGF-ß-induced activation of conjunctival fibroblasts is modulated by FGF-2 and substratum stiffness.

PURPOSE: This study aimed to investigate the effects of substratum stiffness on the sensitivity of human conjunctival fibroblasts to transforming growth factor (TGF)-ß, and to explore the molecular mechanism of action. METHODS: Human conjunctival fibroblasts were cultured on collagen-coated plastic or silicone plates. The stiffness of the silicone plates was 0.2 or 64 kPa. Cells were treated by 2.5 ng/mL TGF-ß2 with or without fibroblast growth factor (FGF)-2 (0-100 ng/mL) for 24 h or 48 h. The protein expression levels were determined by Western blot analysis. Cell proliferation was assessed using the WST-8 assay. RESULTS: FGF-2 suppressed the TGF-ß-induced expression of α-smooth muscle actin (SMA) and collagen type I (Col I), but not fibronectin (FN). Both FGF-2 and TGF-ß2 increased cell proliferation without an additive effect. The induction of α-SMA by TGF-ß2 was decreased on the soft substratum, without any change in the expression level or subcellular location of Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). FGF-2 suppressed TGF-ß-induced α-SMA expression even on the soft substratum. CONCLUSIONS: FGF-2 treatment and a soft substratum suppressed TGF-ß-induced transdifferentiation of conjunctival fibroblasts into myofibroblasts. FGF-2 attenuated the TGF-ß-induced expression of α-SMA, even on a soft substratum.

Emodin ameliorates ovalbumin-induced airway remodeling in mice by suppressing airway smooth muscle cells proliferation.

Increased number of airway smooth muscle cells (ASMCs) is a characteristic of airway remodeling in asthma. In this study we investigated whether emodin alleviated airway remodeling in a murine asthma model and reduced the proliferation of ASMCs in vitro. We provided in vivo evidence suggesting that intraperitoneal injection of emodin (20 mg/kg) 1 h prior to OVA challenge apparently alleviated the thickness of airway smooth muscle, the mass of alpha-smooth muscle actin (α-SMA), collagen deposition, epithelial damage, goblet cell hyperplasia, airway inflammation and airway hyperresponsiveness (AHR) in lung tissue. Meanwhile, we found that emodin suppressed the activation of the Akt pathway in lungtissue of allergic mouse models. Additionally, we found that emodin inhibited cellular proliferation and Akt activation in a dose-dependent manner in vitro. Furthermore, LY294002, an inhibitor for PI3K, abrogated serum-induced phosphorylation of Akt, and decreased the proliferation of ASMCs. These findings indicated that emodin alleviated ASMCs proliferation by inhibiting PI3K/Akt pathway in vivo and in vitro, which may provide a potential therapeutic option for airway smooth muscle remodeling in asthma.

VIP Induces Changes in the F-/G-Actin Ratio of Schlemm's Canal Endothelium via LRRK2 Transcriptional Regulation.

Purpose: A previous study reported that vasoactive intestinal peptide (VIP) can regulate the cytoskeleton of Schlemm's canal (SC) endothelium and expand the SC lumen in a rat glaucoma model. In this study, we aimed to investigate the molecular mechanism of VIP on cytoskeleton regulation. Methods: During in vivo experiments in rats, leucine-rich repeat kinase 2 (LRRK2) expression and the ratio of F-actin to G-actin (F-/G-actin) surrounding SC were examined by immunofluorescence after the application of VIP. For in vitro experiments in human umbilical vein endothelial cells, both quantitative PCR (qPCR) and western blotting were performed to evaluate Sp1 and LRRK2 expression after the application of VIP (and Sp1/LRRK2 inhibitor). In addition, the F-/G-actin ratio was examined by both immunofluorescence and western blotting after the application of VIP (and LRRK2 inhibitor). Results: VIP induced increases in the expression of LRRK2 both in vivo and in vitro and the nuclear translocation of Sp1 in vitro. The application of Sp1 inhibitor abolished the increase in LRRK2 expression induced by VIP in vitro. In addition, VIP changed the F-/G-actin ratio, and this effect was abolished by the LRRK2 inhibitor both in vivo and in vitro. Conclusions: VIP increased the expression of LRRK2, and this regulation was due to the nuclear translocation of Sp1. VIP further changed the F-/G-actin ratio and regulated the balance between the stabilization and destabilization of the F-actin architecture. This study elucidates a novel mechanism by which VIP regulates the actin cytoskeleton of SC endothelium via the Sp1-LRRK2 pathway, suggesting a potential novel treatment strategy for glaucoma.

Spatio-temporal patterning of different connexins in developing and postnatal human kidneys and in nephrotic syndrome of the Finnish type (CNF).

Connexins (Cxs) are membrane-spanning proteins which enable flow of information important for kidney homeostasis. Changes in their spatiotemporal patterning characterize blood vessel abnormalities and chronic kidney diseases (CKD). We analysed spatiotemporal expression of Cx37, Cx40, Cx43 and Cx45 in nephron and glomerular cells of developing, postnatal kidneys, and nephrotic syndrome of the Finnish type (CNF) by using immunohistochemistry, statistical methods and electron microscopy. During kidney development, strong Cx45 expression in proximal tubules and decreasing expression in glomeruli was observed. In developing distal nephron, Cx37 and Cx40 showed moderate-to-strong expression, while weak Cx43 expression gradually increased. Cx45/Cx40 co-localized in mesangial and granular cells. Cx43 /Cx45 co-localized in podocytes, mesangial and parietal epithelial cells, and with podocyte markers (synaptopodin, nephrin). Different Cxs co-expressed with endothelial (CD31) and VSMC (α -SMA) markers in vascular walls. Peak signalling of Cx37, Cx43 and Cx40 accompanied kidney nephrogenesis, while strongest Cx45 signalling paralleled nephron maturation. Spatiotemporal Cxs patterning indicate participation of Cx45 in differentiation of proximal tubules, and Cx43, Cx37 and Cx40 in distal tubules differentiation. CNF characterized disorganized Cx45 expression in proximal tubules, increased Cx43 expression in distal tubules and overall elevation of Cx40 and Cx37, while Cx40 co-localized with increased number of interstitial myofibroblasts.

Adding myofibroblasts to the lacrimal pump.

The lacrimal sac (LS) empties in the nasolacrimal duct to drain the tears in the inferior nasal meatus. Different studies indicated the role of the lacrimal pump in the lacrimal drainage. Although controversial, the lacrimal pump mechanism is an extrinsic one, either active, or passive. An intrinsic contractile potential of the LS was not documented previously. We thus aimed a retrospective immunohistochemical study to test the alpha-smooth muscle actin (α-SMA) and h-caldesmon expression in the LS wall. We used archived paraffin-embedded samples of LS from ten adult patients. The α-SMA + phenotype was detected in basal epithelial cells, in subepithelial ribbons of stromal cells, in vascular smooth muscle cells, as well as in pericytes. H-caldesmon was exclusively expressed in pericytes, vascular smooth muscle cells and myoepithelial cells of the subepithelial glands. The most striking feature we found in all samples was a consistent stromal network of α-SMA+/h-caldesmon- myofibroblasts. This finding supports an intrinsic scaffold useful for the lacrimal pump.

[Effect of high expression of type â collagen α1 chain protein on the prognosis of triple negative breast cancer].

Objective: To investigate the expression of type Ⅰ collagen α1 chain protein (COL1A1) in triple negative breast cancer (TNBC), and its relationship with clinicopathological features and prognosis of TNBC. Methods: A total of 148 TNBC specimens were collected from the Affiliated Cancer Hospital of Xinjiang Medical University from 2013 to 2015. The mRNA expression of COL1A1 was detected by fluorescence quantitative RT-PCR and the protein expression of COL1A1 was detected by Western blot. The expression of COL1A1 and α-smooth muscle actin (α-SMA) in TNBC were detected by immunohistochemistry. The relationship between the expression of COL1A1 and clinicopathological parameters and prognosis of TNBC patients was analyzed. Results: The mRNA and protein expression of COL1A1 in MDA-MB-231 cells were 1.696±0.486 and 0.550±0.088, respectively, which were higher than those in MCF-10A cells (1.020±0.231 and 0.350±0.083, P=0.032, P=0.046). The mRNA and protein expression of COL1A1 in TNBC tissues were 1.632 ±0.598 and 0.733 ±0.068, respectively, which were higher than those in paracancerous tissues (1.041±0.316 and 0.612±0.016, P=0.003, P=0.039). The high expression rates of COL1A1 and α-SMA in TNBC tissues were 35.8% and 56.7% respectively, which were higher than those in paracancerous tissues (16.7% and 30.0%, P=0.041, P=0.037). The expression of COL1A1 was correlated with tumor size, TNM stage, lymph node metastasis, vascular invasion and α-SMA expression (all P<0.05). The median survival time in COL1A1 high expression group was 64 months, which was lower than that in low expression group (73 months, P<0.05). Multivariate analysis of Cox proportional hazard regression model showed that COL1A1 expression was an independent influencing factor for the survival of TNBC patients (HR=3.952, P=0.004). Conclusion: The high expression of COL1A1 in TNBC is an independent prognostic factor of TNBC patients.

Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro.

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo.

A schistosome miRNA promotes host hepatic fibrosis by targeting transforming growth factor beta receptor III.

BACKGROUND & AIMS: MicroRNAs (MiRNAs) derived from parasites, and even from plants, have been detected in body fluids and are known to modulate host genes. In this study, we aimed to investigate if the schistosome miRNAs are involved in the occurrence and progression of hepatic fibrosis during Schistosoma japonicum (S. japonicum) infection. METHODS: The presence of miRNAs from S. japonicum (sja-miRNAs) in hepatic stellate cells (HSCs) was detected by RNA sequencing. sja-miRNAs were screened by transfecting HSCs with sja-miRNA mimics. The role of sja-miR-2162 in hepatic fibrosis was evaluated by either elevating its expression in naïve mice or by inhibiting its activity in infected mice, through administration of recombinant adeno-associated virus serotype 8 vectors expressing sja-miR-2162 or miRNA sponges, respectively. RESULTS: We identified a miRNA of S. japonicum, sja-miR-2162, that was consistently present in the HSCs of infected mice. Transfection of sja-miR-2162 mimics led to activation of HSC cells in vitro, characterized by elevation of collagens and α-SMA. The rAAV8-mediated delivery of sja-miR-2162 to naïve mice induced hepatic fibrosis, while sustained inhibition of sja-miR-2162 in infected mice attenuated hepatic fibrosis. The transforming growth factor beta receptor III (TGFBR3), a negative regulator of TGF-ß signaling, was a direct target of sja-miR-2162 in HSCs. CONCLUSIONS: This study demonstrated that pathogen-derived miRNAs directly promote hepatic fibrogenesis in a cross-species manner, and their efficient and sustained inhibition might present a promising therapeutic intervention for infectious diseases. LAY SUMMARY: A schistosome-specific microRNA, sja-miR-2162, is consistently present in the hepatic stellate cells of mice infected with S. japonicum, where it promotes hepatic fibrosis in the host through cross-species regulation of host fibrosis-related genes. The efficient and sustained inhibition of pathogen-derived micRNAs may represent a novel therapeutic intervention for infectious diseases.

The effects of bosentan on hyperoxia-induced lung injury in neonatal rats.

BACKGROUND: Bronchopulmonary dysplasia (BPD) remains an important cause of morbidity and mortality in premature infants. There is currently no proven effective treatment modality for BPD, and inflammation and oxidative injury play an important role in the pathogenesis of this disease. This study investigated the histopathological and biochemical effects of bosentan, which is a non-specific endothelin receptor antagonist with known antioxidant and anti-inflammatory properties, on hyperoxia-induced lung injury (HILI) in neonatal rats. METHODS: The experiment was performed on newborn rats from the 3rd to the 13th postnatal day. The rats were randomly divided into six groups: Group 1 (air-exposed + saline, n = 6); Group 2 (HILI, n = 8); Group 3 (air-exposed + bosentan, n = 7); Group 4 (HILI + saline, n = 7); Group 5 (HILI + early bosentan-treated group, n = 6), and Group 6 (HILI + late bosentan-treated group, n = 7). Bosentan was administered (30 mg/kg/day) intraperitoneally. The histopathological effects of bosentan on lung tissue were assessed by their alveolar surface area, fibrosis, and smooth muscle actin (SMA) scores, and the biochemical effects on lung tissue were assessed by interleukin-1 beta (IL-1ß), IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α). RESULTS: The alveolar surface area and fibrosis scores were found to be significantly higher in HILI groups compared with Group 1 (P < 0.01). The SMA scores in HILI groups were also significantly higher than Group 1 (P < 0.01). Bosentan treatment, especially late therapy, reduced all of these histopathological scores and the levels of IL-6 and TNF-α in the hyperoxia groups (P < 0.01). CONCLUSION: This experimental study showed that bosentan had a protective effect on hyperoxic lung injury through its anti-inflammatory properties.

Blockade of lncRNA-ASLNCS5088-enriched exosome generation in M2 macrophages by GW4869 dampens the effect of M2 macrophages on orchestrating fibroblast activation.

In hypertrophic scar (HS) formation, the type 2 immune response induces the alternatively activated macrophages (M2), which manipulate fibroblasts to differentiate into myofibroblasts with active biologic functions and proliferation. Myofibroblasts express α-smooth muscle actin (α-SMA) and synthesize and produce additional collagen type I and collagen type III, inducing HS formation. However, studies on the mechanism of M2 macrophage modulation are only based on the recognition of profibrotic factors such as TGF-ß1 secreted by macrophages. The influence of exosomes from M2 macrophages on scar formation is still unknown. Both M2 macrophages and myofibroblasts highly express glutaminases (GLSs). GLS is a critical enzyme in glutaminolysis and is important for M2 macrophage and fibroblast polarization. In this study, we found that in a TGF-ß1-stimulated coculture system, a long noncoding RNA (lncRNA) named lncRNA-ASLNCS5088 was enriched in M2 macrophage-derived exosomes. This lncRNA could be transferred with high efficiency to fibroblasts and acted as an endogenous sponge to adsorb microRNA-200c-3p, resulting in increased GLS and α-SMA expression. Pretreatment with GW4869, which impairs M2 macrophage exosome synthesis, ameliorated these pathologic changes in fibroblasts in vitro. Local injection in the late scar formation period with GW4869 reduced α-SMA+ fibroblasts and alleviated the fibrosis of tissue after wound healing in vivo.-Chen, J., Zhou, R., Liang, Y., Fu, X., Wang, D., Wang, C. Blockade of lncRNA-ASLNCS5088-enriched exosome generation in M2 macrophages by GW4869 dampens the effect of M2 macrophages on orchestrating fibroblast activation.

Indoxyl sulfate-induced calcification of vascular smooth muscle cells via the PI3K/Akt/NF-κB signaling pathway.

Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD) and contributes to their high rate of cardiovascular mortality. Indoxyl sulfate (IS) is a representative protein-bound uremic toxin in CKD patients, which has been recognized as a major risk factor for VC. Recent studies have demonstrated that nuclear factor-kappa B (NK-κB) is highly activated in the chronic inflammation conditions of CKD patients and participated in the pathogenesis of VC. However, whether NK-κB is involved in the progression of IS-induced VC remains without elucidation. Here, we showed that NK-κB activity was increased in the IS-induced calcification of human aortic smooth muscle cells (HASMCs). Blocking the NK-κB with a selective inhibitor (Bay-11-7082) significantly relieved the osteogenic transdifferentiation of HASMCs, characterized by the downregulation of early osteogenic-specific marker, core-binding factor alpha subunit 1 (Cbfα1), and upregulation of smooth muscle α-actin (α-SMA), a specific vascular smooth muscle cell marker. Besides, IS stimulated the activation of PI3K/Akt signaling. Furthermore, LY294002, a specific inhibitor of PI3K/Akt pathway, attenuated the activation of NK-κB and osteogenic differentiation of HASMCs. Together, these results suggest that PI3K/Akt/NK-κB signaling plays an important role in the pathogenesis of osteogenic transdifferentiation induced by IS.