Simultaneous Positron Emission Tomography and Molecular Magnetic Resonance Imaging of Cardiopulmonary Fibrosis in a Mouse Model of Left Ventricular Dysfunction.

BACKGROUND: Aging-associated left ventricular dysfunction promotes cardiopulmonary fibrogenic remodeling, Group 2 pulmonary hypertension (PH), and right ventricular failure. At the time of diagnosis, cardiac function has declined, and cardiopulmonary fibrosis has often developed. Here, we sought to develop a molecular positron emission tomography (PET)-magnetic resonance imaging (MRI) protocol to detect both cardiopulmonary fibrosis and fibrotic disease activity in a left ventricular dysfunction model. METHODS AND RESULTS: Left ventricular dysfunction was induced by transverse aortic constriction (TAC) in 6-month-old senescence-accelerated prone mice, a subset of mice that received sham surgery. Three weeks after surgery, mice underwent simultaneous PET-MRI at 4.7 T. Collagen-targeted PET and fibrogenesis magnetic resonance (MR) probes were intravenously administered. PET signal was computed as myocardium- or lung-to-muscle ratio. Percent signal intensity increase and Δ lung-to-muscle ratio were computed from the pre-/postinjection magnetic resonance images. Elevated allysine in the heart (P=0.02) and lungs (P=0.17) of TAC mice corresponded to an increase in myocardial magnetic resonance imaging percent signal intensity increase (P<0.0001) and Δlung-to-muscle ratio (P<0.0001). Hydroxyproline in the heart (P<0.0001) and lungs (P<0.01) were elevated in TAC mice, which corresponded to an increase in heart (myocardium-to-muscle ratio, P=0.02) and lung (lung-to-muscle ratio, P<0.001) PET measurements. Pressure-volume loop and echocardiography demonstrated adverse left ventricular remodeling, function, and increased right ventricular systolic pressure in TAC mice. CONCLUSIONS: Administration of collagen-targeted PET and allysine-targeted MR probes led to elevated PET-magnetic resonance imaging signals in the myocardium and lungs of TAC mice. The study demonstrates the potential to detect fibrosis and fibrogenesis in cardiopulmonary disease through a dual molecular PET-magnetic resonance imaging protocol.

Raspberry Ketone Attenuates Hepatic Fibrogenesis and Inflammation via Regulating the Crosstalk of FXR and PGC-1α Signaling.

Hepatic fibrosis is a compensatory response to chronic liver injury and inflammation, and dietary intervention is recommended as one of the fundamental prevention strategies. Raspberry ketone (RK) is an aromatic compound first isolated from raspberry and widely used to prepare food flavors. The current study investigated the hepatoprotection and potential mechanism of RK against hepatic fibrosis. In vitro, hepatic stellate cell (HSC) activation was stimulated with TGF-ß and cultured with RK, farnesoid X receptor (FXR), or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) agonist or inhibitor, respectively. In vivo, C57BL/6 mice were injected intraperitoneally with thioacetamide (TAA) at 100/200 mg/kg from the first to the fifth week. Mice were intragastrically administrated with RK or Cur once a day from the second to the fifth week. In activated HSCs, RK inhibited extracellular matrix (ECM) accumulation, inflammation, and epithelial-mesenchymal transition (EMT) process. RK both activated FXR/PGC-1α and regulated their crosstalk, which were verified by their inhibitors and agonists. Deficiency of FXR or PGC-1α also attenuated the effect of RK on the reverse of activated HSCs. RK also decreased serum ALT/AST levels, liver histopathological change, ECM accumulation, inflammation, and EMT in mice caused by TAA. Double activation of FXR/PGC-1α might be the key targets for RK against hepatic fibrosis. Above all, these discoveries supported the potential of RK as a novel candidate for the dietary intervention of hepatic fibrosis.

Hepatitis C virus NS5A and core protein induce fibrosis-related genes regulation on Huh7 cells through activation of LX2 cells.

INTRODUCTION AND OBJECTIVES: Liver fibrosis remains a complication derived from a chronic Hepatitis C Virus (HCV) infection even when it is resolved, and no liver antifibrotic drug has been approved. Molecular mechanisms on hepatocytes and activation of hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. To elucidate molecular mechanisms, it is important to analyze pathway regulation during HSC activation and HCV infection. MATERIALS AND METHODS: We evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2. RESULTS: We observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFß1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix. CONCLUSIONS: We demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.

Oxidative stress promotes liver fibrosis by modulating the microRNA-144 and SIN3A-p38 pathways in hepatic stellate cells.

Background & Aims: Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro. Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A, and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis.

Altered Serum Proteins Suggest Inflammation, Fibrogenesis and Angiogenesis in Adult Patients with a Fontan Circulation.

Previous omics research in patients with complex congenital heart disease and single-ventricle circulation (irrespective of the stage of palliative repair) revealed alterations in cardiac and systemic metabolism, inter alia abnormalities in energy metabolism, and inflammation, oxidative stress or endothelial dysfunction. We employed an affinity-proteomics approach focused on cell surface markers, cytokines, and chemokines in the serum of 20 adult Fontan patients with a good functioning systemic left ventricle, and we 20 matched controls to reveal any specific processes on a cellular level. Analysis of 349 proteins revealed 4 altered protein levels related to chronic inflammation, with elevated levels of syndecan-1 and glycophorin-A, as well as decreased levels of leukemia inhibitory factor and nerve growth factor-ß in Fontan patients compared to controls. All in all, this means that Fontan circulation carries specific physiological and metabolic instabilities, including chronic inflammation, oxidative stress imbalance, and consequently, possible damage to cell structure and alterations in translational pathways. A combination of proteomics-based biomarkers and the traditional biomarkers (uric acid, γGT, and cholesterol) performed best in classification (patient vs. control). A metabolism- and signaling-based approach may be helpful for a better understanding of Fontan (patho-)physiology. Syndecan-1, glycophorin-A, leukemia inhibitory factor, and nerve growth factor-ß, especially in combination with uric acid, γGT, and cholesterol, might be interesting candidate parameters to complement traditional diagnostic imaging tools and the determination of traditional biomarkers, yielding a better understanding of the development of comorbidities in Fontan patients, and they may play a future role in the identification of targets to mitigate inflammation and comorbidities in Fontan patients.

H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c.

Objective: Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT. Methods: To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFß1. The expression of molecular markers of EndMT and fibrogenesis were analysed. Results: In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c. Conclusion: our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.

The natural history of CVB3 myocarditis in C57BL/6J mice: an extended in-depth characterization.

BACKGROUND AND AIMS: Viral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course. METHODS AND RESULTS: C57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 105 (regular dose) (RD) or 5 × 106 (high dose) (HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8, and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<.001). In male CVB mice, premature mortality occurred between days 8 and 23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups. CONCLUSION: CVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease.

Aflatoxin B1-exposed hepatocyte-derived extracellular vesicles: Initiating hepatic stellate cell-mediated liver fibrosis through a p53-Parkin-dependent mitophagy pathway.

Environmental aflatoxin B1 (AFB1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB1-exposed hepatocyte-derived EVs (AFB1-EVs) were extracted, and the functional effects of AFB1-EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB1-EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB1-EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB1-EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB1 exposure was rescued by genetic intervention with siPARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB1-EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis.

Effect of adalimumab as an anti-inflammatory agent on gene expression of retinal pigment epithelial cells.

Adalimumab (ADA) is an anti-inflammatory antibody that has FDA approval as a systemic medication for treating noninfectious uveitis. It is also provisionally being investigated as an intravitreal injection for various retinal conditions. This study aimed to assess the effect of ADA on apoptotic, inflammatory, and fibrogenesis gene expression at mRNA and protein levels in retinal pigment epithelial (RPE) cells. RPEs were treated with serial concentrations of ADA (0.5x, x, 2x, and 4x; [x = 250 µg/mL]) for 24 hours. MTT assay was done and the mRNA and protein expressions were quantified using real-time PCR and ELISA assay, respectively. The mRNA levels of IL-1b and IL-6 were significantly increased in ADA-treated RPEs at 0.5x and x concentrations. However, the increase in cytokine secretion was observed only in IL-1b at x concentration. TGF-ß was significantly upregulated in the 0.5x and 4x doses of ADA both at mRNA and protein levels. MTT assay, along with an unchanged BCL-2/BAX ratio confirmed the safety of ADA on RPEs at all studied concentrations. In conclusion, despite its safety, the 2x concentration of ADA was the only dose that did not ignite the expression of any of the studied inflammatory and fibrogenesis genes. This dosage, which is roughly equal to 2 mg intravitreal dose in a clinical setting, might be referred to as a reference starting point for future in-vivo studies in ocular conditions.

Curcumin prevents high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation via mitigating intracellular free radicals and TGF-ß secretion.

Diabetic kidney disease (DKD) is a leading cause of kidney failure. However, the involvement of renal fibroblasts and their communications with renal epithelial cells during DKD remain poorly understood. We investigated the potential role of renal proximal tubular epithelial cells (PTECs) in renal fibroblast activation that might lead to DKD. Additionally, the protective effects of curcumin, a known antioxidant, against renal fibroblast activation induced by high glucose-treated PTECs were investigated. Secretome was collected from HK-2 PTECs under normal glucose, high glucose, high glucose pretreated/cotreated with curcumin, or osmotic control condition for 24 h. Such secretome was then used to treat BHK-21 renal fibroblasts for 24 h. BHK-21 cells treated with high glucose-induced secretome had increased levels of fibroblast activation markers, including spindle index, F-actin, α-smooth muscle actin (α-SMA), fibronectin, collagen I, matrix metalloproteinase-2 (MMP-2) and MMP-9, as compared with normal glucose and osmotic control conditions. However, all these increases were successfully mitigated by curcumin. In addition, high glucose markedly increased intracellular reactive oxygen species (ROS) and transforming growth factor-ß (TGF-ß) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1ß (IL-1ß), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-ß levels were successfully mitigated by curcumin. Therefore, curcumin prevents the high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation, at least in part, via mitigating intracellular ROS and TGF-ß secretion.

Histidine-rich glycoprotein in metabolic dysfunction-associated steatohepatitis-related disease progression and liver carcinogenesis.

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD), previously non-alcoholic fatty liver disease (NAFLD), is a leading cause of chronic liver disease worldwide. In 20%-30% of MASLD patients, the disease progresses to metabolic dysfunction-associated steatohepatitis (MASH, previously NASH) which can lead to fibrosis/cirrhosis, liver failure as well as hepatocellular carcinoma (HCC). Here we investigated the role of histidine-rich glycoprotein (HRG), a plasma protein produced by hepatocytes, in MASLD/MASH progression and HCC development. Methods: The role of HRG was investigated by morphological, cellular, and molecular biology approaches in (a) HRG knock-out mice (HRG-/- mice) fed on a CDAA dietary protocol or a MASH related diethyl-nitrosamine/CDAA protocol of hepatocarcinogenesis, (b) THP1 monocytic cells treated with purified HRG, and (c) well-characterized cohorts of MASLD patients with or without HCC. Results: In non-neoplastic settings, murine and clinical data indicate that HRG increases significantly in parallel with disease progression. In particular, in MASLD/MASH patients, higher levels of HRG plasma levels were detected in subjects with extensive fibrosis/cirrhosis. When submitted to the pro-carcinogenic protocol, HRG-/- mice showed a significant decrease in the volume and number of HCC nodules in relation to decreased infiltration of macrophages producing pro-inflammatory mediators, including IL-1ß, IL-6, IL-12, IL-10, and VEGF as well as impaired angiogenesis. The histopathological analysis (H-score) of MASH-related HCC indicate that the higher HRG positivity in peritumoral tissue significantly correlates with a lower overall patient survival and an increased recurrence. Moreover, a significant increase in HRG plasma levels was detected in cirrhotic (F4) patients and in patients carrying HCC vs. F0/F1 patients. Conclusion: Murine and clinical data indicate that HRG plays a significant role in MASLD/MASH progression to HCC by supporting a specific population of tumor-associated macrophages with pro-inflammatory response and pro-angiogenetic capabilities which critically support cancer cell survival. Furthermore, our data suggest HRG as a possible prognostic predictor in HCC patients with MASLD/MASH-related HCCs.

Milk fat globule-epidermal growth factor 8 (MFGE8) prevents intestinal fibrosis.

OBJECTIVE: Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN: Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS: We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvß5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.

Bone ingrowth into a porous structure is achieved by preceding fibrogenesis and vascularization.

Porous structures are frequently used in surgical implants to strengthen the interlocking power produced by bone ingrowth. Therefore, we aimed to elucidate the mechanism underlying bone ingrowth into a porous structure accompanied by vascularization. A nonbioactive polyetheretherketone implant with a 3D-printed porous structure was prepared and implanted in a bone hole created in the tibias of rabbits. We observed bone ingrowth in the same individual specimens immediately and at 2, 4, 8, and 12 weeks post-implantation using in-vivo computed tomography (CT). Furthermore, a detailed evaluation with blood vessels of each specimen at 2, 4, and 12 weeks was performed with ex-vivo CT and histological specimen. Additional histological evaluation was performed using thin sections of an implant made with thermoplastic polyurethane having the same structure. As a result, the bone invasion began after four weeks, when the construction of fibrous tissue and the spread of new blood vessels within the voids matured. As the bone matured in the load-bearing area, new blood vessels outside the bone matrix regressed. This longitudinal evaluation study suggests that preceding fibrogenesis and vascularization may be key in developing bone ingrowth. STATEMENT OF SIGNIFICANCE: A porous structure is an essential structure for dental and orthopedic implants because it provides strong fixation through bone invasion. Although it was known that vascularization was involved in this, the details were not known. This in vivo study revealed that in order for bone ingrowth to begin, a preparatory period of approximately 4 weeks was required to establish blood flow inside and outside the implant. Furthermore, it was confirmed that by spreading the fibrous structure in advance, it has an advantageous effect on the migration of cells involved in the formation of bones and blood vessels. We pointed out that it is necessary to consider fibrogenesis and vascularization when creating future implants.

Folate Deficiency and/or the Genetic Variant Mthfr677C >T Can Drive Hepatic Fibrosis or Steatosis in Mice, in a Sex-Specific Manner.

SCOPE: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr677C >T variant on NAFLD. METHODS AND RESULTS: This study uses the new Mthfr677C >T mouse model for the human MTHFR677C >T variant. Mthfr677CC and Mthfr677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr677TT, provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. CONCLUSION: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr677TT, whereas Mthfr677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders.

HIV coinfection exacerbates HBV-induced liver fibrogenesis through a HIF-1α- and TGF-ß1-dependent pathway.

BACKGROUND & AIMS: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-ß1 signaling. METHODS: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. RESULTS: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-ß1 (TGF-ß1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-ß1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-ß1-induced fibrogenesis. CONCLUSIONS: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-ß1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. IMPACT AND IMPLICATIONS: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-ß1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection.

Pro-Fibrotic Effects of CCL18 on Human Lung Fibroblasts Are Mediated via CCR6.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. METHODS: We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. RESULTS: We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. CONCLUSION: Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.

Omental coating attenuates implant-induced foreign body reaction in rats.

The objective of this study is to clarify whether the omental coating can effectively attenuate foreign body reaction (FBR) induced by implanted materials. Male Sprague-Dawley rats were injected with polydextran particle slurry intraperitoneally to activate the omentum. 7 days later, polyether polyurethane sponge discs were implanted subcutaneously on each side of the rat's back as the foreign implants to induce FBR. The next day, omental transposition were performed. The disc on the left side of each rat's back was wrapped with omental flap (omental group); the disc on the right side was untreated (control group). All discs were removed 21 days after implantation and assessed by determining the components of the fibrovascular tissue (angiogenesis, inflammation, foreign body giant cells (FBGCs) aggregation and fibrogenesis). In implants in omental group, micro vessel density (MVD), Hemoglobin (Hb) content and VEGF levels (pro-angiogenic cytokine) were increased when compared with implants from control group. Inflammatory parameters (IL-1ß; macrophage accumulation-NAG activity; neutrophil accumulation- MPO levels) were decreased in implants after omental coating. Also, collagen deposition, fibrous capsule thickness, and FBGCs decreased in implants from omental group. However, intra-implant levels of TNF-α and TGF-ß1 were not different after omental coating. Our findings showed for the first time that the omental coating around the implants attenuate the adverse FBR, it may be critical in developing new strategies to control FBR and improve the function and performance of the implanted materials.

An alleviative effect of Lonicerae japonicae flos water extract against liver fibrogenesis in vitro and in vivo.

Lonicerae japonicae (L. japonicae) flos is a medical and food homology herb. This study investigated the phenolic acid and flavonoid contents in L. japonicae flos water extract solution (LJWES) and the preventive effects of LJWES against liver fibrogenesis via FL83B cells and rats. LJWES contains many polyphenols, such as chlorogenic acid, morin, and epicatechin. LJWES increased cell viability and decreased cytotoxicity in thioacetamide (TAA)-treated FL83B cells (75 mM) (p < .05). LJWES decreased (p < .05) gene expressions of Tnf-α, Tnfr1, Bax, and cytochrome c but upregulated Bcl-2 and Bcl-xl in TAA-treated cells; meanwhile, increased protein levels of P53, cleaved caspase 3, and cleaved caspase 9 in TAA treated cells were downregulated (p < .05) by LJWES supplementation. In vivo, results indicated that TAA treatment increased serum liver damage indices (alanine aminotransferase [ALT] and alkaline phosphatase [ALP]) and cytokines (interleukin-6 and transforming growth factor-ß1) levels and impaired liver antioxidant capacities (increased thiobarbituric acid reactive substance value but decreased catalase/glutathione peroxidase activities) in rats (p < .05) while LJWES supplementation amended (p < .05) them. Liver fibrosis scores, collagen deposition, and alpha-smooth muscle actin deposition in TAA-treated rats were also decreased by LJWES supplementation (p < .05). To sum up, LJWES could be a potential hepatoprotective agent against liver fibrogenesis by enhancing antioxidant ability, downregulating inflammation in livers, and reducing apoptosis in hepatocytes.

Targeted delivery of galunisertib using machine perfusion reduces fibrogenesis in an integrated ex vivo renal transplant and fibrogenesis model.

BACKGROUND AND PURPOSE: Fibrosis in kidney allografts is a major post-transplant complication that contributes to graft failure. Lately, multiple potent inhibitors of fibrosis-related pathways have been developed such as galunisertib, an inhibitor of the transforming growth factor-beta (TGF-ß/TGFß1) signalling pathway. This drug, however, poses risks for adverse effects when administered systemically. Therefore, we devised a new repurposing strategy in which galunisertib is administered ex vivo. We combined machine perfusion and tissue slices to explore the antifibrotic effects of galunisertib in renal grafts. EXPERIMENTAL APPROACH: Porcine kidneys were subjected to 30 min of warm ischaemia, 24 h of oxygenated hypothermic machine perfusion and 6 h of normothermic machine perfusion with various treatments (i.e. untreated control, TGFß1, galunisertib or TGFß1 + galunisertib; n = 8 kidneys per group). To determine whether effects persisted upon ceasing treatment, kidney slices were prepared from respective kidneys and incubated for 48 h. KEY RESULTS: Galunisertib treatment improved general viability without negatively affecting renal function or elevating levels of injury markers or by-products of oxidative stress during perfusion. Galunisertib also reduced inflammation and, more importantly, reduced the onset of fibrosis after 48 h of incubation. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate the value of using machine perfusion for administering antifibrotic drugs such as galunisertib, proving it to be an effective example of repurposing.

The transcriptomic profile shows the protective effects of celecoxib on cirrhotic splenomegaly.

BACKGROUND: Splenomegaly can exacerbate liver cirrhosis and portal hypertension. We have previously demonstrated that cyclooxygenase-2 (COX-2) inhibitor can attenuate cirrhotic splenomegaly. However, the mechanism of cirrhotic splenomegaly remains unclear, thus becoming the focus of the present study. MATERIALS AND METHODS: Thioacetamide (TAA) intraperitoneal injection was used to induce cirrhotic splenomegaly. Rats were randomized into the control, TAA and TAA + celecoxib groups. Histological analysis and high-throughput RNA sequencing of the spleen were conducted. Splenic collagen III, α-SMA, Ki-67, and VEGF were quantified. RESULTS: A total of 1461 differentially expressed genes (DEGs) were identified in the spleens of the TAA group compared to the control group. The immune response and immune cell activation might be the major signaling pathways involved in the pathogenesis of cirrhotic splenomegaly. With its immunoregulatory effect, celecoxib presents to ameliorate cirrhotic splenomegaly and liver cirrhosis. Furthermore, 304 coexisting DEGs were obtained between TAA vs. control and TAA + celecoxib vs. TAA. Gene ontology (GO) and KEGG analyses collectively indicated that celecoxib may attenuate cirrhotic splenomegaly through the suppression of splenic immune cell proliferation, inflammation, immune regulation, and fibrogenesis. The impacts on these factors were subsequently validated by the decreased splenic Ki-67-positive cells, macrophages, fibrotic areas, and mRNA levels of collagen III and α-SMA. CONCLUSIONS: Celecoxib attenuates cirrhotic splenomegaly by inhibiting splenic immune cell proliferation, inflammation, and fibrogenesis. The current study sheds light on the therapeutic strategy of liver cirrhosis by targeting splenic abnormalities and provides COX-2 inhibitors as a novel medical treatment for cirrhotic splenomegaly.