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.
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.

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-β 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.

OBJECTIVE: 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.
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, TGFβ1, 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.

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.

UNASSIGNED: 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.
UNASSIGNED: 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 TGFβ1. The expression of molecular markers of EndMT and fibrogenesis were analysed.
UNASSIGNED: 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.
UNASSIGNED: 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.

OBJECTIVE: 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.
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.
CONCLUSIONS: 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.

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-β 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.

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-β (TGF-β) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1β (IL-1β), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-β 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-β secretion.

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.
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.
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-1β, 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.
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.

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.
METHODS: 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 αvβ5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo.
CONCLUSIONS: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.