TGF-ß downstream of Smad3 and MAPK signaling antagonistically regulate the viability and partial epithelial-mesenchymal transition of liver progenitor cells.

BACKGROUND: Liver progenitor cells (LPCs) are a subpopulation of cells that contribute to liver regeneration, fibrosis and liver cancer initiation under different circumstances. RESULTS: By performing adenoviral-mediated transfection, CCK-8 analyses, F-actin staining, transwell analyses, luciferase reporter analyses and Western blotting, we observed that TGF-ß promoted cytostasis and partial epithelial-mesenchymal transition (EMT) in LPCs. In addition, we confirmed that TGF-ß activated the Smad and MAPK pathways, including the Erk, JNK and p38 MAPK signaling pathways, and revealed that TGFß-Smad signaling induced growth inhibition and partial EMT, whereas TGFß-MAPK signaling had the opposite effects on LPCs. We further found that the activity of Smad and MAPK signaling downstream of TGF-ß was mutually restricted in LPCs. Mechanistically, we found that TGF-ß activated Smad signaling through serine phosphorylation of both the C-terminal and linker regions of Smad2 and 3 in LPCs. Additionally, TGFß-MAPK signaling inhibited the phosphorylation of Smad3 but not Smad2 at the C-terminus, and it reinforced the linker phosphorylation of Smad3 at T179 and S213. We then found that overexpression of mutated Smad3 at linker phosphorylation sites intensifies TGF-ß-induced cytostasis and EMT, mimicking the effects of MAPK inhibition in LPCs, whereas mutation of Smad3 at the C-terminus caused LPCs to blunt TGF-ß-induced cytostasis and partial EMT. CONCLUSION: These results suggested that TGF-ß downstream of Smad3 and MAPK signaling were mutually antagonistic in regulating the viability and partial EMT of LPCs. This antagonism may help LPCs overcome the cytostatic effect of TGF-ß under fibrotic conditions and maintain partial EMT and progenitor phenotypes.

GRIN2A mutation is a novel indicator of stratifying beneficiaries of immune checkpoint inhibitors in multiple cancers.

Glutamate-NMDAR receptors (GRINs) have been reported to influence cancer immunogenicity; however, the relationship between GRIN alterations and the response to immune checkpoint inhibitors (ICIs) has not been determined. This study combined clinical characteristics and mutational profiles from multiple cohorts to form a discovery cohort (n = 901). The aim of this study was to investigate the correlation between the mutation status of the GRIN gene and the response to ICI therapy. Additionally, an independent ICI-treated cohort from the Memorial Sloan Kettering Cancer Center (MSKCC, N = 1513) was used for validation. Furthermore, this study explored the associations between GRIN2A mutations and intrinsic and extrinsic immunity using multiomics analysis. In the discovery cohort, patients with GRIN2A-MUTs had improved clinical outcomes, as indicated by a higher objective response rate (ORR: 36.8% vs 25.8%, P = 0.020), durable clinical benefit (DCB: 55.2% vs 38.7%, P = 0.005), prolonged progression-free survival (PFS: HR = 0.65; 95% CI 0.49 to 0.87; P = 0.003), and increased overall survival (OS: HR = 0.67; 95% CI 0.50 to 0.89; P = 0.006). Similar results were observed in the validation cohort, in which GRIN2A-MUT patients exhibited a significant improvement in overall survival (HR = 0.66; 95% CI = 0.49 to 0.88; P = 0.005; adjusted P = 0.045). Moreover, patients with GRIN2A-MUTs exhibited an increase in tumor mutational burden, high expression of costimulatory molecules, increased activity of antigen-processing machinery, and infiltration of various immune cells. Additionally, gene sets associated with cell cycle regulation and the interferon response were enriched in GRIN2A-mutated tumors. In conclusion, GRIN2A mutation is a novel biomarker associated with a favorable response to ICIs in multiple cancers.

EGFR-mediated activation of adipose tissue macrophages promotes obesity and insulin resistance.

Obesity and obesity-related health complications are increasing in prevalence. Adipose tissue from obese subjects has low-grade, chronic inflammation, leading to insulin resistance. Adipose tissue macrophages (ATMs) are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. In response to a high fat diet (HFD), ATM numbers initially increase by proliferation of resident macrophages, but subsequent increases also result from infiltration in response to chemotactic signals from inflamed adipose tissue. To elucidate the underlying mechanisms regulating the increases in ATMs and their proinflammatory phenotype, we investigated the role of activation of ATM epidermal growth factor receptor (EGFR). A high fat diet increased expression of EGFR and its ligand amphiregulin in ATMs. Selective deletion of EGFR in ATMs inhibited both resident ATM proliferation and monocyte infiltration into adipose tissue and decreased obesity and development of insulin resistance. Therefore, ATM EGFR activation plays an important role in adipose tissue dysfunction.

Clinical course of patients on maintenance hemodialysis and COVID-19: a retrospective longitudinal study.

Coronavirus Disease 2019 (COVID-19) emerges as a global pandemic and there is a lack of evidence about the clinical course and outcome of patients on maintenance hemodialysis (MHD). Here we conducted a retrospective longitudinal study aimed to analyze the clinical features and outcome of MHD patients hospitalized with COVID-19. Of 3126 inpatients with COVID-19 at 3 Branches of Wuhan Tongji Hospital from Jan 18th to Mar 9th, 2020, 19 patients were undergoing maintenance hemodialysis. Among the 19 MHD patients with COVID-19, 6 patients (31.6%) died, and 13 patients (68.4%) were able to be discharged. Baseline characteristics, clinical courses, laboratory findings, and dynamic trajectories of major laboratory markers were compared between survivors and nonsurvivors. According to our findings, MHD patients with COVID-19 who experienced non-surviving outcome had more elevated CRP, IL6 and procalcitonin as well as fibrinogen levels at various points compared to survivors. Thus the dysregulation of immune response as well as coagulation abnormalities might be highly involved in the pathological process of COVID-19, contributing to the poor prognosis in MHD patients.

Association of liver abnormalities with in-hospital mortality in patients with COVID-19.

BACKGROUND & AIMS: The evolution and clinical significance of abnormal liver chemistries and the impact of hepatitis B infection on outcome in patients with COVID-19 is not well characterized. This study aimed to explore these issues. METHODS: This large retrospective cohort study included 2,073 patients with coronavirus disease 2019 (COVID-19) and definite outcomes in Wuhan, China. Longitudinal liver function tests were conducted, with associated factors and risk of death determined by multivariate regression analyses. A prognostic nomogram was formulated to predict the survival of patients with COVID-19. The characteristics of liver abnormalities and outcomes of patients with COVID-19, with and without hepatitis B, were compared after 1:3 propensity score matching. RESULTS: Of the 2,073 patients, 1,282 (61.8%) had abnormal liver chemistries during hospitalization, and 297 (14.3%) had a liver injury. The mean levels of aspartate aminotransferase (AST) and direct bilirubin (D-Bil) increased early after symptom onset in deceased patients and showed disparity compared to levels in discharged patients throughout the clinical course of the disease. Abnormal AST (adjusted hazard ratio [HR] 1.39; 95% CI 1.04-1.86, p = 0.027) and D-Bil (adjusted HR 1.66; 95% CI 1.22-2.26; p = 0.001) levels at admission were independent risk factors for mortality due to COVID-19. A nomogram was established based on the results of multivariate analysis and showed sufficient discriminatory power and good consistency between the prediction and the observation. HBV infection in patients did not increase the risk of poor COVID-19-associated outcomes. CONCLUSIONS: Abnormal AST and D-Bil levels at admission were independent predictors of COVID-19-related mortality. Therefore, monitoring liver chemistries, especially AST and D-Bil levels, is necessary in hospitalized patients with COVID-19. LAY SUMMARY: Liver test abnormalities (in particular elevations in the levels of aspartate aminotransferase [AST] and direct bilirubin [D-Bil]) were observed after symptom onset in patients who went on to die of coronavirus disease 2019 (COVID-19). Abnormal levels of AST and D-Bil at admission were independent predictors of COVID-19-related mortality. HBV infection in patients did not increase the risk of poor COVID-19-associated outcomes.

The Role of the EGF Receptor in Sex Differences in Kidney Injury.

BACKGROUND: Sex differences mediating predisposition to kidney injury are well known, with evidence indicating lower CKD incidence rates and slower decline in renal function in nondiabetic CKD for premenopausal women compared with men. However, signaling pathways involved have not been elucidated to date. The EGF receptor (EGFR) is widely expressed in the kidney in glomeruli and tubules, and persistent and dysregulated EGFR activation mediates progressive renal injury. METHODS: To investigate the sex differences in response to renal injury, we examined EGFR expression in mice, in human kidney tissue, and in cultured cell lines. RESULTS: In wild type mice, renal mRNA and protein EGFR levels were comparable in males and females at postnatal day 7 but were significantly lower in age-matched adult females than in adult males. Similar gender differences in renal EGFR expression were detected in normal adult human kidneys. In Dsk5 mutant mice with a gain-of-function allele that increases basal EGFR kinase activity, males had progressive glomerulopathy, albuminuria, loss of podocytes, and tubulointerstitial fibrosis, but female Dsk5 mice had minimal kidney injury. Oophorectomy had no effect on renal EGFR levels in female Dsk5 mice, while castration protected against the kidney injury in male Dsk5 mice, in association with a reduction in EGFR expression to levels seen in females. Conversely, testosterone increased EGFR expression and renal injury in female Dsk5 mice. Testosterone directly stimulated EGFR expression in cultured kidney cells. CONCLUSIONS: These studies indicate that differential renal EGFR expression plays a role in the sex differences in susceptibility to progressive kidney injury that may be mediated at least in part by testosterone.

TGF-ß promotes fibrosis after severe acute kidney injury by enhancing renal macrophage infiltration.

TGF-ß signals through a receptor complex composed of 2 type I and 2 type II (TGF-ßRII) subunits. We investigated the role of macrophage TGF-ß signaling in fibrosis after AKI in mice with selective monocyte/macrophage TGF-ßRII deletion (macrophage TGF-ßRII-/- mice). Four weeks after injury, renal TGF-ß1 expression and fibrosis were higher in WT mice than macrophage TGF-ßRII-/- mice, which had decreased renal macrophages. The in vitro chemotactic response to f-Met-Leu-Phe was comparable between bone marrow-derived monocytes (BMMs) from WT and macrophage TGF-ßRII-/- mice, but TGF-ßRII-/- BMMs did not respond to TGF-ß. We then implanted Matrigel plugs suffused with either f-Met-Leu-Phe or TGF-ß1 into WT or macrophage TGF-ßRII-/- mice. After 6 days, f-Met-Leu-Phe induced similar macrophage infiltration into the Matrigel plugs of WT and macrophage TGF-ßRII-/- mice, but TGF-ß induced infiltration only in WT mice. We further determined the number of labeled WT or TGF-ßRII-/- BMMs infiltrating into WT kidneys 20 days after ischemic injury. There were more labeled WT BMMs than TGF-ßRII-/- BMMs. Therefore, macrophage TGF-ßRII deletion protects against the development of tubulointerstitial fibrosis following severe ischemic renal injury. Chemoattraction of macrophages to the injured kidney through a TGF-ß/TGF-ßRII axis is a heretofore undescribed mechanism by which TGF-ß can mediate renal fibrosis during progressive renal injury.

Taurocholate Induces Connective Tissue Growth Factor Expression in Hepatocytes Through ERK-YAP Signaling.

BACKGROUND/AIMS: Cholestasis is characterized by intrahepatic accumulation of cytotoxic bile acids (BAs), ultimately leading to fibrosis and cirrhosis, but the precise role of BAs in cholestasis-induced liver fibrosis remains largely elusive. In this study, we investigated the role and the potential mechanisms of BAs during cholestasis in vivo and in vitro. METHODS: The effect of BAs during cholestasis was studied in bile duct ligation (BDL) rat models in vivo. We performed immunohistochemistry, Western blotting, and quantitative RT-PCR to investigate the expression of connective tissue growth factor (CTGF/CCN2) in rat liver during cholestasis. The hepatic cell lines AML12 and BRL were stimulated with taurocholate (TC) and the level of CTGF/CCN2, and activation of ERK, Akt, p38 MAPK, JNK, YAP, and TGF-ß/Smad signaling were examined using Western blotting. Next, to elucidate the mechanism underlying bile acid-induced CTGF/CCN2, we treated the cells with MEK1/2 inhibitor (U0126), YAP function inhibitor (verteporfin), p38 kinase inhibitor (SB203580), Akt inhibitor (MK2206), and small interfering RNA (siRNA) targeting mek1, erk, and yap in cooperation with TC. Besides, we confirmed the activation of these signaling pathways in BDL and sham rat livers by immunohistochemistry, Western blotting, and quantitative RT-PCR. RESULTS: In this study, we confirmed that the expression of CTGF/CCN2 was increased in BDL-induced rodent cholestatic liver fibrosis. In addition, we showed that TC, the main component of BAs, enhanced the synthesis of CTGF/ CCN2 in AML12 and BRL hepatic cell lines. Moreover, we demonstrated that TC activated ERK, Akt, and YAP signaling in hepatocytes, but the precise roles of these signaling cascades in the expression of CTGF/CCN2 were different: TC-induced expression of CTGF/CCN2 was mediated by ERK-YAP signaling, whereas Akt signaling inhibited ERK signaling and YAP and subsequently the expression of CTGF/CCN2 in hepatocytes. Furthermore, YAP functioned as a downstream regulator of ERK signaling in TC-induced CTGF/CCN2 expression in hepatocytes. CONCLUSION: Our report provides evidence for the role of conjugated BAs in liver fibrosis and suggests that the production of CTGF/CCN2, induced by conjugated BAs via ERK-YAP axis activation, may be a therapeutic target in cholestasis-induced liver fibrosis.

Autocrine transforming growth factor-ß/activin A-Smad signaling induces hepatic progenitor cells undergoing partial epithelial-mesenchymal transition states.

Hepatic progenitor cells (HPCs) are a subpopulation of cells which was usually expanded in chronic liver injury and are contributed to liver regeneration through differentiating into hepatocytes and cholangiocytes. Epithelial-mesenchymal transition is a dynamic process which is important for the progression of liver fibrosis and cancer initiation. This study demonstrated that LE/6 and WB-F344 cells, both of which were HPC derived cell lines, were undergoing partial epithelial-mesenchymal transition states, which was indicated by the co-expression of epithelial markers (E-cadherin and zona occludin 1), and mesenchymal markers (vimentin, fibronectin, collagen 1and α-SMA). Furthermore, autocrine TGF-ß and activin A signaling contributed to the maintenance of partial EMT in HPCs. In addition, Smad signaling, a classic downstream signaling cascade of both TGF-ß and activin A, also participated in the partial EMT. These findings revealed the existence of partial EMT states in HPCs and confirmed some partial EMT related autocrine signaling cascades, and may help to further the understanding and explore the functional role of HPCs in the process of hepatic fibrosis and liver cancer initiation.

Endovascular Treatment of Behcet Disease With Recurrent Infrainguinal Arterial Pseudoaneurysms: A Case Report and Literature Review.

Aneurysm or pseudoaneurysm formation is one of the vascular complications of Behcet disease. At present, the optimal treatment for the disease has not been established.The authors report a case of vasculo-Behcet disease (v-BD) with recurrent pseudoaneurysms in the left infrainguinal arteries (common femoral artery, superficial femoral artery, and popliteal artery), as well as thrombosis in the popliteal vein and posterior tibial vein. The patient underwent 3 rounds of surgery, but developed a new pseudoaneurysm several months after each surgery. Eventually, the patient was successfully treated with a combination of endovascular repair, using a fully covered stent graft, and prednisone. The pseudoaneurysm regressed without recurrence for more than 1 year.For v-BD, treatment with immunosuppressive therapy alone may not be sufficient to prevent the recurrence of pseudoaneurysms. For the endovascular treatment of pseudoaneurysms affecting the infrainguinal arteries in v-BD, a fully covered stent graft without oversizing is essential to prevent the recurrence of pseudoaneurysms.

Activin A-Smad Signaling Mediates Connective Tissue Growth Factor Synthesis in Liver Progenitor Cells.

Liver progenitor cells (LPCs) are activated in chronic liver damage and may contribute to liver fibrosis. Our previous investigation reported that LPCs produced connective tissue growth factor (CTGF/CCN2), an inducer of liver fibrosis, yet the regulatory mechanism of the production of CTGF/CCN2 in LPCs remains elusive. In this study, we report that Activin A is an inducer of CTGF/CCN2 in LPCs. Here we show that expression of both Activin A and CTGF/CCN2 were upregulated in the cirrhotic liver, and the expression of Activin A positively correlates with that of CTGF/CCN2 in liver tissues. We go on to show that Activin A induced de novo synthesis of CTGF/CCN2 in LPC cell lines LE/6 and WB-F344. Furthermore, Activin A contributed to autonomous production of CTGF/CCN2 in liver progenitor cells (LPCs) via activation of the Smad signaling pathway. Smad2, 3 and 4 were all required for this induction. Collectively, these results provide evidence for the fibrotic role of LPCs in the liver and suggest that the Activin A-Smad-CTGF/CCN2 signaling in LPCs may be a therapeutic target of liver fibrosis.

Reduced expression of transcriptional intermediary factor 1 gamma promotes metastasis and indicates poor prognosis of hepatocellular carcinoma.

UNLABELLED: Transcriptional intermediary factor 1 gamma (TIF1γ) may play either a potential tumor-suppressor or -promoter role in cancer. Here we report on a critical role of TIF1γ in the progression of hepatocellular carcinoma (HCC). Reduced expression of TIF1γ was detected in HCC, especially in advanced HCC tissues, compared to adjacent noncancerous tissues. HCC patients with low TIF1γ expression had shorter overall survival times and higher recurrence rates than those with high TIF1γ expression. Reduced TIF1γ expression was an independent and significant risk factor for recurrence and survival after curative resection. In HCC cells, TIF1γ played a dual role: It promoted tumor growth in early-stage HCC, but not in advanced-stage HCC, whereas it inhibited invasion and metastasis in both early- and advanced-stage HCC. Mechanistically, we confirmed that TIF1γ inhibited transforming growth factor-ß/ Drosophila mothers against decapentaplegic protein (TGF-ß/Smad) signaling through monoubiquitination of Smad4 and suppressed the formation of Smad2/3/4 complex in HCC cells. TGF-ß-inducing cytostasis and metastasis were both inhibited by TIF1γ in HCC. We further proved that TIF1γ suppressed cyotstasis-related TGF-ß/Smad downstream c-myc down-regulation, as well as p21/cip1 and p15/ink4b up-regulation in early-stage HCC. Meanwhile, TGF-ß inducible epithelial-mesenchymal transition and TGF-ß/Smad downstream metastatic cascades, including phosphatase and tensin homolog deleted on chromosome ten down-regulation, chemokine (CXC motif) receptor 4 and matrix metalloproteinase 1 induction, and epidermal growth factor receptor- and protein kinase B-signaling transactivation, were inhibited by TIF1γ. In addition, we found that the down-regulation of TIF1γ in HCC was caused by hypermethylation of CpG islands in the TIF1γ promoter, and demonstrated that the combination of TIF1γ and phosphorylated Smad2 was a more powerful predictor of poor prognosis. CONCLUSION: TIF1γ regulates tumor growth and metastasis through inhibition of TGF-ß/Smad signaling and may serve as a novel prognostic biomarker in HCC.

Smad6 suppresses the growth and self-renewal of hepatic progenitor cells.

Activation of hepatic progenitor cells (HPCs) is commonly observed in chronic liver disease and Wnt/ß-catenin signaling plays a crucial role in the expansion of HPCs. However, the molecular mechanisms that regulate the activation of Wnt/ß-catenin signaling in the liver, especially in HPCs, remain largely elusive. Here, we reported that ectopic expression of Smad6 suppressed the proliferation and self-renewal of WB-F344 cells, a HPC cell line. Mechanistically, we found that Smad6 inhibited Wnt/ß-catenin signaling through promoting the interaction of C-terminal binding protein (CtBP) with ß-catenin/T-cell factor (TCF) complex to inhibit ß-catenin mediated transcriptional activation in WB-F344 cells. We used siRNA targeting ß-catenin to demonstrate that Wnt/ß-catenin signaling was required for the proliferation and self-renewal of HPCs. Taken together, these results suggest that Smad6 is a regulatory molecule which regulates the proliferation, self-renewal and Wnt/ß-catenin signaling in HPCs.

Transforming growth factor ß induces expression of connective tissue growth factor in hepatic progenitor cells through Smad independent signaling.

Hepatic progenitor cells (HPCs) are activated in the chronic liver injury and are found to participate in the progression of liver fibrosis, while the precise role of HPCs in liver fibrosis remains largely elusive. In this study, by immunostaining of human liver sections, we confirmed that HPCs were activated in the cirrhotic liver and secreted transforming growth factor ß (TGF-ß) and connective tissue growth factor (CTGF), both of which were important inducers of liver fibrosis. Besides, we used HPC cell lines LE/6 and WB-F344 as in vitro models and found that TGF-ß induced secretion of CTGF in HPCs. Moreover, TGF-ß signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. Furthermore, we found that TGF-ß induced expression of CTGF was not mediated by TGF-ß activated Smad signaling but mediated by TGF-ß activated Erk, JNK and p38 MAPK signaling. Taken together, our results provide evidence for the role of HPCs in liver fibrosis and suggest that the production of CTGF by TGF-ß activated MAPK signaling in HPCs may be a therapeutic target of liver fibrosis.

Fluorescent in situ hybridization diagnosis of extramedullary nodal blast crisis.

The t(9;22)(q34;q11) translocation between bcr and abl genes plays a pivotal role in the pathogenesis and diagnosis of chronic myelogenous leukemia (CML). Fluorescence in situ hybridization (FISH) using specific DNA probes provides a useful and accurate way for the detection of bcr/abl fusion gene in single cell. Here, we report an unusual case of a patient with no prior hematologic disease who initially manifested lymphadenopathy. The lymph node findings were suspicious for T-lineage lymphoblastic lymphoma, however, his blood and bone marrow at that time were in chronic phase of CML. This presented difficulty for accurate discrimination between CML blast crisis (BC) and non-Hodgkin's lymphomas (NHLs). To discern where the extramedullary nodal malignancy originated from, we cytologically analyzed lymph node biopsies and bone marrow with FISH to detect bcr/abl fusion signals. Together with the morphology, immunohistochemistry, cytogenetics as well as molecular analysis, the patient was diagnosed as extramedullary T-lymphoid BC of Ph+ CML. In conclusion, this case is unusual at three levels: first, extramedullary nodal BC as a presenting manifestation of CML is rare and the blasts are of precursor T lymphoblastic lineage, rather than the more common B-cell lineage; second, this case suggests that extramedullary lymphoid nodal BC of CML can exist independently without the bone marrow developing into BC; and third, FISH analysis on the single neoplastic cell is an accurate way to confirm that the neoplasm is either extramedullary localized blasts of CML or genetically distinct neoplasm.