Browsing by Author "Zhang, Wenjun"

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    Activation of the Hedgehog signaling pathway leads to fibrosis in aortic valves
    (BMC, 2023-03-02) Gu, Dongsheng; Soepriatna, Arvin H.; Zhang, Wenjun; Li, Jun; Zhao, Jenny; Zhang, Xiaoli; Shu, Xianhong; Wang, Yongshi; Landis, Benjamin J.; Goergen, Craig J.; Xie, Jingwu; Pediatrics, School of Medicine
    Background: Fibrosis is a pathological wound healing process characterized by excessive extracellular matrix deposition, which interferes with normal organ function and contributes to ~ 45% of human mortality. Fibrosis develops in response to chronic injury in nearly all organs, but the a cascade of events leading to fibrosis remains unclear. While hedgehog (Hh) signaling activation has been associated with fibrosis in the lung, kidney, and skin, it is unknown whether hedgehog signaling activation is the cause or the consequence of fibrosis. We hypothesize that activation of hedgehog signaling is sufficient to drive fibrosis in mouse models. Results: In this study, we provide direct evidence to show that activation of Hh signaling via expression of activated smoothened, SmoM2, is sufficient to induce fibrosis in the vasculature and aortic valves. We showed that activated SmoM2 -induced fibrosis is associated with abnormal function of aortic valves and heart. The relevance of this mouse model to human health is reflected in our findings that elevated GLI expression is detected in 6 out of 11 aortic valves from patients with fibrotic aortic valves. Conclusions: Our data show that activating hedgehog signaling is sufficient to drive fibrosis in mice, and this mouse model is relevant to human aortic valve stenosis.
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    BMP10 preserves cardiac function through its dual activation of SMAD-mediated and STAT3-mediated pathways
    (Elsevier, 2019-12-27) Qu, Xiuxia; Liu, Ying; Cao, Dayan; Chen, Jinghai; Liu, Zhuo; Ji, Hongrui; Chen, Yuwen; Zhang, Wenjun; Zhu, Ping; Xiao, Deyong; Li, Xiaohui; Shou, Weinian; Chen, Hanying; Pediatrics, School of Medicine
    Bone morphogenetic protein 10 (BMP10) is a cardiac peptide growth factor belonging to the transforming growth factor β superfamily that critically controls cardiovascular development, growth, and maturation. It has been shown that BMP10 elicits its intracellular signaling through a receptor complex of activin receptor-like kinase 1 with morphogenetic protein receptor type II or activin receptor type 2A. Previously, we generated and characterized a transgenic mouse line expressing BMP10 from the α-myosin heavy chain gene promoter and found that these mice have normal cardiac hypertrophic responses to both physiological and pathological stimuli. In this study, we report that these transgenic mice exhibit significantly reduced levels of cardiomyocyte apoptosis and cardiac fibrosis in response to a prolonged administration of the β-adrenoreceptor agonist isoproterenol. We further confirmed this cardioprotective function with a newly generated conditional Bmp10 transgenic mouse line, in which Bmp10 was activated in adult hearts by tamoxifen. Moreover, the intraperitoneal administration of recombinant human BMP10 was found to effectively protect hearts from injury, suggesting potential therapeutic utility of using BMP10 to prevent heart failure. Gene profiling and biochemical analyses indicated that BMP10 activates the SMAD-mediated canonical pathway and, unexpectedly, also the signal transducer and activator of transcription 3 (STAT3)-mediated signaling pathway both in vivo and in vitro Additional findings further supported the notion that BMP10's cardioprotective function likely is due to its dual activation of SMAD- and STAT3-regulated signaling pathways, promoting cardiomyocyte survival and suppressing cardiac fibrosis.
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    Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation
    (Frontiers Media, 2023-07-17) Cross-Najafi, Arthur A.; Farag, Kristine; Isidan, Abdulkadir; Li, Wei; Zhang, Wenjun; Lin, Zhansong; Walsh, Julia R.; Lopez, Kevin; Park, Yujin; Higgins, Nancy G.; Cooper, David K. C.; Ekser, Burcin; Li, Ping; Surgery, School of Medicine
    Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.
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    Comparison of Artificial Intelligence and Eyeball Method in the Detection of Fatty Liver Disease
    (2023-07-26) Catron, Evan J.; Passarelli, Robert P.; Danielle, Wilmes; Wei, Barry; Le, Thi M.U.; Li, Ping; Zhang, Wenjun; Lin, Jingmei; Melcher, Mark L.; Mihaylov, Plamen V.; Kubal, Chandrashekhar A.; Mangus, Robert S.; Ekser, Burcin
    Background: Quantification of liver fat content relies on visual microscopic inspection of liver biopsies by pathologists. Their percent macrosteatosis (%MaS) estimation is vital in determining donor liver transplantability; however, the eyeball method may vary between observers. Overestimations of %MaS can potentially lead to the discard of viable donor livers. We hypothesize that artificial intelligence (AI) could be helpful in providing a more objective and accurate measurement of %MaS. Methods: Literature review identified HALO (image analysis) and U-Net (deep-learning) as high-accuracy AI programs capable of calculating %MaS in liver biopsies. We compared (i) an experienced pathologist’s and (ii) a transplant surgeon’s eyeball %MaS estimations from de-novo liver transplant (LT) biopsy samples taken 2h post-reperfusion to (iii) the HALO-calculated %MaS (Fig.1). 250 patients had undergone LT at Indiana University between 2020-2021, and 211 had sufficient data for inclusion. Each biopsy was digitized into 5 random non-overlapping tiles at 20x magnification (a total of 1,055 images). We used HALO software for analysis and set the minimum vacuole area to 10μm² to avoid the inclusion of microsteatosis. Microsteatosis was excluded by the pathologist and the surgeon by the eyeball method using the same 1,055 images. Each %MaS estimation was compared with early allograft dysfunction (EAD). EAD is defined by the presence of at least one of the following: INR >1.6 on postoperative day (POD) 7, total bilirubin >10mg/dL on POD7, or AST/ALT >2000IU/L within the first 7 days following LT. Results: Of 211 LTs, 42 (19.9%) had EAD. The mean %MaS estimation of pathologist and transplant surgeon were 6.3% (SD: 11.9%) and 3.2% (SD: 6.4%), respectively. HALO yielded a significantly lower mean %MaS of 2.6% (SD: 2.6%) than the pathologist’s eyeball method (p<0.001). The mean %MaS calculated by HALO was higher in EAD patients than in non-EAD (p=0.032), but this difference did not reach statistical significance in the pathologist’s estimation (p=0.069). Conclusions: Although mean %MaS measurements from all parties were mild (<10%), human eyeball estimations of %MaS were significantly higher than HALO’s %MaS. The HALO-calculated %MaS differed significantly between the EAD and non-EAD LTs which might suggest a possible correlation between the AI’s steatosis analysis and EAD outcomes. However, pathologic variables other than %MaS (necrosis or cholestasis) should be included in future analyses to determine whether %MaS is the dominant parameter predicting EAD. AI is a promising tool to quantify liver steatosis and will help pathologists and transplant surgeons predict liver transplant viability.
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    Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization
    (PLOS, 2021-03-05) Isidan, Abdulkadir; Liu, Shaohui; Chen, Angela M.; Zhang, Wenjun; Li, Ping; Smith, Lester J.; Hara, Hidetaka; Cooper, David K. C.; Ekser, Burcin; Surgery, School of Medicine
    Background: The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. Methods: Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. Results: All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. Conclusion: Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.
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    Critical Roles of STAT3 in β-Adrenergic Functions in the Heart
    (American Heart Association, 2016-01-05) Zhang, Wenjun; Qu, Xiuxia; Chen, Biyi; Snyder, Marylynn; Wang, Meijing; Li, Baiyan; Tang, Yue; Chen, Hanying; Zhu, Wuqiang; Zhan, Li; Yin, Ni; Li, Deqiang; Li, Xie; Liu, Ying; Zhang, J. Jillian; Fu, Xin-Yuan; Rubart, Michael; Song, Long-Sheng; Huang, Xin-Yun; Shou, Weinian; Department of Pediatrics, IU School of Medicine
    BACKGROUND: β-Adrenergic receptors (βARs) play paradoxical roles in the heart. On one hand, βARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of βARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac βAR-mediated signaling and function. METHODS AND RESULTS: We observed that STAT3 can be directly activated in cardiomyocytes by β-adrenergic agonists. To follow up this finding, we analyzed βAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute βAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic β-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for βAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of βAR pathway, including β1AR, protein kinase A, and T-type Ca(2+) channels. CONCLUSIONS: Our data demonstrate for the first time that STAT3 has a fundamental role in βAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for βAR-mediated cardiac stress adaption, pathological remodeling, and heart failure.
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    Current Advances in Basic and Translational Research of Cholangiocarcinoma
    (MDPI, 2021-06-01) Sato, Keisaku; Baiocchi, Leonardo; Kennedy, Lindsey; Zhang, Wenjun; Ekser, Burcin; Glaser, Shannon; Francis, Heather; Alpini, Gianfranco; Medicine, School of Medicine
    Cholangiocarcinoma (CCA) is a type of biliary tract cancer emerging from the biliary tree. CCA is the second most common primary liver cancer after hepatocellular carcinoma and is highly aggressive resulting in poor prognosis and patient survival. Treatment options for CCA patients are limited since early diagnosis is challenging, and the efficacy of chemotherapy or radiotherapy is also limited because CCA is a heterogeneous malignancy. Basic research is important for CCA to establish novel diagnostic testing and more effective therapies. Previous studies have introduced new techniques and methodologies for animal models, in vitro models, and biomarkers. Recent experimental strategies include patient-derived xenograft, syngeneic mouse models, and CCA organoids to mimic heterogeneous CCA characteristics of each patient or three-dimensional cellular architecture in vitro. Recent studies have identified various novel CCA biomarkers, especially non-coding RNAs that were associated with poor prognosis or metastases in CCA patients. This review summarizes current advances and limitations in basic and translational studies of CCA.
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    Current Barriers to Clinical Liver Xenotransplantation
    (Frontiers Media, 2022-02-23) Cross-Najafi, Arthur A.; Lopez, Kevin; Isidan, Abdulkadir; Park, Yujin; Zhang, Wenjun; Li, Ping; Yilmaz, Sezai; Akbulut, Sami; Ekser, Burcin; Surgery, School of Medicine
    Preclinical trials of pig-to-nonhuman primate liver xenotransplantation have recently achieved longer survival times. However, life-threatening thrombocytopenia and coagulation dysregulation continue to limit preclinical liver xenograft survival times to less than one month despite various genetic modifications in pigs and intensive pharmacological support. Transfusion of human coagulation factors and complex immunosuppressive regimens have resulted in substantial improvements in recipient survival. The fundamental biological mechanisms of thrombocytopenia and coagulation dysregulation remain incompletely understood. Current studies demonstrate that porcine von Willebrand Factor binds more tightly to human platelet GPIb receptors due to increased O-linked glycosylation, resulting in increased human platelet activation. Porcine liver sinusoidal endothelial cells and Kupffer cells phagocytose human platelets in an asialoglycoprotein receptor 1-dependent and CD40/CD154-dependent manner, respectively. Porcine Kupffer cells phagocytose human platelets via a species-incompatible SIRPα/CD47 axis. Key drivers of coagulation dysregulation include constitutive activation of the extrinsic clotting cascade due to failure of porcine tissue factor pathway inhibitor to repress recipient tissue factor. Additionally, porcine thrombomodulin fails to activate human protein C when bound by human thrombin, leading to a hypercoagulable state. Combined genetic modification of these key genes may mitigate liver xenotransplantation-induced thrombocytopenia and coagulation dysregulation, leading to greater recipient survival in pig-to-nonhuman primate liver xenotransplantation and, potentially, the first pig-to-human clinical trial.
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    Cyclic AMP Signaling in Biliary Proliferation: A Possible Target for Cholangiocarcinoma Treatment?
    (MDPI, 2021-07-04) Baiocchi, Leonardo; Lenci, Ilaria; Milana, Martina; Kennedy, Lindsey; Sato, Keisaku; Zhang, Wenjun; Ekser, Burcin; Ceci, Ludovica; Meadows, Vik; Glaser, Shannon; Alpini, Gianfranco; Francis, Heather; Medicine, School of Medicine
    Cholangiocarcinoma is a lethal disease with scarce response to current systemic therapy. The rare occurrence and large heterogeneity of this cancer, together with poor knowledge of its molecular mechanisms, are elements contributing to the difficulties in finding an appropriate cure. Cholangiocytes (and their cellular precursors) are considered the liver component giving rise to cholangiocarcinoma. These cells respond to several hormones, neuropeptides and molecular stimuli employing the cAMP/PKA system for the translation of messages in the intracellular space. For instance, in physiological conditions, stimulation of the secretin receptor determines an increase of intracellular levels of cAMP, thus activating a series of molecular events, finally determining in bicarbonate-enriched choleresis. However, activation of the same receptor during cholangiocytes' injury promotes cellular growth again, using cAMP as the second messenger. Since several scientific pieces of evidence link cAMP signaling system to cholangiocytes' proliferation, the possible changes of this pathway during cancer growth also seem relevant. In this review, we summarize the current findings regarding the cAMP pathway and its role in biliary normal and neoplastic cell proliferation. Perspectives for targeting the cAMP machinery in cholangiocarcinoma therapy are also discussed.
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    Development and Characterization of Human Primary Cholangiocarcinoma Cell Lines
    (Elsevier, 2022) Isidan, Abdulkadir; Yenigun, Ali; Soma, Daiki; Aksu, Eric; Lopez, Kevin; Park, Yujin; Cross-Najafi, Arthur; Li, Ping; Kundu, Debjyoti; House, Michael G.; Chakraborty, Sanjukta; Glaser, Shannon; Kennedy, Lindsey; Francis, Heather; Zhang, Wenjun; Alpini, Gianfranco; Ekser, Burcin; Medicine, School of Medicine
    Cholangiocarcinoma (CCA) is the second most common primary liver tumor and is associated with late diagnosis, limited treatment options, and a 5-year survival rate of around 30%. CCA cell lines were first established in 1971, and since then, only 70 to 80 CCA cell lines have been established. These cell lines have been essential in basic and translational research to understand and identify novel mechanistic pathways, biomarkers, and disease-specific genes. Each CCA cell line has unique characteristics, reflecting a specific genotype, sex-related properties, and patient-related signatures, making them scientifically and commercially valuable. CCA cell lines are crucial in the use of novel technologies, such as three-dimensional organoid models, which help to model the tumor microenvironment and cell-to-cell crosstalk between tumor-neighboring cells. This review highlights crucial information on CCA cell lines, including: i) type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) chemical digestion method (eg, trypsin or collagenase), iv) cell-sorting method (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or Dulbecco's modified Eagle's medium), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.