Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in HSCs.

BACKGROUND AND AIMS: Fibrosis is the common end point for all forms of chronic liver injury, and the progression of fibrosis leads to the development of end-stage liver disease. Activation of HSCs and their transdifferentiation into myofibroblasts results in the accumulation of extracellular matrix proteins that form the fibrotic scar. Long noncoding RNAs regulate the activity of HSCs and provide targets for fibrotic therapies. APPROACH AND RESULTS: We identified long noncoding RNA TILAM located near COL1A1 , expressed in HSCs, and induced with liver fibrosis in humans and mice. Loss-of-function studies in human HSCs and human liver organoids revealed that TILAM regulates the expression of COL1A1 and other extracellular matrix genes. To determine the role of TILAM in vivo, we annotated the mouse ortholog ( Tilam ), generated Tilam- deficient green fluorescent protein-reporter mice, and challenged these mice in 2 different models of liver fibrosis. Single-cell data and analysis of single-data and analysis of Tilam-deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Tilam -deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Furthermore, loss of Tilam expression attenuated the development of fibrosis in the setting of in vivo liver injury. Finally, we found that TILAM interacts with promyelocytic leukemia nuclear body scaffold protein to regulate a feedback loop by which TGF-ß2 reinforces TILAM expression and nuclear localization of promyelocytic leukemia nuclear body scaffold protein to promote the fibrotic activity of HSCs. CONCLUSIONS: TILAM is activated in HSCs with liver injury and interacts with promyelocytic leukemia nuclear body scaffold protein to drive the development of fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end-stage liver disease.

AMPK stimulation inhibits YAP/TAZ signaling to ameliorate hepatic fibrosis.

Hepatic fibrosis is driven by the activation of hepatic stellate cells (HSCs). The Hippo pathway and its effectors, YAP and TAZ, are key regulators of HSC activation and fibrosis. However, there is a lack of mechanistic understanding of YAP/TAZ regulation in HSCs. Here we show that AMPK activation leads to YAP/TAZ inhibition and HSC inactivation in vitro, while the expression of a kinase-inactive mutant reversed these effects compared to wild type AMPKɑ1. Notably, the depletion of LATS1/2, an upstream kinase of YAP/TAZ signaling, rescues YAP/TAZ activation, suggesting that AMPK may be mediating YAP/TAZ inhibition via LATS1/2. In the carbon tetrachloride mouse model of fibrosis, pharmacologic activation of AMPK in HSCs inhibits YAP/TAZ signaling and reduces fibrosis. The findings implicate AMPK as a critical regulator of YAP/TAZ signaling and HSC inactivation and highlight AMPK activation as a therapeutic target for the treatment of hepatic fibrosis.

Eosinophilic gastritis in adult complicated by gastric ulcer perforation.

Eosinophilic gastritis is a rare type of eosinophilic gastrointestinal diseases. Patients with eosinophilic gastritis usually present with symptoms such as nausea, emesis, abdominal pain, and weight loss. In severe cases, patients can suffer rare complications such as gastric outlet obstruction and spontaneous perforation. Here, we present the case of a young adult male who presented with acute onset abdominal pain for 1 day. The patient was found to have significant mural thickening of gastric antrum with pneumoperitoneum on abdominal CT scan, consistent with a perforated gastric ulcer. The patient underwent exploratory laparotomy and required modified graham patch repair. The diagnosis of eosinophilic gastritis was made based on the pathology review of intraoperative endoscopic biopsy specimens.

Problematic alcohol use and its impact on liver disease quality of life in a multicenter study of patients with cirrhosis.

BACKGROUND: Management of cirrhosis is challenging and has been complicated by the COVID-19 pandemic due to decreased access to care, increased psychological distress, and alcohol misuse. Recently, The National Institute on Alcohol Abuse and Alcoholism has broadened the definition of recovery from alcohol use disorder to include quality of life (QoL) as an indicator of recovery. This study examined the associations of alcohol-associated cirrhosis etiology and problematic drinking with liver disease QoL (LDQoL). METHODS: Patients with cirrhosis (N=329) were recruited from 3 sites (63% from 2 Veterans Affairs Health Care Systems and 37% from 1 safety net hospital) serving populations that are economically or socially marginalized. Cirrhosis etiology was ascertained by chart review of medical records. Problematic drinking was defined by ≥8 on the Alcohol Use Disorders Identification Test. Multivariable general linear modeling adjusting for age, sex, race/ethnicity, site, pandemic-related stress, and history of anxiety/depressive disorder were conducted. Sensitivity analyses further adjusted for indicators of liver disease severity. RESULTS: Participants were on average 64.6 years old, 17% female, 58% non-White, 44% with alcohol-associated cirrhosis, and 17% with problematic drinking. Problematic drinking was significantly associated with worse LDQoL scores in the overall scale and in the memory/concentration and health distress subscales. These associations remained significant after adjusting for indicators of liver disease severity, including Model for End-Stage Liver Disease-Sodium score and decompensated cirrhosis status. CONCLUSIONS: Among patients with cirrhosis, problematic drinking was associated with worse LDQoL, especially in the domains of memory/concentration and health distress. Assessment and awareness of cognitive deficits and negative emotionality within the context of cirrhosis and problematic drinking may help clinicians provide better integrated care for this population.

Telehepatology Satisfaction Is Associated with Ethnicity: The Real-World Experience of a Vulnerable Population with Fatty Liver Disease.

BACKGROUND: Since the coronavirus disease 2019 (COVID-19) pandemic began, telemedicine use has transformed healthcare delivery. Yet there is concern that telemedicine may widen care disparities for vulnerable populations, and patient experience data are limited. AIMS: We aimed to assess patient satisfaction with hepatology-related telemedicine (telehepatology) for delivery of fatty liver disease (FLD) care in a safety-net healthcare system. METHODS: Adult patients with FLD were surveyed regarding satisfaction with telehepatology. Clinical, demographic, resources, and social determinants of health (SDoH) data were collected to identify factors associated with satisfaction through multivariable modeling. RESULTS: From June 2020 to March 2022, 220 participants were enrolled: the median age was 52 years, 37% were men, and 68% were Hispanic. One hundred nineteen (54%) had prior telehepatology experience. Overall, satisfaction was high; 70% reported being somewhat or very satisfied. On univariate analysis, Hispanic ethnicity (versus non-Hispanic, OR 0.34, 95% CI 0.1-0.9, p = 0.03) and limited access to personal cellphone/internet (OR 0.16, 95% CI 0.04-0.6, p = 0.01) were associated with lower satisfaction. On multivariable logistic regression modeling adjusted for pandemic duration, age, sex, severity of liver disease, and coexisting liver disease, Hispanic ethnicity and lack of personal cellphone/internet remained independently associated with lower telehepatology satisfaction (OR 0.24, 95% CI 0.07-0.9, p = 0.03 and OR 0.2, 95% CI 0.04-0.9, p = 0.04, respectively). The association remained statistically significant after inclusion of various SDoH in the multivariable model. CONCLUSIONS: Satisfaction with telehepatology among FLD patients in a safety-net clinical setting was high overall. However, Hispanic ethnicity and lack of personal cellphone/internet were independently associated with lower telehepatology satisfaction. A better understanding of patients' experience with telehepatology is needed to identify reasons for dissatisfaction, and in-person visits should remain an option for patients to ensure equitable care.

Integrins in biliary injury and fibrosis.

PURPOSE OF REVIEW: Current treatment options for cholangiopathies are severely limited and there is thus a critical need to identify and develop therapies. This review discusses the role of integrins in biliary injury and fibrosis and their potential as therapeutic targets. RECENT FINDINGS: There are a diverse set of roles that integrins play in biliary injury and fibrosis. Some integrins activate TGF-ß signaling or are involved in sensing of the extracellular matrix, making them attractive targets for biliary fibrosis. In recent work, autoantibodies to α v ß 6 were identified in patients with PSC, supporting the relevance of this integrin in the disease. In addition, a role for α 2 ß 1 in cyst formation was identified in a mouse model of polycystic liver disease. Leukocyte integrins (e.g. α E ß 7 and α 4 ß 7 ) contribute to lymphocyte trafficking, making them potential targets for biliary inflammation; however, this has not yet translated to the clinic. SUMMARY: While all members of the same family of proteins, integrins have diverse roles in the pathogenesis of biliary disease. Targeting one or multiple of these integrins may slow or halt the progression of biliary injury and fibrosis by simultaneously impacting different pathologic cells and processes.

Cellular stress in the pathogenesis of nonalcoholic steatohepatitis and liver fibrosis.

The burden of chronic liver disease is rising substantially worldwide. Fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the common pathway leading to cirrhosis, and limited treatment options are available. There is increasing evidence suggesting the role of cellular stress responses contributing to fibrogenesis. This Review provides an overview of studies that analyse the role of cellular stress in different cell types involved in fibrogenesis, including hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells and macrophages.

Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in hepatic stellate cells.

Background & Aims: Fibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. Methods: We identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analyses of HSCs isolated from mice defined the murine ortholog of TILAM . We then generated Tilam -deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl 4 ) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM . Results: TILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in HSCs during the development of fibrosis in vivo . In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl 4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. Conclusion: TILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease.

Targeting acid ceramidase ameliorates fibrosis in mouse models of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is a common cause of liver disease worldwide, and is characterized by the accumulation of fat in the liver. Non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD, is a leading cause of liver transplantation. Fibrosis is the histologic feature most associated with liver-related morbidity and mortality in patients with NASH, and treatment options remain limited. In previous studies, we discovered that acid ceramidase (aCDase) is a potent antifibrotic target using human hepatic stellate cells (HSCs) and models of hepatic fibrogenesis. Using two dietary mouse models, we demonstrate that depletion of aCDase in HSC reduces fibrosis without worsening metabolic features of NASH, including steatosis, inflammation, and insulin resistance. Consistently, pharmacologic inhibition of aCDase ameliorates fibrosis but does not alter metabolic parameters. The findings suggest that targeting aCDase is a viable therapeutic option to reduce fibrosis in patients with NASH.

Controlled fabrication of functional liver spheroids with microfluidic flow cytometric printing.

Multicellular liver spheroids are 3D culture models useful in the development of therapies for liver fibrosis. While these models can recapitulate fibrotic disease, current methods for generating them via random aggregation are uncontrolled, yielding spheroids of variable size, function, and utility. Here, we report fabrication of precision liver spheroids with microfluidic flow cytometric printing. Our approach fabricates spheroids cell-by-cell, yielding structures with exact numbers of different cell types. Because spheroid function depends on composition, our precision spheroids have superior functional uniformity, allowing more accurate and statistically significant screens compared to randomly generated spheroids. The approach produces thousands of spheroids per hour, and thus affords a scalable platform by which to manufacture single-cell precision spheroids for disease modeling and high throughput drug testing.

Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells.

Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.

Stress testing reveals gaps in clinic readiness of image-based diagnostic artificial intelligence models.

Artificial intelligence models match or exceed dermatologists in melanoma image classification. Less is known about their robustness against real-world variations, and clinicians may incorrectly assume that a model with an acceptable area under the receiver operating characteristic curve or related performance metric is ready for clinical use. Here, we systematically assessed the performance of dermatologist-level convolutional neural networks (CNNs) on real-world non-curated images by applying computational "stress tests". Our goal was to create a proxy environment in which to comprehensively test the generalizability of off-the-shelf CNNs developed without training or evaluation protocols specific to individual clinics. We found inconsistent predictions on images captured repeatedly in the same setting or subjected to simple transformations (e.g., rotation). Such transformations resulted in false positive or negative predictions for 6.5-22% of skin lesions across test datasets. Our findings indicate that models meeting conventionally reported metrics need further validation with computational stress tests to assess clinic readiness.

Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.

Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein ß-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.

Brief Report: High Rates of Adverse Birth Outcomes in HIV and Syphilis Coinfected Women in Botswana.

BACKGROUND: Little is known about the combined impact of HIV/syphilis coinfection on birth outcomes. METHODS: Antenatal HIV and syphilis test results, obstetric history, and infant birth outcomes were collected from obstetric records in maternity wards in Botswana between 2008 and 2011 (5 sites) and 2014 and 2016 (8 sites). We used logistic regression to compare adverse birth outcomes by HIV and syphilis status. Outcomes included stillbirth, preterm delivery, low birth weight, and in-hospital neonatal death. RESULTS: Of 76,466 women, 75,770 (99.1%) had HIV test results, and 20,520 (27.1%) were HIV positive. Syphilis test results were available for 67,290 (88.0%), and 697 (1.0%) had reactive rapid plasma reagin. Among 692 women with syphilis and an HIV test result, 261 (37.7%) were coinfected. HIV-infected women were more likely to be infected with syphilis than HIV-uninfected women [odds ratio (OR) = 1.68; 95% confidence interval (CI): 1.44 to 1.96]. From 2008-2011 to 2014-2016, the proportion of women with syphilis remained constant (1.1% vs. 1.0%, P = 0.41), but HIV/syphilis coinfection declined from 45% to 27% (P < 0.0001). Stillbirth occurred in 5.8% of coinfected women, compared with 1.9% with no HIV/syphilis (OR = 3.09; 95% CI: 1.83 to 5.23); 3.4% with HIV alone (OR = 1.75; 95% CI: 1.03 to 2.97), or 3.7% with syphilis alone (OR = 1.58; 95% CI: 0.77 to 3.25). Low birth weight occurred in 24.1% of coinfected women, compared with 12.1% with no HIV/syphilis (OR 2.31; 95% CI: 1.74 to 3.08; 20% with HIV alone (OR = 1.27; 95% CI: 0.96 to 1.69); or 14.6% with syphilis alone (OR = 1.85; 95% CI: 1.26 to 2.74). CONCLUSIONS: Although HIV/syphilis coinfection in pregnancy has declined in the past decade, coinfection was associated with adverse birth outcomes.

Examining the feasibility of a "top-down" approach to enhancing the keratinocyte-implant adhesion.

The adhesion of human epidermal keratinocytes to the implant surface is one of the most critical steps during the patient's recovery from implantation of transcutaneous prosthesis. To improve the success rate of transcutaneous prosthetic implants, we explored a new "top-down" approach to promoting this dynamic adhering process through modulation of upstream cell signaling pathways. To examine the feasibility of this novel approach, we first established an in vitro platform that is capable of providing a non-invasive, real-time, quantitative characterization of the keratinocyte-implant interaction. This platform is based on the dissipation monitoring function of the quartz crystal microbalance with dissipation monitoring (QCM-D) in conjunction with the open-module setup of the QCM-D. We then employed this platform to assess the effects of various pathways-specific modulators on the adhering process of keratinocytes. We demonstrated that this "top-down" approach is as effective in enhancing the adhesion of keratinocytes as the conventional "bottom-up" approach that relies on modifying the substrate surface with the adhesion protein such as fibronectin. We envision that this new "top-down" approach combined with the QCM-D-based in vitro platform will help facilitate the future development of new therapies for enhancing osseointegration and promoting wound healing.

On the Measurement of Energy Dissipation of Adhered Cells with the Quartz Microbalance with Dissipation Monitoring.

We previously reported the finding of a linear correlation between the change of energy dissipation (Δ D) of adhered cells measured with the quartz crystal microbalance with dissipation monitoring (QCM-D) and the level of focal adhesions of the cells. To account for this correlation, we have developed a theoretical framework for assessing the Δ D-response of adhered cells. We rationalized that the mechanical energy of an oscillating QCM-D sensor coupled with a cell monolayer is dissipated through three main processes: the interfacial friction through the dynamic restructuring (formation and rupture) of cell-extracellular matrix (ECM) bonds, the interfacial viscous damping by the liquid trapped between the QCM-D sensor and the basal membrane of the cell layer, and the intracellular viscous damping through the viscous slip between the cytoplasm and stress fibers as well as among stress fibers themselves. Our modeling study shows that the interfacial viscous damping by the trapped liquid is the primary process for energy dissipation during the early stage of the cell adhesion, whereas the dynamic restructuring of cell-ECM bonds becomes more prevalent during the later stage of the cell adhesion. Our modeling study also establishes a positive linear correlation between the Δ D-response and the level of cell adhesion quantified with the number of cell-ECM bonds, which corroborates our previous experimental finding. This correlation with a wide well-defined linear dynamic range provides a much needed theoretical validation of the dissipation monitoring function of the QCM-D as a powerful quantitative analytical tool for cell study.

Quartz crystal microbalance: Sensing cell-substrate adhesion and beyond.

Cell adhesion is an essential aspect of cellular behavior. Finding innovative methods to probe the adhesion of cells in their native state can greatly advance the understanding of control and regulation of cellular behavior and their impact on human health. The quartz crystal microbalance (QCM) is a label-free, biosensing system that has, in the past fifty years, evolved from a simple acoustic based mass sensor to a powerful bioanalytical tool. Its unique capability of monitoring the cell-substrate interaction non-invasively in real time has led to the emergence of its applications in areas that are relevant to fundamental cell biology and medical research. This review is intended to provide readers an overview of the use of the QCM for examination of cell-substrate adhesion. It also describes how this innovative approach can be extended to the study of other aspects of cellular behavior, such as cell morphology, cell mechanics, cell motility, cell signaling, all of which can potentially be applied to medical diagnosis and/or pharmaceutical development. In this review a major emphasis is placed on informing readers about some of the most important practical aspects of the QCM-based cell study including data acquisition and analysis, the substrate surface manipulation, and cell manipulation.

Emulating a target trial of antiretroviral therapy regimens started before conception and risk of adverse birth outcomes.

OBJECTIVE: To compare the effect of preconception initiation of zidovudine, lamivudine, nevirapine (ZDV/3TC/NVP) versus tenofovir, emtricitabine, efavirenz (TDF/FTC/EFV) on adverse birth outcomes. DESIGN: Emulation of a hypothetical (target) trial using a birth surveillance study in Botswana during an era of CD4-based antiretroviral therapy (ART) initiation. METHODS: In women who initiated ART less than 3 years from HIV diagnosis, conceived 0.5-5 years after ART initiation, and delivered at least 24-week gestation, we estimated risk ratios for stillbirth, preterm delivery (<37 weeks), very preterm delivery (<32 weeks), small-for-gestational-age (SGA) (<10 percentile), very SGA (<3 percentile), and any adverse or severe birth outcome for first-line ZDV/3TC/NVP versus TDF/FTC/EFV. We conducted a historical comparison in women who initiated TDF/FTC/EFV in 2012-2015 and ZDV/3TC/NVP in 2004-2011, and a contemporaneous comparison in an era of overlapping use from 2009 to 2013. RESULTS: In the historical comparison, 1108 women initiated TDF/FTC/EFV and 637 initiated ZDV/3TC/NVP. In the contemporaneous comparison, 1052 initiated TDF/FTC/EFV and 298 initiated ZDV/3TC/NVP. TDF/FTC/EFV initiators were younger and more likely to be nulliparous than ZDV/3TC/NVP initiators in both comparisons. In the historical comparison, the adjusted risk ratios (95% confidence interval) comparing ZDV/3TC/NVP with TDF/FTC/EFV were 2.95 (1.76, 4.96) for stillbirth, 1.40 (1.17, 1.67) for preterm delivery, 2.58 (1.70, 3.91) for very preterm delivery, 1.96 (1.64, 2.34) for SGA, 2.32 (1.73, 3.09) for very SGA, 1.54 (1.38, 1.72) for any adverse birth outcome, and 2.20 (1.76, 2.75) for any severe birth outcome, and were similar in the contemporaneous comparison. CONCLUSION: Preconception initiation of ZDV/3TC/NVP compared with TDF/FTC/EFV may increase the risk of adverse birth outcomes.

Cardiovascular pressor effects of orexins in the dorsomedial hypothalamus.

Orexins are important regulators of cardiovascular functions in various physiological and pathological conditions. The dorsomedial hypothalamus (DMH), an essential mediator of cardiovascular responses to stress, contains dense orexinergic innervations and receptors. We examined whether orexins can regulate cardiovascular functions through their actions in the DMH in anesthetized rats. An intra-DMH injection of orexin A (30pmol) produced elevation of arterial pressure and heart rate. Orexin A-sensitive sites were located within or immediately adjacent to the DMH and larger responses were induced at the compact part of the dorsomedial hypothalamic nucleus. Orexin A-induced responses were attenuated by intra-DMH pretreatment with an orexin receptor 1 (OX1R) antagonist, SB-334867 (15nmol) (17.7 ± 2.8 vs. 5.2 ± 1.0mmHg; 54.6 ± 10.0 vs. 22.8 ± 7.4 beats/min). Intra-DMH applied [Ala11,D-Leu15]-orexin B (300 pmol), an orexin receptor 2 (OX2R) agonist, elicited cardiovascular responses mimicking the responses of orexin A, except for a smaller pressor response (7.4 ± 1.7 vs. 16.4 ± 1.8mmHg). In a series of experiment, effects of orexin B (100pmol) and then orexin A (30pmol), were examined at a same site. Two patterns of responses were observed in 12 intra-DMH sites: (1) both orexin A and B (9 sites), and (2) only orexin A (3 sites) induced cardiovascular responses, respectively suggesting OX1R/OX2R-mediated and OX1R-predominant mechanisms. In conclusion, orexins regulated cardiovascular functions through OX1R/OX2R- or OX1R-mediated mechanisms at different locations in the DMH.

Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells.

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.