Disparities in Visual Field Testing Frequency Among Subjects With Glaucoma.

Purpose: Prior evidence suggests racial disparities in the utilization of visual field testing (VFT) for the diagnosis and monitoring of glaucoma. In this study, we considered the effect of baseline glaucoma severity and socioeconomic disadvantage along with other potential confounders such as test reliability, ancillary tests, and glaucoma surgeries on racial disparity in the frequency of VFT. Methods: The records of all subjects with a diagnosis of glaucoma who received VFT at an academic, tertiary care facility from January 2018 to December 2021 were accessed. Analysis was performed to compare VFT frequency, the total number of office visits (DoS), and the ratio of VFT frequency to DoS (VFT/DoS) across self-reported races while controlling for sex, age, socioeconomic disadvantage (Area Deprivation Index), VF reliability indicators and baseline mean deviation, optical coherence tomography frequency, and glaucoma surgeries. Results: Among the 2654 subjects (1515 White, 782 Black, and 357 Asian) included in this study, Black subjects had the worst socioeconomic status and disease severity at baseline. They also experienced a 3% lower VFT/DoS ratio compared to White subjects (P = 0.031). Asian subjects had a 5% lower VFT/DoS ratio compared to White subjects (P = 0.015). Discussion: We identified racial disparity in performing VFT in subjects with glaucoma even when multiple confounders were considered. Further investigation is necessary to identify other race-associated factors to work toward reducing racial disparities in VFT. Translational Relevance: Black and Asian subjects with glaucoma receive fewer VFT per visit compared to White subjects even when considering socioeconomic disadvantage and disease severity.

Investigating the Utility of Near-Infrared Reflectance Imaging for Diabetic Retinopathy Screening.

BACKGROUND AND OBJECTIVE: We investigated the reliability of near-infrared reflectance (NIR) imaging as a method of assessing severity of diabetic retinopathy (DR). PATIENTS AND METHODS: One hundred ninety-five NIR images were reviewed by two graders for the number of hyporeflective foci, presence or absence of vascular abnormalities, and presumptive DR stage; these were correlated to fundus photography-defined DR stage. Interrater reliability was confirmed via one-way random effects model of intraclass correlation coefficients. Analysis of variance was used in subgroup analysis, receiver operating characteristic (ROC) curves were created to validate reliability of the model, and logistic regression was used to model foci and vascular abnormalities as predictors for moderate or worse disease. RESULTS: A statistically significant difference in mean number of hyporeflective foci was found between no DR and moderate non-proliferative DR (NPDR; P < 0.0001), no DR and severe NPDR (P < 0.001), no DR and proliferative DR (PDR; P < 0.0001), mild and moderate NPDR (P = 0.008), mild and severe NPDR (P < 0.001), and mild NPDR and PDR (P < 0.001). The area under the ROC curve was 0.849 (CI: 0.792 to 0.905). The threshold for detection of moderate NPDR or worse was 4.75 foci, with a sensitivity of 79.0% and a false positive rate of 20.0%. Multivariate logistic regression model incorporating hyporeflective foci with vascular abnormalities (odds ratio [OR] = 1.592, 95% CI: 1.381 to 1.835; P < 0.001) was able to accurately predict moderate disease or worse, just moderate disease (OR = 1.045, 95% CI: 1.003 to 1.089; P = 0.035), severe disease (OR = 1.050, 95% CI: 1.006 to 1.096; P = 0.027), and proliferative disease (OR = 1.050, 95% CI: 1.008 to 1.095; P = 0.018). CONCLUSIONS: NIR imaging may be an adjunct tool in screening for DR. [Ophthalmic Surg Lasers Imaging Retina 2024;55:318-325.].

Peripapillary Atrophy Area as an Indicator of Glaucomatous Structural and Functional Progression.

Purpose: To determine whether peripapillary atrophy (PPA) area is an indicator of glaucomatous structural and functional damage and progression. Methods: In this retrospective longitudinal analysis from ongoing prospective study we qualified 71 eyes (50 subjects) with glaucoma. All subjects had a comprehensive ophthalmic examination, visual field (VF), and spectral-domain optical coherence tomography (OCT) testing in at least three visits. PPA was manually delineated on en face OCT optic nerve head scans, while observing the corresponding cross-sectional images, as the hyper-reflective area contiguous with the optic disc. Results: The mean follow-up duration was 4.4 ± 1.4 years with an average of 6.8 ± 2.2 visits. At baseline, PPA area was significantly associated only with VF's mean deviation (MD; P = 0.041), visual field index (VFI; P = 0.041), superior ganglion cell inner plexiform layer (GCIPL; P = 0.011), and disc area (P = 0.011). Longitudinally, PPA area was negatively and significantly associated with MD (P = 0.015), VFI (P = 0.035), GCIPL (P = 0.009), superior GCIPL (P = 0.034), and disc area (P = 0.007, positive association). Conclusions: Longitudinal change in PPA area is an indicator of glaucomatous structural and functional progression but PPA area at baseline cannot predict future progression. Translational Relevance: Longitudinal changes in peripapillary atrophy area measured by OCT can be an indicator of structural and functional glaucoma progression.

LIMBARE: An Advanced Linear Mixed-Effects Breakpoint Analysis With Robust Estimation Method With Applications to Longitudinal Ophthalmic Studies.

Purpose: Broken stick analysis is a widely used approach for detecting unknown breakpoints where the association between measurements is nonlinear. We propose LIMBARE, an advanced linear mixed-effects breakpoint analysis with robust estimation, especially designed for longitudinal ophthalmic studies. LIMBARE accommodates repeated measurements from both eyes and over time, and it effectively addresses the presence of outliers. Methods: The model setup of LIMBARE and the computing algorithm for point and confidence interval estimates of the breakpoint were introduced. The performance of LIMBARE and other competing methods was assessed via comprehensive simulation studies and application to a longitudinal ophthalmic study with 216 eyes (145 subjects) followed for an average of 3.7 ± 1.3 years to examine the longitudinal association between structural and functional measurements. Results: In simulation studies, LIMBARE showed the smallest bias and mean squared error for estimating the breakpoint, with an empirical coverage probability of corresponding confidence interval estimates closest to the nominal level for scenarios with and without outlier data points. In the application to the longitudinal ophthalmic study, LIMBARE detected two breakpoints between visual field mean deviation (MD) and retinal nerve fiber layer thickness and one breakpoint between MD and cup-to-disc ratio, whereas the cross-sectional analysis approach detected only one and none, respectively. Conclusions: LIMBARE enhances breakpoint estimation accuracy in longitudinal ophthalmic studies, and the cross-sectional analysis approach is not recommended for future studies. Translational Relevance: Our proposed method and companion R package provide a valuable computational tool for advancing longitudinal ophthalmology research and exploring the association relationships among ophthalmic variables.

Reducing Ophthalmic Health Disparities Through Transfer Learning: A Novel Application to Overcome Data Inequality.

Purpose: Race disparities in the healthcare system and the resulting inequality in clinical data among different races hinder the ability to generate equitable prediction results. This study aims to reduce healthcare disparities arising from data imbalance by leveraging advanced transfer learning (TL) methods. Method: We examined the ophthalmic healthcare disparities at a population level using electronic medical records data from a study cohort (N = 785) receiving care at an academic institute. Regression-based TL models were usesd, transferring valuable information from the dominant racial group (White) to improve visual field mean deviation (MD) rate of change prediction particularly for data-disadvantaged African American (AA) and Asian racial groups. Prediction results of TL models were compared with two conventional approaches. Results: Disparities in socioeconomic status and baseline disease severity were observed among the AA and Asian racial groups. The TL approach achieved marked to comparable improvement in prediction accuracy compared to the two conventional approaches as evident by smaller mean absolute errors or mean square errors. TL identified distinct key features of visual field MD rate of change for each racial group. Conclusions: The study introduces a novel application of TL that improved reliability of the analysis in comparison with conventional methods, especially in small sample size groups. This can improve assessment of healthcare disparity and subsequent remedy approach. Translational Relevance: TL offers an equitable and efficient approach to mitigate healthcare disparities analysis by enhancing prediction performance for data-disadvantaged group.

Tumor-Reactive CD8+ T Cells Enter a TCF1+PD-1- Dysfunctional State.

T cells recognize several types of antigens in tumors, including aberrantly expressed, nonmutated proteins, which are therefore shared with normal tissue and referred to as self/shared-antigens (SSA), and mutated proteins or oncogenic viral proteins, which are referred to as tumor-specific antigens (TSA). Immunotherapies such as immune checkpoint blockade (ICB) can activate T-cell responses against TSA, leading to tumor control, and also against SSA, causing immune-related adverse events (irAE). To improve anti-TSA immunity while limiting anti-SSA autoreactivity, we need to understand how tumor-specific CD8+ T cells (TST) and SSA-specific CD8+ T (SST) cells differentiate in response to cognate antigens during tumorigenesis. Therefore, we developed a genetic cancer mouse model in which we can track TST and SST differentiation longitudinally as liver cancers develop. We found that both TST and SST lost effector function over time, but while TST persisted long term and had a dysfunctional/exhausted phenotype (including expression of PD1, CD39, and TOX), SST exited cell cycle prematurely and disappeared from liver lesions. However, SST persisted in spleens in a dysfunctional TCF1+PD-1- state: unable to produce effector cytokines or proliferate in response to ICB targeting PD-1 or PD-L1. Thus, our studies identify a dysfunctional T-cell state occupied by T cells reactive to SSA: a TCF1+PD-1- state lacking in effector function, demonstrating that the type/specificity of tumor antigen may determine tumor-reactive T-cell differentiation.

Hepatic stellate cells maintain liver homeostasis through paracrine neurotrophin-3 signaling that induces hepatocyte proliferation.

Organ size is maintained by the controlled proliferation of distinct cell populations. In the mouse liver, hepatocytes in the midlobular zone that are positive for cyclin D1 (CCND1) repopulate the parenchyma at a constant rate to preserve liver mass. Here, we investigated how hepatocyte proliferation is supported by hepatic stellate cells (HSCs), pericytes that are in close proximity to hepatocytes. We used T cells to ablate nearly all HSCs in the murine liver, enabling the unbiased characterization of HSC functions. In the normal liver, complete loss of HSCs persisted for up to 10 weeks and caused a gradual reduction in liver mass and in the number of CCND1+ hepatocytes. We identified neurotrophin-3 (Ntf-3) as an HSC-produced factor that induced the proliferation of midlobular hepatocytes through the activation of tropomyosin receptor kinase B (TrkB). Treating HSC-depleted mice with Ntf-3 restored CCND1+ hepatocytes in the midlobular region and increased liver mass. These findings establish that HSCs form the mitogenic niche for midlobular hepatocytes and identify Ntf-3 as a hepatocyte growth factor.

Automated 360-degree goniophotography with the NIDEK Gonioscope GS-1 for glaucoma.

This study was registered with ClinicalTrials.gov (ID: NCT03715231). A total of 20 participants (37 eyes) who were 18 or older and had glaucoma or were glaucoma suspects were enrolled from the NYU Langone Eye Center and Bellevue Hospital. During their usual ophthalmology visit, they were consented for the study and underwent 360-degree goniophotography using the NIDEK Gonioscope GS-1. Afterwards, the three ophthalmologists separately examined the images obtained and determined the status of the iridocorneal angle in four quadrants using the Shaffer grading system. Physicians were masked to patient names and diagnoses. Inter-observer reproducibility was determined using Fleiss' kappa statistics. The interobserver reliability using Fleiss' statistics was shown to be significant between three glaucoma specialists with fair overall agreement (Fleiss' kappa: 0.266, p < .0001) in the interpretation of 360-degree goniophotos. Automated 360-degree goniophotography using the NIDEK Gonioscope GS-1 have quality such that they are interpreted similarly by independent expert observers. This indicates that angle investigation may be performed using this automated device and that interpretation by expert observers is likely to be similar. Images produced from automated 360-degree goniophotography using the NIDEK Gonioscope GS-1 are similarly interpreted amongst glaucoma specialists, thus supporting use of this technique to document and assess the anterior chamber angle in patients with, or suspected of, glaucoma and iridocorneal angle abnormalities.

LIMBARE: an Advanced Linear Mixed-effects Breakpoint Analysis with Robust Estimation Method with Applications to Longitudinal Ophthalmic Studies.

Purpose: Broken stick analysis is a widely used approach for detecting unknown breakpoints where association between measurements is non-linear. We propose LIMBARE, an advanced li near m ixed-effects b reakpoint a nalysis with r obust e stimation, especially designed for longitudinal ophthalmic studies. LIMBARE accommodates repeated measurements from both eyes and overtime, and effectively address the presence of outliers. Methods: The model setup of LIMBARE and computing algorithm for point and confidence interval estimates of the breakpoint was introduced. The performance of LIMBARE and other competing methods was assessed via comprehensive simulation studies and application to a longitudinal ophthalmic study with 216 eyes (145 subjects) followed for an average of 3.7±1.3 years to examine the longitudinal association between structural and functional measurements. Results: In simulation studies, LIMBARE showed the smallest bias and mean squared error (MSE) for estimating the breakpoint, with empirical coverage probability of corresponding CI estimate closest to the nominal level for scenarios with and without outlier data points. In the application to the longitudinal ophthalmic study, LIMBARE detected two breakpoints between visual field mean deviation (MD) and retinal nerve fiber layer thickness (RNFL) and one breakpoint between MD and cup to disc ratio (CDR), while the cross-sectional analysis approach only detected one and none, respectively. Conclusions: LIMBARE enhances breakpoint estimation accuracy in longitudinal ophthalmic studies, while cross-sectional analysis approach is not recommended for future studies. Translational Relevance: Our proposed method and companion software R package provides a valuable computational tool for advancing longitudinal ophthalmology research and exploring the association relationships between ophthalmic variables.

Evaluating the Mediating Role of Recall of Intervention Knowledge in the Relationship Between a Peer-Driven Intervention and HIV Risk Behaviors Among People Who Inject Drugs.

Peer-driven interventions can be effective in reducing HIV injection risk behaviors among people who inject drugs (PWID). We employed a causal mediation framework to examine the mediating role of recall of intervention knowledge in the relationship between a peer-driven intervention and subsequent self-reported HIV injection-related risk behavior among PWID in the HIV Prevention Trials Network (HPTN) 037 study. For each intervention network, the index participant received training at baseline to become a peer educator, while non-index participants and all participants in the control networks received only HIV testing and counseling; recall of intervention knowledge was measured at the 6-month visit for each participant, and each participant was followed to ascertain HIV injection-related risk behaviors at the 12-month visit. We used inverse probability weighting to fit marginal structural models to estimate the total effect (TE) and controlled direct effect (CDE) of the intervention on the outcome. The proportion eliminated (PE) by intervening to remove mediation by the recall of intervention knowledge was computed. There were 385 participants (47% in intervention networks) included in the analysis. The TE and CDE risk ratios for the intervention were 0.47 [95% confidence interval (CI): 0.28, 0.78] and 0.73 (95% CI: 0.26, 2.06) and the PE was 49%. Compared to participants in the control networks, the peer-driven intervention reduced the risk of HIV injection-related risk behavior by 53%. The mediating role of recall of intervention knowledge accounted for less than 50% of the total effect of the intervention, suggesting that other potential causal pathways between the intervention and the outcome, such as motivation and skill, self-efficacy, social norms and behavior modeling, should be considered in future studies.

Estimating Causal Effects of HIV Prevention Interventions with Interference in Network-based Studies among People Who Inject Drugs.

Evaluating causal effects in the presence of interference is challenging in network-based studies of hard-to-reach populations. Like many such populations, people who inject drugs (PWID) are embedded in social networks and often exert influence on others in their network. In our setting, the study design is observational with a non-randomized network-based HIV prevention intervention. Information is available on each participant and their connections that confer possible HIV risk through injection and sexual behaviors. We considered two inverse probability weighted (IPW) estimators to quantify the population-level spillover effects of non-randomized interventions on subsequent health outcomes. We demonstrated that these two IPW estimators are consistent, asymptotically normal, and derived a closed-form estimator for the asymptotic variance, while allowing for overlapping interference sets (groups of individuals in which the interference is assumed possible). A simulation study was conducted to evaluate the finite-sample performance of the estimators. We analyzed data from the Transmission Reduction Intervention Project, which ascertained a network of PWID and their contacts in Athens, Greece, from 2013 to 2015. We evaluated the effects of community alerts on subsequent HIV risk behavior in this observed network, where the connections or links between participants were defined by using substances or having unprotected sex together. In the study, community alerts were distributed to inform people of recent HIV infections among individuals in close proximity in the observed network. The estimates of the risk differences for spillover using either IPW estimator demonstrated a protective effect. The results suggest that HIV risk behavior could be mitigated by exposure to a community alert when an increased risk of HIV is detected in the network.

A maximum-type microbial differential abundance test with application to high-dimensional microbiome data analyses.

Background: High-throughput metagenomic sequencing technologies have shown prominent advantages over traditional pathogen detection methods, bringing great potential in clinical pathogen diagnosis and treatment of infectious diseases. Nevertheless, how to accurately detect the difference in microbiome profiles between treatment or disease conditions remains computationally challenging. Results: In this study, we propose a novel test for identifying the difference between two high-dimensional microbiome abundance data matrices based on the centered log-ratio transformation of the microbiome compositions. The test p-value can be calculated directly with a closed-form solution from the derived asymptotic null distribution. We also investigate the asymptotic statistical power against sparse alternatives that are typically encountered in microbiome studies. The proposed test is maximum-type equal-covariance-assumption-free (MECAF), making it widely applicable to studies that compare microbiome compositions between conditions. Our simulation studies demonstrated that the proposed MECAF test achieves more desirable power than competing methods while having the type I error rate well controlled under various scenarios. The usefulness of the proposed test is further illustrated with two real microbiome data analyses. The source code of the proposed method is freely available at https://github.com/Jiyuan-NYU-Langone/MECAF. Conclusions: MECAF is a flexible differential abundance test and achieves statistical efficiency in analyzing high-throughput microbiome data. The proposed new method will allow us to efficiently discover shifts in microbiome abundances between disease and treatment conditions, broadening our understanding of the disease and ultimately improving clinical diagnosis and treatment.

Targeting cancer stemness mediated by BMI1 and MCL1 for non-small cell lung cancer treatment.

Lung cancer is the leading cause of cancer-associated death, with a global 5-year survival rate <20%. Early metastasis and recurrence remain major challenges for lung cancer treatment. The stemness property of cancer cells has been suggested to play a key role in cancer plasticity, metastasis and drug-resistance, and is a potential target for drug development. In this study, we found that in non-small cell lung cancer (NSCLC), BMI1 and MCL1 play crucial roles of cancer stemness including invasion, chemo-resistance and tumour initiation. JNK signalling serves as a link between oncogenic pathway or genotoxicity to cancer stemness. The activation of JNK, either by mutant EGFR or chemotherapy agent, stabilized BMI1 and MCL1 proteins through suppressing the expression of E3-ubiquitin ligase HUWE1. In lung cancer patient samples, high level of BMI1 is correlated with poor survival, and the expression of BMI1 is positively correlated with MCL1. A novel small-molecule, BI-44, was developed, which effectively suppressed BMI1/MCL1 expressions and inhibited tumour formation and progression in preclinical models. Targeting cancer stemness mediated by BMI1/MCL1 with BI-44 provides the basis for a new therapeutic approach in NSCLC treatment.

Microstructural Deformations Within the Depth of the Lamina Cribrosa in Response to Acute In Vivo Intraocular Pressure Modulation.

Purpose: The lamina cribrosa (LC) is a leading target for initial glaucomatous damage. We investigated the in vivo microstructural deformation within the LC volume in response to acute IOP modulation while maintaining fixed intracranial pressure (ICP). Methods: In vivo optic nerve head (ONH) spectral-domain optical coherence tomography (OCT) scans (Leica, Chicago, IL, USA) were obtained from eight eyes of healthy adult rhesus macaques (7 animals; ages = 7.9-14.4 years) in different IOP settings and fixed ICP (8-12 mm Hg). IOP and ICP were controlled by cannulation of the anterior chamber and the lateral ventricle of the brain, respectively, connected to a gravity-controlled reservoir. ONH images were acquired at baseline IOP, 30 mm Hg (H1-IOP), and 40 to 50 mm Hg (H2-IOP). Scans were registered in 3D, and LC microstructure measurements were obtained from shared regions and depths. Results: Only half of the eyes exhibited LC beam-to-pore ratio (BPR) and microstructure deformations. The maximal BPR change location within the LC volume varied between eyes. BPR deformer eyes had a significantly higher baseline connective tissue volume fraction (CTVF) and lower pore aspect ratio (P = 0.03 and P = 0.04, respectively) compared to BPR non-deformer. In all eyes, the magnitude of BPR changes in the anterior surface was significantly different (either larger or smaller) from the maximal change within the LC (H1-IOP: P = 0.02 and H2-IOP: P = 0.004). Conclusions: The LC deforms unevenly throughout its depth in response to IOP modulation at fixed ICP. Therefore, analysis of merely the anterior LC surface microstructure will not fully capture the microstructure deformations within the LC. BPR deformer eyes have higher CTVF than BPR non-deformer eyes.

TAZ negatively regulates the novel tumor suppressor ANKRD52 and promotes PAK1 dephosphorylation in lung adenocarcinomas.

Lung cancer is the leading cause of cancer death, and therefore the discovery of novel therapeutic targets is crucial. P21-activated kinase (PAK1) is an important oncogene involved in the signaling of actin cytoskeleton organization. Although PAK1 inhibition has been shown to suppress cancer progression, specific PAK1 inhibitors are not available due to the complex structure and insufficient understanding of this kinase. The Hippo signaling effector TAZ is known to be elevated in multiple human cancers and to promote cancer metastasis. This study aimed to explore the role of TAZ in regulating the tumor suppressor ankyrin repeat domain 52 (ANKRD52) and PAK1 activity. A negative correlation between TAZ and ANKRD52 was observed, with knockdown of TAZ leading to enhanced ANKRD52 promoter activity and increased mRNA levels. Moreover, reduced ANKRD52 levels were associated with late-stage lung cancer. Knockdowns of ANKRD52 resulted in elevated cell mobility, while forced ANKRD52 expression attenuated cell mobility. ANKRD52 is a subunit of the protein phosphatase 6 (PP6) holoenzyme. Mass spectrometry analysis revealed the interaction between PAK1 and the ANKRD52-PP6 complex. Knockdown of ANKRD52 or PP6c resulted in upregulated PAK1 phosphorylation. Our study demonstrates that the novel tumor suppressor protein ANKRD52 is transcriptionally inhibited by TAZ, regulating cell mobility through interactions with PP6c and dephosphorylation of PAK1.

Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap.

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.

Enhanced YAP expression leads to EGFR TKI resistance in lung adenocarcinomas.

Epidermal growth factor receptor (EGFR) mutation is prevalently expressed in lung adenocarcinoma cases and acts as one of the major driving oncogenes. EGFR tyrosine kinase inhibitors (TKIs) have been used in patients with EGFR-mutant as an effective targeted therapy in lung adenocarcinoma, but drug resistance and tumor recurrence inevitably occurs. Recently, Yes-associate protein (YAP) has been reported to promote multiple cancer cell properties, such as promoting cell proliferation, epithelial-mesenchymal transition and drug resistance. This study investigated the roles of YAP in TKI-resistant lung adenocarcinoma. In TKI-sensitive cells, enhanced YAP expression leads to TKI resistant. Also, upregulated YAP expression and activation were detected in long-term TKI-induced resistant cells. With reduced YAP expression using shRNA or YAP inhibitors, TKI-resistant cells become TKI-sensitive. reduced xenograft tumor size in nude mice and Moreover, combined EGFR TKI and a YAP inhibitor, statin, prolonged survival among lung cancer patients analyzed by Taiwan National Health Insurance Research database. These observations revealed the importance of YAP in promoting TKI-resistance and combined YAP inhibition can be a potential therapy delaying the occurrence of TKI-resistance in lung adenocarcinoma.

YAP1 is essential for tumor growth and is a potential therapeutic target for EGFR-dependent lung adenocarcinomas.

Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP1) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP1 in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. In this study, we detected a correlation between YAP1 level and EGFR mutation status in lung adenocarcinoma tissues. Using lung adenocarcinoma cell lines, enhanced YAP1 expression and activity mediated by EGFR signaling was detected through enhanced protein stability. A SRC family protein, YES, was involved in EGFR-regulated YAP1 expression and this pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP1 levels by mechanisms bypassing EGFR signaling were effective in reducing viability in EGFR-dependent cells including those with EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP1 in EGFR mutant lung adenocarcinomas and identified YAP1 as a promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance.

Fluvastatin attenuates hepatic steatosis-induced fibrogenesis in rats through inhibiting paracrine effect of hepatocyte on hepatic stellate cells.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu). METHODS: Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 (phox) subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-ß1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes. RESULTS: In vitro, Flu (1-20 µM) inhibited PA-induced free-radical production, gp91 (phox) expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats. CONCLUSIONS: We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.

Codistribution of collagen type IV and laminin in liver fibrosis of elderly cadavers: immunohistochemical marker of perisinusoidal basement membrane formation.

Liver sinusoids are lined by a fenestrated endothelium that lacks a basement membrane. Formation of perisinusoidal basement membranes beneath the endothelium is an integral feature of capillarization of sinusoids that is a significant pathology found in advanced fibrosis. Liver fibrosis is prevalent in elderly cadavers; however, basement membrane formation in these liver samples has yet to be studied. Collagen type IV and laminin are major basement membrane proteins and their codistribution around sinusoids provides an immunohistochemical marker of basement membrane formation. Here, we examined the intralobular sites of perisinusoidal basement membrane formation in elderly cadaveric livers having various stages of fibrosis. Collagen IV and laminin codistributed in basement membranes of portal and septal ductular and vascular structures, providing a positive control. In the parenchyma, collagen IV immunostaining of sinusoids was panlobular in all stages of fibrosis, and the stain was continuous along the sinusoids. In contrast, laminin was not detected in livers, showing minimal fibrotic change. It was rarely seen in perisinusoidal/pericellular fibrosis, but frequently in septa formation, bridging fibrosis, and cirrhosis. The laminin stain was patchy, occurring principally in sinusoids of periportal and periseptal areas, less commonly in mid-lobular and rarely in centrilobular areas. Consecutive sections revealed that laminin codistributed with collagen IV in these sinusoidal locations, thus marking the sites of perisinusoidal basement membrane formation in aged fibrotic livers. This development is presumably related to aging of the liver and exacerbated by liver injury caused by advanced liver fibrosis, possibly resulting in sinusoidal capillarization.