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:XX-XX.].

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.

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.

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.

Paeoniae radix reduces PDGF-stimulated hepatic stellate cell migration.

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. In chronic liver injury, HSCs undergo transdifferentiation to an activated myofibroblastic phenotype and migrate to injured areas in response to chemotactic factors, producing extracellular matrix proteins such as collagen type I to repair the damage as well as overexpression of α-smooth muscle actin (α-SMA). Paeoniae Radix, the root of Paeonia lactiflora Pall, was investigated for PDGF-BB-induced HSC chemotaxis. Rat HSCs and LX-2, a human HSC cell line, were used for the in vitro experiments. Cell migration was analyzed by wound-healing and transwell assays. An ELISA and a Sircol collagen assay kit were used to detect the expressions of α-SMA and of collagen, respectively. Phosphorylations of mitogen-activated protein kinases, including ERK 1/2, p38, and JNK, were evaluated with immunoblotting. Results indicated that PDGF-BB increased migration as well as α-SMA and collagen expression in HSCs. Paeoniae Radix extracts and its active components, paeonol and 1,2,3,4,6-penta- O-galloyl- ß-D-glucose (PGG), inhibited PDGF-BB-induced HSC migration and α-SMA and collagen expressions in a concentration-dependent manner. The inhibitory effects were associated with downregulation of PDGF receptor- α, ERK, p38, and JNK activation. Both paeonol and PGG participate in HSC migration, but via differential mechanisms.

Protective effects of kaerophyllin against liver fibrogenesis in rats.

BACKGROUND: We previously demonstrated that kaerophyllin, a lignan, isolated from a widely used traditional Chinese herb, Bupleurum scorzonerifolium, leading to the inhibition of hepatic stellate cells (HSCs) activation in vitro. This current study evaluated the in vivo role of kaerophyllin in protecting the liver against injury and fibrogenesis caused by thioacetamide (TAA) in rats and further explored the underlying mechanisms. MATERIALS AND METHODS: Liver fibrosis in Sprague-Dawley rats was induced by intraperitoneal injection of TAA (200 mg/kg) twice per week for 6 weeks. Animals were divided into five groups: vehicle control, TAA control, TAA + low dose kaerophyllin, TAA + high dose kaerophyllin and TAA + curcumin groups. Kaerophyllin (10 or 30 mg/kg) or curcumin (150 mg/mL) was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. Rat HSCs were used to investigate the anti-inflammatory role of kaerophyllin against tumour necrosis factor α (TNF-α) in vitro. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression was knocked down in rat HSCs using PPAR-γ small interfering RNAs. RESULTS: Kaerophyllin significantly protected liver from injury by reducing serum aspartate transaminase and alanine transaminase levels and by improving the histological architecture and fibrosis score. In addition, kaerophyllin suppressed inflammation by reducing the mRNA of TNF-α, interleukin-1ß (IL-1ß) and monocyte chemoattractant protein-1 (MCP-1) genes. In HSCs, kaerophyllin elevated PPAR-γ activity and reduced TNF-α-stimulated mRNA levels of intracellular adhesion molecule-1 (ICAM-1), MCP-1 and IL-1ß genes, which were reversed by small interfering RNA knockdown of PPAR-γ gene. CONCLUSIONS: Our results demonstrated that kaerophyllin protected the rat liver from TAA-caused injury and fibrogenesis by suppressing hepatic inflammation and inhibiting HSC activation, possibly through upregulation of PPAR-γ expression.

Kaerophyllin inhibits hepatic stellate cell activation by apoptotic bodies from hepatocytes.

BACKGROUND: Hepatic stellate cells (HSCs), the key cell type for hepatic fibrosis, become activated and profibrogenic in the presence of hepatocyte apoptotic bodies (ABs). Bupleurum scorzonerifolium (BS), a widely used traditional Chinese herb for liver diseases, was fractionated, and the inhibitory effects of BS extracts on AB-induced HSC migration were screened. The activity-guided fractionation led to a lignan, kaerophyllin. In this study, the anti-fibrotic effects of kaerophyllin were studied in the presence of ABs. METHODS: LX-2 cells phagocytosing ultraviolet (UV)-induced HepG2 ABs were investigated by confocal microscopy and flow cytometry. AB-induced HSC activation was evaluated by immunoblotting and real-time PCR analyses. HSC migration was measured by wound-healing assays. RESULTS: HepG2 ABs induced LX-2 activation, with the production of collagen I and α-smooth muscle actin, upregulated profibrogenic gene transcriptions and increased NF-κB activity, cell migration and phagocytosis. Kaerophyllin from BS antagonized AB-induced HSC migration and activation. CONCLUSIONS: Kaerophyllin inhibited AB-induced LX-2 activation and migration with downregulation of Akt/ERK phosphorylations and NF-κB activity. Our study suggests a novel platform for screening anti-fibrotic compounds with ABs.

Downregulation of hepatic stellate cell activation by retinol and palmitate mediated by adipose differentiation-related protein (ADRP).

Hepatic stellate cells (HSCs) store retinoids and triacylglycerols in cytoplasmic lipid droplets. Two prominent features of HSC activation in liver fibrosis are loss of lipid droplets along with increase of alpha-smooth muscle actin (alpha-SMA), but the link between these responses and HSC activation remains elusive. In non-adipose cells, adipose differentiation-related protein (ADRP) coats lipid droplets and regulates their formation and lipolysis; however its function in HSCs is unknown. Here, we observed, in human liver sections or primary HSC culture, ADRP localization to lipid droplets of HSCs, and reduced staining coincident with loss of lipid droplets in liver fibrosis and in culture-activated HSCs, consistent with HSC activation. In the LX-2 human immortalized HSCs, with scant lipid droplets and features of activated HSCs, we found that the upregulation of ADRP mRNA by palmitate is potentiated by retinol, accompanied by increased ADRP protein, generation of retinyl palmitate, and lipid droplet formation. ADRP induction also led to decreased expression of alpha-SMA mRNA and its protein, while ADRP knockdown with small interfering RNA (siRNA) normalized alpha-SMA expression. Furthermore, ADRP induction by retinol and palmitate resulted in decreased expression of collagen I and matrix metalloproteinase-2 mRNA, fibrogenic genes associated with activated HSCs, while increasing matrix metalloproteinase-1 mRNA; ADRP knockdown with siRNA reversed these changes. Tissue inhibitor of metalloproteinase-1 was not affected. Thus, ADRP upregulation mediated by retinol and palmitate promotes downregulation of HSC activation and is functionally linked to the expression of fibrogenic genes.

Hepatic stellate cells express functional CXCR4: role in stromal cell-derived factor-1alpha-mediated stellate cell activation.

UNLABELLED: Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell-derived factor-1alpha (SDF-1alpha), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF-1alpha/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF-1alpha is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture-induced activation. Treatment of stellate cells with recombinant SDF-1alpha increases expression of alpha-smooth muscle actin and collagen I and stimulates a dose-dependent increase in HSC proliferation. Inhibitor studies suggest that SDF-1alpha/CXCR4-dependent extracellular signal-regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation. CONCLUSION: HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF-1alpha is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase pathways mediate SDF-1alpha-induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches.

Studies on antiproliferative effects of phthalides from Ligusticum chuanxiong in hepatic stellate cells.

Suppression of hepatic stellate cell (HSC) growth and activation, and induction of apoptosis, have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. Our previous study showed that the Chinese herb Ligusticum chuanxiong (LC) inhibits platelet-derived growth factor (PDGF-BB)-induced HSC proliferation. The present study was designed to investigate the active principles and their action mechanisms. With a bioactivity-directed fractionation approach, DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of active principles of LC. Two phthalides, Z,Z'-6,8',7,3'-diligustilide (1) and levistolide A (2), from LC significantly abrogated PDGF-BB-induced proliferation in both rat and human HSC lines. These inhibitory effects of compounds 1 and 2 were associated with reduction of alpha-smooth muscle actin and collagen expressions. The cell cycle promoting proteins, cyclins D1, D2, E, A and B1, were downregulated while the inhibitory proteins p21 and 27 were up-regulated. JNK phosphorylation was up-regulated by compounds 1 and 2. In HSC-T6, the two compounds induced apoptosis through the activation of caspases 9 and 3, increase in cytosolic cytochrome c release, and downregulation of Bcl-2 and Akt phosphorylation. Moreover, neither phthalides caused direct cytotoxicity to either HSCs or rat primary hepatocytes under experimental concentrations. These results indicate that two phthalides from LC inhibited PDGF-BB-activated HSC proliferation possibly through cell cycle inhibition and apoptosis mechanisms. They might be potential anti-fibrotic drugs for the treatment and prevention of hepatic fibrosis.

Inhibitory effects of Ligusticum chuanxiong on the proliferation of rat hepatic stellate cells.

BACKGROUND AND AIMS: Platelet-derived growth factor (PDGF) is a very potent mitogen for hepatic stellate cells (HSC) in hepatic fibrogenesis. Ligusticum chuanxiong Hort. (LC), a traditional Chinese herb used for cerebrovascular diseases, has been shown to exert anti-inflammatory and free radical scavenging effects. The aims of the present study were to investigate the effects of LC extract on the proliferation-related biomarkers in a rat HSC cell line (HSC-T6) stimulated with PDGF. METHODS: DNA synthesis via bromodeoxyuridine (BrdU) incorporation, cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of LC. RESULTS: The results revealed that LC extract (25-100 microg/mL) concentration-dependently decreased the PDGF-induced cell proliferation as well as alpha-smooth muscle actin expression in HSC. The inhibitory activity of LC on HSC was associated with: (i) inhibition of BrdU incorporation; (ii) induction of apoptosis with the activation of caspase-3, up-regulation of cell cycle inhibitory proteins p21 and p27, and down-regulation of cell cycle stimulatory proteins cyclins D1 and D2; and (iii) increased phosphorylation of mitogen-activated protein kinases (JNK). LC at the studied concentrations showed no direct cytotoxicity on primary hepatocytes. CONCLUSION: The results suggest that LC significantly inhibited PDGF-activated HSC proliferation, possibly through apoptotic mechanisms and the potential of LC as an antifibrotic agent warrants further investigation.

Antiproliferative effect of salvianolic acid A on rat hepatic stellate cells.

Suppression of activation or proliferation, or induction of apoptosis in hepatic stellate cells (HSCs) have been proposed as therapeutic strategies against liver fibrosis. Salvia miltiorrhiza has been reported to exert antifibrotic effects in rats with hepatic fibrosis, but its mechanisms of action remain to be clarified. We have investigated the effects of salvianolic acid A (Sal A), an active principle from S. miltiorrhiza, on the proliferation-related biomarkers in a cell line of rat HSCs (HSC-T6) stimulated with platelet-derived growth factor-BB homodimer (PDGF-BB). DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of Sal A. The results showed that Sal A (1-10 microM) concentration-dependently attenuated PDGF-BB-stimulated proliferation (BrdU incorporation) in HSC-T6 cells. Sal A at 10 microM induced cell apoptosis in PDGF-BB-incubated HSCs, together with a reduction of Bcl-2 protein expression, induction of cell cycle inhibitory proteins p21 and p27, and down-regulation of cyclins D1 and E, suppression of Akt phosphorylation, reduction in PDGF receptor phosphorylation, and an increase in caspase-3 activity. Sal A exerted no direct cytotoxicity on primary hepatocytes and HSC-T6 cells under experimental concentrations. Our results suggested that Sal A inhibited PDGF-BB-activated HSC proliferation, partially through apoptosis induction.