A Synthetic Analog of Resveratrol Inhibits the Proangiogenic Response of Liver Sinusoidal Cells during Hepatic Metastasis.

We utilized Fas21, a resveratrol analog, to modulate the function of hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) during the angiogenic phase of murine liver metastasis by B16 melanoma and 51b colorectal carcinoma. Preangiogenic micrometastases were treated with Fas21 (1 mg/kg/day) or vehicle during the development of intra-angiogenic tracts. Mice treated with Fas21 showed reduced liver tumor foci in both liver metastasis models. Micrometastases were classified immunohistochemically, as well as according to their position coordinates and connection to local microvasculature. The volume of liver occupied by sinusoidal-type foci, containing infiltrating angiogenic capillaries, decreased by ~50% in Fas21-treated mice compared to vehicle-treated ones in both tumor metastasis models. The volume of portal foci, containing peripheral neoangiogenesis within a discontinuous layer of myofibroblasts, was similar in all experimental groups in both tumor metastasis models, but displayed enhanced necrotic central areas devoid of angiogenesis following Fas21 treatment. As a result, sinusoidal tumors from mice treated with Fas21 showed a 50% reduction in desmin(+)/asma(+) HSCs and CD31(+) vessel density, and a 45% reduction in intrametastatic VEGF mRNA compared with sinusoidal tumors from vehicle-treated mice. Necrotic portal metastases increased 2-4-fold in treated mice. In vitro, Fas21 reduced VEGF secretion by HSCs and 51b cells dose-dependently. Additionally, HSCs migration in response to tumor soluble factors was dose-dependently diminished by Fas21, as was LSEC migration in response to HSCs and tumor soluble factors. Resveratrol analog Fas21 inhibits the proangiogenic response of HSCs and LSECs during the development of murine liver metastasis.

Frontline Science: Specialized proresolving lipid mediators inhibit the priming and activation of the macrophage NLRP3 inflammasome.

The prototypic proinflammatory cytokine IL-1ß plays a central role in innate immunity and inflammatory disorders. The formation of mature IL-1ß from an inactive pro-IL-1ß precursor is produced via nonconventional multiprotein complexes called the inflammasomes, of which the most common is the nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3) inflammasome composed by NLRP3, (ASC) apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD), and caspase-1. Specialized proresolving mediators (SPMs) promote resolution of inflammation, which is an essential process to maintain host health. SPMs prevent excessive inflammation by terminating the inflammatory response and returning to tissue homeostasis without immunosupression. This study tested the hypothesis that modulation of the NLRP3 inflammasome in macrophages is one mechanism involved in the SPM-regulated processes during resolution. Our findings demonstrate that the SPM resolvin D2 (RvD2) suppressed the expression of pro-IL-1ß and reduced the secretion of mature IL-1ß in bone marrow-derived macrophages challenged with LPS+ATP (classical NLRP3 inflammasome model) or LPS+palmitate (lipotoxic model). Similar findings were observed in thioglycolate-elicited peritoneal macrophages, in which RvD2 remarkably reduced ASC oligomerization, inflammasome assembly, and caspase-1 activity. In vivo, in a self-resolving zymosan A-induced peritonitis model, RvD2 blocked the NLRP3 inflammasome leading to reduced release of IL-1ß into the exudates, repression of osteopontin, and MCP-1 expression and induction of M2 markers of resolution (i.e., CD206 and arginase-1) in peritoneal macrophages. RvD2 inhibitory actions were receptor mediated and were abrogated by a selective GPR18 antagonist. Together, these findings support the hypothesis that SPMs have the ability to inhibit the priming and to expedite the deactivation of the NLRP3 inflammasome in macrophages during the resolution process.

The specialized proresolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are hallmarks of nonalcoholic fatty liver disease (NAFLD), which is the hepatic manifestation of the metabolic syndrome associated with obesity. The specialized proresolving lipid mediator maresin 1 (MaR1) preserves tissue homeostasis by exerting cytoprotective actions, dampening inflammation, and expediting its timely resolution. Here, we explored whether MaR1 protects liver cells from lipotoxic and hypoxia-induced ER stress. Mice were rendered obese by high-fat diet feeding, and experiments were performed in primary hepatocytes, Kupffer cells, and precision-cut liver slices (PCLSs). Palmitate-induced lipotoxicity increased ER stress and altered autophagy in hepatocytes, effects that were prevented by MaR1. MaR1 protected hepatocytes against lipotoxicity-induced apoptosis by activating the UPR prosurvival mechanisms and preventing the excessive up-regulation of proapoptotic pathways. Protective MaR1 effects were also seen in hepatocytes challenged with hypoxia and TNF-α-induced cell death. High-throughput microRNA (miRNA) sequencing revealed that MaR1 actions were associated with specific miRNA signatures targeting both protein folding and apoptosis. MaR1 also prevented lipotoxic-triggered ER stress and hypoxia-induced inflammation in PCLSs and enhanced Kupffer cell phagocytic capacity. Together, these findings describe the ability of MaR1 to oppose ER stress in liver cells under conditions frequently encountered in NAFLD.-Rius, B., Duran-Güell, M., Flores-Costa, R., López-Vicario, C., Lopategi, A., Alcaraz-Quiles, J., Casulleras, M., Lozano, J. J., Titos, E., Clària, J. The specialized proresolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress.

Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.

OBJECTIVE: Liver fibrosis is associated with significant collagen-I deposition largely produced by activated hepatic stellate cells (HSCs); yet, the link between hepatocyte damage and the HSC profibrogenic response remains unclear. Here we show significant induction of osteopontin (OPN) and high-mobility group box-1 (HMGB1) in liver fibrosis. Since OPN was identified as upstream of HMGB1, we hypothesised that OPN could participate in the pathogenesis of liver fibrosis by increasing HMGB1 to upregulate collagen-I expression. DESIGN AND RESULTS: Patients with long-term hepatitis C virus (HCV) progressing in disease stage displayed enhanced hepatic OPN and HMGB1 immunostaining, which correlated with fibrosis stage, whereas it remained similar in non-progressors. Hepatocyte cytoplasmic OPN and HMGB1 expression was significant while loss of nuclear HMGB1 occurred in patients with HCV-induced fibrosis compared with healthy explants. Well-established liver fibrosis along with marked induction of HMGB1 occurred in CCl4-injected OpnHep transgenic yet it was less in wild type and almost absent in Opn-/- mice. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep) protected mice from CCl4-induced liver fibrosis. Coculture with hepatocytes that secrete OPN plus HMGB1 and challenge with recombinant OPN (rOPN) or HMGB1 (rHMGB1) enhanced collagen-I expression in HSCs, which was blunted by neutralising antibodies (Abs) and by Opn or Hmgb1 ablation. rOPN induced acetylation of HMGB1 in HSCs due to increased NADPH oxidase activity and the associated decrease in histone deacetylases 1/2 leading to upregulation of collagen-I. Last, rHMGB1 signalled via receptor for advanced glycation end-products and activated the PI3K-pAkt1/2/3 pathway to upregulate collagen-I. CONCLUSIONS: During liver fibrosis, the increase in OPN induces HMGB1, which acts as a downstream alarmin driving collagen-I synthesis in HSCs.

Polymorphisms in the IL-1 gene cluster influence systemic inflammation in patients at risk for acute-on-chronic liver failure.

Acute-on-chronic liver failure (ACLF) in cirrhosis is an increasingly recognized syndrome characterized by acute decompensation, organ failure(s) and high short-term mortality. Recent findings suggest that an overexuberant systemic inflammation plays a primary role in ACLF progression. In this study, we examined whether genetic factors shape systemic immune responses in patients with decompensated cirrhosis. Six single-nucleotide polymorphisms (SNPs) in inflammation-related genes (interleukin [IL]-1 beta [IL-1ß], rs1143623; IL-1 receptor antagonist [IL-1ra], rs4251961; IL-10, rs1800871; suppressor of cytokine signaling-3, rs4969170; nucleotide-binding oligomerization domain-containing protein 2, rs3135500; and chemerin chemokine-like receptor 1, rs1878022) were genotyped in 279 patients with cirrhosis with (n = 178) and without (n = 101) ACLF from the CANONIC study of the CLIF consortium. Among these SNPs, we identified two polymorphisms belonging to the IL-1 gene cluster (IL-1ß and IL-1ra) in strong association with ACLF. Both SNPs were protective against ACLF; IL-1ß (odds ratio [OR], 0.34, 95% confidence interval [CI], 0.13-0.89; P < 0.05) and IL-1ra (OR, 0.58; 95% CI, 0.35-0.95; P < 0.05) under the recessive and overdominant inheritance models, respectively. These protective SNPs translated into reduced circulating levels of IL-1ß, IL-1α, IL-6, granulocyte-colony stimulating factor, granulocyte-macrophage colony-stimulating factor, and C-reactive protein at enrollment as well as after 7-14 days of admission. These findings were confirmed in vitro in leukocytes incubated with plasma from patients with decompensated cirrhosis carrying the protective SNP genotypes. Notably, a higher frequency of the protective genotypes was observed in patients without (80%) than in those with (20%) ACLF. Consistently, patients carrying the combined protective genotypes showed a lower 28-day mortality rate. CONCLUSION: These data identify two common functional polymorphisms in the IL-1 gene cluster, which are associated with the inflammatory process related to development of ACLF. (Hepatology 2017;65:202-216).

Signaling and Immunoresolving Actions of Resolvin D1 in Inflamed Human Visceral Adipose Tissue.

Persistent activation of the innate immune system greatly influences the risk for developing metabolic complications associated with obesity. In this study, we explored the therapeutic potential of the specialized proresolving mediator (SPM) resolvin D1 (RvD1) to actively promote the resolution of inflammation in human visceral adipose tissue from obese (Ob) patients. Using liquid chromatography-tandem mass spectrometry-based metabololipidomic analysis, we identified unbalanced production of SPMs (i.e., D- and E-series resolvins, protectin D1, maresin 1, and lipoxins) with respect to inflammatory lipid mediators (i.e., leukotriene B4 and PGs) in omental adipose tissue from Ob patients. In parallel, high-throughput transcriptomic analysis revealed a unique signature in this tissue that was characterized by overactivation of the IL-10 signaling pathway. Incubation of inflamed Ob visceral adipose tissues and human macrophages with RvD1 limited excessive activation of the IL-10 pathway by reducing phosphorylation of STAT proteins. Of interest, RvD1 blocked STAT-1 and its target inflammatory genes (i.e., CXCL9), as well as persistent STAT3 activation, without affecting the IL-10 anti-inflammatory response characterized by inhibition of IL-6, IL-1ß, IL-8, and TNF-α. Furthermore, RvD1 promoted resolution by enhancing expression of the IL-10 target gene heme oxygenase-1 by mechanisms dependent on p38 MAPK activity. Together, our data show that RvD1 can tailor the quantitative and qualitative responses of human inflamed adipose tissue to IL-10 and provide a mechanistic basis for the immunoresolving actions of RvD1 in this tissue. These findings may have potential therapeutic implications in obesity-related insulin resistance and other metabolic complications.

Prostaglandin E2 Exerts Multiple Regulatory Actions on Human Obese Adipose Tissue Remodeling, Inflammation, Adaptive Thermogenesis and Lipolysis.

Obesity induces white adipose tissue (WAT) dysfunction characterized by unremitting inflammation and fibrosis, impaired adaptive thermogenesis and increased lipolysis. Prostaglandins (PGs) are powerful lipid mediators that influence the homeostasis of several organs and tissues. The aim of the current study was to explore the regulatory actions of PGs in human omental WAT collected from obese patients undergoing laparoscopic bariatric surgery. In addition to adipocyte hypertrophy, obese WAT showed remarkable inflammation and total and pericellular fibrosis. In this tissue, a unique molecular signature characterized by altered expression of genes involved in inflammation, fibrosis and WAT browning was identified by microarray analysis. Targeted LC-MS/MS lipidomic analysis identified increased PGE2 levels in obese fat in the context of a remarkable COX-2 induction and in the absence of changes in the expression of terminal prostaglandin E synthases (i.e. mPGES-1, mPGES-2 and cPGES). IPA analysis established PGE2 as a common top regulator of the fibrogenic/inflammatory process present in this tissue. Exogenous addition of PGE2 significantly reduced the expression of fibrogenic genes in human WAT explants and significantly down-regulated Col1α1, Col1α2 and αSMA in differentiated 3T3 adipocytes exposed to TGF-ß. In addition, PGE2 inhibited the expression of inflammatory genes (i.e. IL-6 and MCP-1) in WAT explants as well as in adipocytes challenged with LPS. PGE2 anti-inflammatory actions were confirmed by microarray analysis of human pre-adipocytes incubated with this prostanoid. Moreover, PGE2 induced expression of brown markers (UCP1 and PRDM16) in WAT and adipocytes, but not in pre-adipocytes, suggesting that PGE2 might induce the trans-differentiation of adipocytes towards beige/brite cells. Finally, PGE2 inhibited isoproterenol-induced adipocyte lipolysis. Taken together, these findings identify PGE2 as a regulator of the complex network of interactions driving uncontrolled inflammation and fibrosis and impaired adaptive thermogenesis and lipolysis in human obese visceral WAT.

Role of bioactive lipid mediators in obese adipose tissue inflammation and endocrine dysfunction.

White adipose tissue is recognized as an active endocrine organ implicated in the maintenance of metabolic homeostasis. However, adipose tissue function, which has a crucial role in the development of obesity-related comorbidities including insulin resistance and non-alcoholic fatty liver disease, is dysregulated in obese individuals. This review explores the physiological functions and molecular actions of bioactive lipids biosynthesized in adipose tissue including sphingolipids and phospholipids, and in particular fatty acids derived from phospholipids of the cell membrane. Special emphasis is given to polyunsaturated fatty acids of the omega-6 and omega-3 families and their conversion to bioactive lipid mediators through the cyclooxygenase and lipoxygenase pathways. The participation of omega-3-derived lipid autacoids in the resolution of adipose tissue inflammation and in the prevention of obesity-associated hepatic complications is also thoroughly discussed.

Pro-resolving mediators produced from EPA and DHA: Overview of the pathways involved and their mechanisms in metabolic syndrome and related liver diseases.

A novel genus of pro-resolving lipid mediators endogenously generated from omega-3 polyunsaturated fatty acids has been identified in exudates obtained during the resolution phase of acute inflammation. The term specialized pro-resolving mediators (SPM) has been coined for these lipid mediators, comprising four novel chemical mediator families designated resolvins of the E series (if derived from eicosapentaenoic acid) and resolvins of the D series, protectins and maresins (if generated from docosahexaenoic acid). These SPM act not only as "stop-signals" of inflammatory response, but also as facilitators of the ability of macrophages to clear apoptotic cells (efferocytosis) and migrate to peripheral lymph nodes (efflux), thus, expediting their removal from sites of inflammation. In this review, we provide an overview of the current efforts to elucidate the structure-function, biosynthesis and actions of these omega-3-derived SPM in the context of inflammatory diseases. We specifically highlight the role of these SPM as endogenous counter-regulators of the persistent inflammatory status present in adipose tissue of obese individuals and describe the potential therapeutic impact of these bioactive lipid autacoids on the prevention of hepatic co-morbidities associated with obesity and the metabolic syndrome.

Inhibition of soluble epoxide hydrolase modulates inflammation and autophagy in obese adipose tissue and liver: role for omega-3 epoxides.

Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in a number of diseases that have inflammation as a common underlying cause. sEH limits tissue levels of cytochrome P450 (CYP) epoxides derived from omega-6 and omega-3 polyunsaturated fatty acids (PUFA) by converting these antiinflammatory mediators into their less active diols. Here, we explored the metabolic effects of a sEH inhibitor (t-TUCB) in fat-1 mice with transgenic expression of an omega-3 desaturase capable of enriching tissues with endogenous omega-3 PUFA. These mice exhibited increased CYP1A1, CYP2E1, and CYP2U1 expression and abundant levels of the omega-3-derived epoxides 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic (19,20-EDP) in insulin-sensitive tissues, especially liver, as determined by LC-ESI-MS/MS. In obese fat-1 mice, t-TUCB raised hepatic 17,18-EEQ and 19,20-EDP levels and reinforced the omega-3-dependent reduction observed in tissue inflammation and lipid peroxidation. t-TUCB also produced a more intense antisteatotic action in obese fat-1 mice, as revealed by magnetic resonance spectroscopy. Notably, t-TUCB skewed macrophage polarization toward an antiinflammatory M2 phenotype and expanded the interscapular brown adipose tissue volume. Moreover, t-TUCB restored hepatic levels of Atg12-Atg5 and LC3-II conjugates and reduced p62 expression, indicating up-regulation of hepatic autophagy. t-TUCB consistently reduced endoplasmic reticulum stress demonstrated by the attenuation of IRE-1α and eIF2α phosphorylation. These actions were recapitulated in vitro in palmitate-primed hepatocytes and adipocytes incubated with 19,20-EDP or 17,18-EEQ. Relatively similar but less pronounced actions were observed with the omega-6 epoxide, 14,15-EET, and nonoxidized DHA. Together, these findings identify omega-3 epoxides as important regulators of inflammation and autophagy in insulin-sensitive tissues and postulate sEH as a druggable target in metabolic diseases.

Osteopontin induces ductular reaction contributing to liver fibrosis.

OBJECTIVE: In human chronic liver disease, there is association between ductular reaction (DR) and fibrosis; yet, the mechanism triggering its onset and its role in scar formation remains unknown. Since we previously showed that osteopontin (OPN) is highly induced during drug-induced liver fibrosis, we hypothesised that OPN could drive oval cells (OC) expansion and DR and signal to hepatic stellate cells (HSC) to promote scarring. RESULTS: In vivo studies demonstrated increased OPN expression in biliary epithelial cells (BEC) and in OC in thioacetamide (TAA)-treated mice. OPN ablation protected mice from TAA and bile duct ligation-induced liver injury, DR and scarring. This was associated with greater hepatocyte proliferation, lower OC expansion and DR along with less fibrosis, suggesting that OPN could activate the OC compartment to differentiate into BEC, which could then signal to HSC to enhance scarring. Since TAA-treated wild-type mice and cirrhotic patients showed TGF-ß(+) BEC, which were lacking in TAA-treated Opn(-/-) mice and in healthy human explants, this suggested that OPN could regulate TGF-ß, a profibrogenic factor. In vitro experiments confirmed that recombinant OPN (rOPN) decreases hepatocyte proliferation and increases OC and BEC proliferation. To evaluate how BEC regulate collagen-I production in HSC, co-cultures were established. Co-cultured BEC upregulated OPN and TGF-ß expression and enhanced collagen-I synthesis by HSC. Lastly, recombinant TGF-ß (rTGFß) and rOPN promoted BEC proliferation and neutralisation of OPN and TGF-ß reduced collagen-I expression in co-cultured HSC. CONCLUSIONS: OPN emerges as a key matricellular protein driving DR and contributing to scarring and liver fibrosis via TGF-ß.

Extracellular matrix and liver disease.

SIGNIFICANCE: The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. CRITICAL ISSUES: This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. RECENT ADVANCES: Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF' apoptosis, senescence, and reversal to quiescence. FUTURE DIRECTIONS: We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new "omics" tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs.

Osteopontin, an oxidant stress sensitive cytokine, up-regulates collagen-I via integrin α(V)ß(3) engagement and PI3K/pAkt/NFκB signaling.

UNLABELLED: A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen-I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSCs), could drive fibrogenesis by modulating the HSC pro-fibrogenic phenotype and Collagen-I expression. Recombinant OPN (rOPN) up-regulated Collagen-I protein in primary HSCs in a transforming growth factor beta (TGFß)-independent fashion, whereas it down-regulated matrix metalloprotease-13 (MMP13), thus favoring scarring. rOPN activated primary HSCs, confirmed by increased α-smooth muscle actin (αSMA) expression and enhanced their invasive and wound-healing potential. HSCs isolated from wild-type (WT) mice were more profibrogenic than those from OPN knockout (Opn(-/-)) mice and infection of primary HSCs with an Ad-OPN increased Collagen-I, indicating correlation between both proteins. OPN induction of Collagen-I occurred via integrin α(v)ß(3) engagement and activation of the phosphoinositide 3-kinase/phosphorylated Akt/nuclear factor kappa B (PI3K/pAkt/NFκB)-signaling pathway, whereas cluster of differentiation 44 (CD44) binding and mammalian target of rapamycin/70-kDa ribosomal protein S6 kinase (mTOR/p70S6K) were not involved. Neutralization of integrin α(v) ß(3) prevented the OPN-mediated activation of the PI3K/pAkt/NFκB-signaling cascade and Collagen-I up-regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN-mediated Collagen-I increase. Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen-I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by CCl(4) injection or thioacetamide (TAA) treatment elevated OPN expression. Reactive oxygen species up-regulated OPN in vitro and in vivo and antioxidants prevented this effect. Transgenic mice overexpressing OPN in hepatocytes (Opn(HEP) Tg) mice developed spontaneous liver fibrosis compared to WT mice. Last, chronic CCl(4) injection and TAA treatment caused more liver fibrosis to WT than to Opn(-/-) mice and the reverse occurred in Opn(HEP) Tg mice. CONCLUSION: OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen-I protein contributing to scarring and liver fibrosis.

Colon carcinoma cell interaction with liver sinusoidal endothelium inhibits organ-specific antitumor immunity through interleukin-1-induced mannose receptor in mice.

UNLABELLED: Mannose receptor (ManR)-mediated liver sinusoidal endothelial cell (LSEC) endocytosis plays a role in antigen presentation and innate immunity, but its role in hepatic metastasis is unknown. We studied ManR-mediated endocytosis during C26 colorectal cancer cell interaction with LSECs and its implications in metastasis. Uptake of labeled ManR ligands (mannan and ovalbumin) and immunohistochemistry were used to study ManR endocytosis and expression. Several interleukin (IL)-1 inhibitors and the cyclooxygenase-2 (COX-2) inhibitor celecoxib were used to analyze the role of IL-1 and COX-2 in ManR regulation. Anti-mouse ManR antibodies and ManR knockout (ManR(-/-)) mice were used to identify ManR-dependent mechanisms during antitumor immune response of liver sinusoidal lymphocytes (LSLs) interacting with tumor-activated LSECs. ManR expression and endocytosis increased in tumor-activated LSECs through a two-step mechanism: (1) Release of COX-2-dependent IL-1-stimulating factors by lymphocyte function-associated antigen-1-expressing C26 cells in response to intercellular adhesion molecule-1 (ICAM-1), which was expressed and secreted by tumor-activated LSECs; and (2) widespread up-regulation of ManR in LSECs through tumor-induced IL-1. In addition, LSLs that had interacted with tumor-activated LSECs in vivo decreased their antitumor cytotoxicity and interferon (IFN)-gamma secretion while they increased IL-10 release ex vivo. IFN-gamma/IL-10 ratio also decreased in the hepatic blood from tumor-injected mice. Immunosuppressant effects of tumor-activated LSECs on LSLs were abrogated in both LSECs from ManR(-/-) mice and tumor-activated LSECs given anti-mouse ManR antibodies. CONCLUSION: ICAM-1-induced tumor COX-2 decreased antitumor activity during hepatic metastasis through IL-1-induced ManR. ManR constituted a common mediator for prometastatic effects of IL-1, COX-2, and ICAM-1. A rise in hepatic IFN-gamma/IL-10 ratio and antitumor cytotoxicity by way of ManR blockade is consistent with the antimetastatic effects of IL-1, COX-2, and ICAM-1 inhibitors. These data support ManR and ManR-stimulating factors as targets for hepatic colorectal metastasis therapy.

Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis.

BACKGROUND: The recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear. METHODS: The proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 mum in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence. RESULTS: Spheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells. CONCLUSION: 3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.