Suppression of Fli-1 protects against pericyte loss and cognitive deficits in Alzheimer's disease.

Brain pericytes regulate cerebral blood flow, maintain the integrity of the blood-brain barrier (BBB), and facilitate the removal of amyloid ß (Aß), which is critical to healthy brain activity. Pericyte loss has been observed in brains from patients with Alzheimer's disease (AD) and animal models. Our previous data demonstrated that friend leukemia virus integration 1 (Fli-1), an erythroblast transformation-specific (ETS) transcription factor, governs pericyte viability in murine sepsis; however, the role of Fli-1 and its impact on pericyte loss in AD remain unknown. Here, we demonstrated that Fli-1 expression was up-regulated in postmortem brains from a cohort of human AD donors and in 5xFAD mice, which corresponded with a decreased pericyte number, elevated inflammatory mediators, and increased Aß accumulation compared with cognitively normal individuals and wild-type (WT) mice. Antisense oligonucleotide Fli-1 Gapmer administered via intrahippocampal injection decelerated pericyte loss, decreased inflammatory response, ameliorated cognitive deficits, improved BBB dysfunction, and reduced Aß deposition in 5xFAD mice. Fli-1 Gapmer-mediated inhibition of Fli-1 protected against Aß accumulation-induced human brain pericyte apoptosis in vitro. Overall, these studies indicate that Fli-1 contributes to pericyte loss, inflammatory response, Aß deposition, vascular dysfunction, and cognitive decline, and suggest that inhibition of Fli-1 may represent novel therapeutic strategies for AD.

Generation of a new immortalized human lung pericyte cell line: a promising tool for human lung pericyte studies.

Pericytes apposed to the capillary endothelium are known to stabilize and promote endothelial integrity. Recent studies indicate that lung pericytes play a prominent role in lung physiology, and they are involved in the development of various lung diseases including lung injury in sepsis, pulmonary fibrosis, asthma, and pulmonary hypertension. Accordingly, human lung pericyte studies are important for understanding the mechanistic basis of lung physiology and pathophysiology; however, human lung pericytes can only be cultured for a few passages and no immortalized human lung pericyte cell line has been established so far. Thus, our study aims to establish an immortalized human lung pericyte cell line. Developed using SV40 large T antigen lentivirus, immortalized pericytes exhibit stable SV40T expression, sustained proliferation, and have significantly higher telomerase activity compared to normal human lung pericytes. In addition, these cells retained pericyte characteristics, marked by similar morphology, and expression of pericyte cell surface markers such as PDGFRß, NG2, CD44, CD146, CD90, and CD73. Furthermore, similar to that of primary pericytes, immortalized pericytes promoted endothelial cell tube formation and responded to different stimuli. Our previous data showed that friend leukemia virus integration 1 (Fli-1), a member of the ETS transcription factor family, is a key regulator that modulates inflammatory responses in mouse lung pericytes. We further demonstrated that Fli-1 regulates inflammatory responses in immortalized human lung pericytes. To summarize, we successfully established an immortalized human lung pericyte cell line, which serves as a promising tool for in vitro pericyte studies to understand human lung pericyte physiology and pathophysiology.

miR-145a Regulation of Pericyte Dysfunction in a Murine Model of Sepsis.

BACKGROUND: Sepsis is a life-threatening systemic disease with severe microvascular dysfunction. Pericytes preserve vascular homeostasis. To our knowledge, the potential roles of microRNAs in sepsis-induced pericyte dysfunction have not been explored. METHODS: We determined lung pericyte expression of miR-145a in cecal ligation and puncture (CLP)-induced sepsis. Mouse lung pericytes were isolated and transfected with a miR-145a mimic, followed by stimulation with lipopolysaccharide (LPS). We measured inflammatory cytokine levels. To assess the functions of miR-145a in vivo, we generated a pericyte-specific miR-145a-knockout mouse and determined sepsis-induced organ injury, lung and renal vascular leakage, and mouse survival rates. We used RNA sequencing and Western blotting to analyze the signaling pathways regulated by miR-145a. RESULTS: CLP led to decreased miR-145a expression in lung pericytes. The miR-145a mimic inhibited LPS-induced increases in cytokines. In CLP-induced sepsis, pericytes lacking miR-145a exhibited increased lung and kidney vascular leakage and reduced survival rates. We found that miR-145a could suppress LPS-induced NF-κB activation. In addition, we confirmed that the transcription factor Friend leukemia virus integration 1 (Fli-1) is a target of miR-145a and that Fli-1 activates NF-κB signaling. CONCLUSION: Our results demonstrated that pericyte miR-145a mediates sepsis-associated microvascular dysfunction, potentially by means of Fli-1-mediated modulation of NF-κB signaling.

Exosomes from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is characterized by disruption of the alveolar-capillary barrier resulting in accumulation of proteinaceous edema and increased inflammatory cells in the alveolar space. We previously found that endothelial progenitor cell (EPC) exosomes prevent endothelial dysfunction and lung injury in sepsis in part due to their encapsulation of miRNA-126. However, the effects of EPC exosomes in acute lung injury (ALI) remain unknown. METHODS: To determine if EPC exosomes would have beneficial effects in ALI, intratracheal administration of lipopolysaccharide (LPS) was used to induce ALI in mice. Lung permeability, inflammation, and the role of miRNA-126 in the alveolar-epithelial barrier function were examined. RESULTS: The intratracheal administration of EPC exosomes reduced lung injury following LPS-induced ALI at 24 and 48 h. Compared to placebo, intratracheal administration of EPC exosomes significantly reduced the cell number, protein concentration, and cytokines/chemokines in the bronchoalveolar lavage fluid (BALF), indicating a reduction in permeability and inflammation. Further, EPC exosomes reduced myeloperoxidase (MPO) activity, lung injury score, and pulmonary edema, demonstrating protection against lung injury. Murine fibroblast (NIH3T3) exosomes, which do not contain abundant miRNA-126, did not provide these beneficial effects. In human small airway epithelial cells (SAECs), we found that overexpression of miRNA-126-3p can target phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2), while overexpression of miRNA-126-5p inhibits the inflammatory alarmin HMGB1 and permeability factor VEGFα. Interestingly, both miR-126-3p and 5p increase the expression of tight junction proteins suggesting a potential mechanism by which miRNA-126 may mitigate LPS-induced lung injury. CONCLUSIONS: Our data demonstrated that human EPC exosomes are beneficial in LPS-induced ALI mice, in part through the delivery of miRNA-126 into the injured alveolus.

Fli-1 transcription factor regulates the expression of caspase-1 in lung pericytes.

Our previous data demonstrated that Friend leukemia virus integration 1 (Fli-1), an ETS transcription factor, governs pericyte loss and vascular dysfunction in cecal ligation and puncture-induced murine sepsis by regulating essential pyroptosis markers including caspase-1. However, whether Fli-1 regulates caspase-1 expression levels in vitro and how Fli-1 regulates caspase-1 remain unknown. Our present work further demonstrated that overexpressed Fli-1 significantly increased caspase-1 and IL-18 expression levels in cultured mouse lung pericytes. Bacterial outer membrane vesicles (OMVs) have been found to induce cell pyroptosis through transferring LPS intracellularly. Using OMVs to induce an in vitro model of pyroptosis, we observed that OMVs significantly increased protein levels of Fli-1 in mouse lung pericytes. Furthermore, knockdown of Fli-1 by siRNA blocked OMVs-induced caspase-1, caspase-11 and IL-18 expression levels. As caspase-1 was predicted as a potential target of Fli-1, we cloned murine caspase-1 promoter into a luciferase construct. Our data demonstrate for the first time that Fli-1 regulates caspase-1 expression by directly binding to its promoter regions measured by chromatin immunoprecipitation (ChIP) assay and luciferase reporter system. In summary, our findings demonstrated a novel role and mechanism of Fli-1 in regulating caspase-1 expression in lung pericytes.

Fli-1 Governs Pericyte Dysfunction in a Murine Model of Sepsis.

Background: Pericytes are vascular mural cells and are embedded in the basement membrane of the microvasculature. Recent studies suggest a role for pericytes in lipopolysaccharide (LPS)-induced microvascular dysfunction and mortality, but the mechanisms of pericyte loss in sepsis are largely unknown. Methods: By using a cecal ligation and puncture (CLP)-induced murine model of sepsis, we observed that CLP led to lung and renal pericyte loss and reduced lung pericyte density and pericyte/endothelial cell (EC) coverage. Results: Up-regulated Friend leukemia virus integration 1 (Fli-1) messenger ribonucleic acid (RNA) and protein levels were found in lung pericytes from CLP mice in vivo and in LPS-stimulated lung pericytes in vitro. Knockout of Fli-1 in Foxd1-derived pericytes prevented CLP-induced pericyte loss, vascular leak, and improved survival. Disrupted Fli-1 expression by small interfering RNA inhibited LPS-induced inflammatory cytokines and chemokines in cultured lung pericytes. Furthermore, CLP-induced pericyte pyroptosis was mitigated in pericyte Fli-1 knockout mice. Conclusions: Our findings suggest that Fli-1 is a potential therapeutic target in sepsis.

The role of miRNAs in cardiovascular disease risk factors.

Coronary artery disease and atherosclerosis are complex pathologies that develop over time due to genetic and environmental factors. Differential expression of miRNAs has been identified in patients with coronary artery disease and atherosclerosis, however, their association with cardiovascular disease risk factors, including hyperlipidemia, hypertension, obesity, diabetes, lack of physical activity and smoking, remains unclear. This review examines the role of miRNAs as either biomarkers or potential contributors to the pathophysiology of these aforementioned risk factors. It is intended to provide an overview of the published literature which describes alterations in miRNA levels in both human and animal studies of cardiovascular risk factors and when known, the possible mechanism by which these miRNAs may exert either beneficial or deleterious effects. The intent of this review is engage clinical, translational, and basic scientists to design future collaborative studies to further elucidate the potential role of miRNAs in cardiovascular diseases.

Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer.

The transcription factor FOXO3 is a well-established tumor suppressor whose activity, stability, and localization are regulated by phosphorylation and acetylation. Previous data by our laboratory demonstrated amplified thromboxane-A2 signaling was associated with poor prognoses in bladder cancer patients and overexpression of the thromboxane-A2 isoform-ß receptor (TPß), but not TPα, induced malignant transformation of immortalized bladder cells in vivo. Here, we describe a mechanism of TP mediated modulation of FOXO3 activity and localization by phosphorylation and deacetylation in a bladder cancer cell model. In vitro gain and loss of function studies performed in non-transformed cell lines, UROsta and SV-HUC, revealed knockdown of FOXO3 expression by shRNA increased cell migration and invasion, while exogenously overexpressing TPß raised basal phosphorylated (p)FOXO3-S294 levels. Conversely, overexpression of ERK-resistant, mutant FOXO3 reduced increases in UMUC3 cell migration and invasion, including that mediated by TP agonist (U46619). Additionally, stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression, which could be attenuated by treatment with a TP antagonist (PTXA2) or ERK inhibitor (U0126). Initially U46619 caused nuclear accumulation of pFOXO3-S294; however, prolonged stimulation increased FOXO3 cytoplasmic localization. U46619 stimulation decreased overall FOXO3 transcriptional activity, but was associated with increased expression of its pro-survival target, manganese superoxide dismutase. The data also shows that TP stimulation increased the expression of the histone deacetylase, SIRT1, and corresponded with decreased acetylated-FOXO3. Collectively, the data suggest a role for TP signaling in the regulation of FOXO3 activity, mediated in part through phosphorylation and deacetylation.

Endothelial progenitor cells and a stromal cell-derived factor-1α analogue synergistically improve survival in sepsis.

RATIONALE: Endothelial progenitor cells (EPCs) have been associated with human sepsis but their role is incompletely understood. Stromal cell-derived factor (SDF)-1α facilitates EPC recruitment and is elevated in murine sepsis models. Previous studies have demonstrated that the SDF-1α analog CTCE-0214 (CTCE) is beneficial in polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. OBJECTIVES: We hypothesized that exogenously administered EPCs are also beneficial in CLP sepsis and that CTCE provides synergistic benefit. METHODS: Mice were subjected to CLP and administered EPCs at varying doses, CTCE, or a combination of the two. Mouse survival, plasma miRNA expression, IL-10 production, and lung vascular leakage were determined. The in vitro effect of CTCE on miRNA expression and EPC function were determined. MEASUREMENTS AND MAIN RESULTS: Survival was improved with EPC therapy at a threshold of 10(6) cells. In coculture studies, EPCs augmented LPS-induced macrophage IL-10 production. In vivo EPC administration in sepsis increased plasma IL-10, suppressed lung vascular leakage, attenuated liver and kidney injury, and augmented miR-126 and -125b expression, which regulate endothelial cell function and/or inflammation. When subthreshold numbers of EPCs were coadministered with CTCE in CLP mice they synergistically improved survival. We demonstrated that CTCE recruits endogenous EPCs in septic mice. In in vitro analysis, CTCE enhanced EPC proliferation, angiogenesis, and prosurvival signaling while inhibiting EPC senescence. These cellular effects were, in part, explained by the effect of CTCE on miR-126, -125b, -34a, and -155 expression in EPCs. CONCLUSIONS: EPCs and CTCE represent important potential therapeutic strategies in sepsis.

Toll-like receptor-induced inflammatory cytokines are suppressed by gain of function or overexpression of Gα(i2) protein.

Previous studies have implicated a role of Gα(i) proteins as co-regulators of Toll-like receptor (TLR) activation. These studies largely derived from examining the effect of Gα(i) protein inhibitors or genetic deletion of Gα(i) proteins. However, the effect of increased Gα(i) protein function or Gα(i) protein expression on TLR activation has not been investigated. We hypothesized that gain of function or increased expression of Gα(i) proteins suppresses TLR2- and TLR4-induced inflammatory cytokines. Novel transgenic mice with genomic "knock-in" of a regulator of G protein signaling (RGS)-insensitive Gnai2 allele (Gα(i2)(G184S/G184S) ; GS/GS) were employed. These mice express essentially normal levels of Gα(i2) protein; however, the Gα(i2) is insensitive to its negative regulator RGS thus rendering more sustained Gα(i2) protein activation following ligand/receptor binding. In subsequent studies, we generated Raw 264.7 cells that stably overexpress Gα(i2) protein (Raw Gα(i2)). Peritoneal macrophages, splenocytes, and mouse embryonic fibroblasts (MEF) were isolated from WT and GS/GS mice and were stimulated with LPS, Pam3CSK4, or Poly (I:C). We also subjected WT and GS/GS mice to endotoxic shock (LPS, 25 mg/kg i.p.) and plasma tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 production were determined. We found that in vitro LPS and Pam3CSK4-induced TNF-α, and IL-6 production are decreased in macrophages from GS/GS mice compared with WT mice (p < 0.05). In vitro, LPS and Pam3CSK4 induced IL-6 production in splenocytes, and in vivo, LPS-induced IL-6 were suppressed in GS/GS mice. Poly (I:C)-induced TNF-α, and IL-6 in vitro demonstrated no difference between GS/GS mice and WT mice. LPS-induced IL-6 production was inhibited in MEFs from GS/GS mice similarly to macrophage and splenocytes. In parallel studies, Raw Gα(i2) cells also exhibit decreased TNF-α and IL-6 production in response to LPS and Pam3CSK4. These studies support our hypothesis that Gα(i2) proteins are novel negative regulators of TLR activation.

Beneficial effect of a CXCR4 agonist in murine models of systemic inflammation.

The chemokine CXC receptor 4 (CXCR4) is activated by stromal cell-derived factor (SDF-1α). CXCR4 may be part of a lipopolysaccharide (LPS) sensing co-clustering complex that modulates TLR4 activation and evidence suggest that SDF-1α can activate anti-inflammatory signaling pathways and suppress inflammation. In the present study we examined the hypothesis that the SDF-1α peptide analog and CXCR4 agonist CTCE-0214 is anti-inflammatory in three distinct models of murine systemic inflammation. Our findings demonstrate that CTCE-0214 in vivo significantly suppressed plasma tumor necrosis factor alpha (TNF-α) increases in acute endotoxemia and following zymosan-induced multiple organ dysfunction syndrome (MODS). In both models, CTCE-0214 did not suppress plasma increases in the anti-inflammatory cytokine interleukin (IL)-10. CTCE-0214 improved survival without antibiotics in a model of severe sepsis induced by cecal ligation and puncture (CLP). CTCE-0214 also decreased plasma increases in IL-6 but not TNF-α and IL-10 in response to CLP-induced inflammation. We demonstrated in a moderately severe model of CLP (one puncture) that IL-6 levels at 24 h were similar to sham controls. However in severe CLP (two punctures) plasma IL-6 levels were markedly elevated. Plasma SDF-1α levels varied inversely with the plasma IL-6. In addition to the beneficial effect of CTCE-0214 in these models of systemic inflammation in vivo, we also demonstrated that the analog dose dependently suppressed LPS-induced IL-6 production in bone marrow-derived macrophages. CTCE-0214 therefore may be beneficial in controlling inflammation sepsis and systemic inflammatory syndromes.

Urinary thromboxane B2 and thromboxane receptors in bladder cancer: opportunity for detection and monitoring.

We have previously found increased expression of thromboxane synthase (TXAS) and thromboxane receptor (TP) beta isoform in the tissues of patients with bladder cancer. Studies in cell lines and mice have indicated a potential significant role of the thromboxane signaling pathway in the pathogenesis of human bladder cancer. This study was designed to determine if the changes observed in the tissues of patients with bladder cancer were mirrored by changes in the urine of these patients. We found increased levels of thromboxane B(2) (TXB(2)) the major metabolite of TXAS and increased levels of the TPß receptor. These results raised the possibility that patients with bladder cancer may be followed for progression or remission of their disease by quantitation of these substances in their urine.

Increased expression of beta-arrestin 1 and 2 in murine models of rheumatoid arthritis: isoform specific regulation of inflammation.

Pro-inflammatory cytokines and chemokines play critical roles in autoimmune diseases including rheumatoid arthritis (RA). Recently, it has been reported that ß-arrestin 1 and 2 are involved in the regulation of inflammation. We hypothesized that ß-arrestin 1 and 2 play critical roles in murine models of RA. Using a collagen-induced arthritis (CIA) and a human TNFα transgenic (TNFtg) mouse model, we demonstrated that ß-arrestin 1 and 2 expression are significantly increased in joint tissue of CIA mice and TNFtg mice. In fibroblast-like synoviocytes (FLS) isolated from hind knee joint of CIA mice, we observed an increase of ß-arrestin 1 and 2 protein and mRNA levels in the early stage of arthritis. In FLS, low molecular weight hyaluronan (HA)-induced TNFα and IL-6 production was increased by overexpression of ß-arrestin 1 but decreased by overexpression of ß-arrestin 2 demonstrating isoform specific regulation. TNFα and HA induced an increase of ß-arrestin 1 and 2 expression in FLS, while high mobility group box (HMGB)-1 only stimulated ß-arrestin 1 expression. TNFα- or HA-induced ß-arrestin 2 expression was blocked by a p38 inhibitor. To examine the in vivo role of ß-arrestin 2 in the pathogenesis of arthritis, WT and ß-arrestin 2 KO mice were subjected to collagen antibody-induced arthritis (CAIA). ß-Arrestin 2 KO mice exhibited more severe arthritis in CAIA. Thus ß-arrestin 2 is anti-inflammatory in CAIA. These composite observations suggest that ß-arrestin 1 and 2 differentially regulate FLS inflammation and increased ß-arrestin 2 may reduce experimental arthritis severity.

Heterotrimeric Gα(i) proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis.

Previous studies have implicated a role of heterotrimeric Gα(i) proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gα(i) proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gα(i)-GTP protein complex was immunoprecipitated with a Gα(i) protein activation assay. In subsequent in vivo studies, the Gα(i) protein inhibitor pertussis toxin (PTx) or G(i) protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gα(i2) in sepsis, Gα(i2) (-/-) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gα(i) proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα(i) proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gα(i2) (-/-) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gα(i2) (-/-) compared to WT mice. Gα(i2) (-/-) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα(i) proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gα(i2) protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.

Beta-arrestin 2 negatively regulates sepsis-induced inflammation.

SUMMARY: Beta-arrestins 1 and 2 are ubiquitously expressed proteins that alter signalling by G-protein-coupled receptors. beta-arrestin 2 plays an important role as a signalling adaptor and scaffold in regulating cellular inflammatory responses. We hypothesized that beta-arrestin 2 is a critical modulator of inflammatory response in experimental sepsis. beta-arrestin 2(-/-) and wild-type (WT) mice were subjected to caecal ligation and puncture (CLP). The survival rate was significantly decreased (P < 0.05) in beta-arrestin 2(-/-) mice (13% survival) compared with WT mice (53% survival). A second group of mice were killed 18 hr after CLP for blood, peritoneal lavage and tissue sample collection. CLP-induced plasma interleukin (IL)-6 was significantly increased 25 +/- 12 fold and caecal myeloperoxidase (MPO) activity was increased 2.4 +/- 0.3 fold in beta-arrestin 2(-/-) compared with WT mice. beta-arrestin 2(-/-) mice exhibited more severe lung damage and higher bacterial loads compared with WT mice post CLP challenge as measured by histopathology and colony-forming unit count. In subsequent experiments, splenocytes, peritoneal macrophages and bone marrow-derived macrophages (BMDMs) were isolated and cultured from beta-arrestin 2(-/-) and WT mice and stimulated in vitro with lipopolysaccharide (LPS). Tumour necrosis factor (TNF)-alpha, IL-6 and IL-10 production induced by LPS was significantly augmented (2.2 +/- 0.2 fold, 1.8 +/- 0.1 fold, and 2.2 +/- 0.4 fold, respectively; P < 0.05) in splenocytes from beta-arrestin 2(-/-) mice compared with WT mice. The splenocyte response was different from that of peritoneal macrophages or BMDMs, which exhibited no difference in TNF-alpha and IL-6 production upon LPS stimulation between WT and beta-arrestin 2(-/-) mice. Our data demonstrate that beta-arrestin 2 functions to negatively regulate the inflammatory response in polymicrobial sepsis.

Novel role of thromboxane receptors beta isoform in bladder cancer pathogenesis.

These studies were undertaken to determine the potential role of thromboxane receptors (TP) in bladder cancer. The data reported herein show that expression of the TP-beta receptor protein is increased in tissue obtained from patients with bladder cancer and associated with a significantly poorer prognosis (P < 0.005). Bladder cancer cell lines express the TP-beta isoform, unlike immortalized nontransformed urothelial cells (SV-HUC) that express only the TP-alpha isoform. TP-beta receptor expression, but not TP-alpha, promoted cell proliferation, migration, and invasion in vitro, and also resulted in malignant transformation of SV-HUC cells in vivo. Agonist-mediated phosphorylation of extracellular signal-regulated kinase and FAK was dependent on the expression of TP-beta. Furthermore, TP-beta mediated multiple biological effects by signaling through either G-protein alpha subunit 12 or beta-arrestin 2. Treatment of mice with the TP receptor antagonist GR32191, alone or in combination with cisplatin, significantly delayed tumor onset and prolonged survival of mice transplanted with TCC-SUP bladder cancer cells compared with vehicle or cisplatin alone. These results support the model that the TP-beta receptor isoform plays a unique role in bladder cancer progression and its expression may have predictive value and provide a novel therapeutic target.

Lysophosphatidic acid inhibits bacterial endotoxin-induced pro-inflammatory response: potential anti-inflammatory signaling pathways.

Previous studies have demonstrated that heterotrimeric guanine nucleotide-binding regulatory (Gi) protein-deficient mice exhibit augmented inflammatory responses to lipopolysaccharide (LPS). These findings suggest that Gi protein agonists will suppress LPS-induced inflammatory gene expression. Lysophosphatidic acid (LPA) activates G protein-coupled receptors leading to Gi protein activation. We hypothesized that LPA will inhibit LPS-induced inflammatory responses through activation of Gi-coupled anti-inflammatory signaling pathways. We examined the anti-inflammatory effect of LPA on LPS responses both in vivo and in vitro in CD-1 mice. The mice were injected intravenously with LPA (10 mg/kg) followed by intraperitoneal injection of LPS (75 mg/kg for survival and 25 mg/kg for other studies). LPA significantly increased the mice survival to endotoxemia (P < 0.05). LPA injection reduced LPS-induced plasma TNF-alpha production (69 +/- 6%, P < 0.05) and myeloperoxidase (MPO) activity in lung (33 +/- 9%, P < 0.05) as compared to vehicle injection. LPS-induced plasma IL-6 was unchanged by LPA. In vitro studies with peritoneal macrophages paralleled results from in vivo studies. LPA (1 and 10 microM) significantly inhibited LPS-induced TNFalpha production (61 +/- 9% and 72 +/- 9%, respectively, P < 0.05) but not IL-6. We further demonstrated that the anti-inflammatory effect of LPA was reversed by ERK 1/2 and phosphatase inhibitors, suggesting that ERK 1/2 pathway and serine/threonine phosphatases are involved. Inhibition of phosphatidylinositol 3 (PI3) kinase signaling pathways also partially reversed the LPA anti-inflammatory response. However, LPA did not alter NFkappaB and peroxisome proliferator-activated receptor gamma (PPARgamma) activation. Inhibitors of PPARgamma did not alter LPA-induced inhibition of LPS signaling. These studies demonstrate that LPA has significant anti-inflammatory activities involving activation of ERK 1/2, serine/threonine phosphatases, and PI3 kinase signaling pathways.

Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in macrophages by Galpha(i) proteins.

Heterotrimeric G(i) proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of G(i) proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS induced production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 and interferon-gamma-inducible protein-10 (IP-10); SA induced TNF-alpha, and IL-1beta production; and GBS induced TNF-alpha, IL-6, IL-1beta, macrophage inflammatory protein-1alpha (MIP-1alpha) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. In contrast to the role of G(i) proteins as a positive regulator of mediators, LPS-induced production of MIP-1alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Galpha(i1/3) (-/-) mice, and SA-induced MIP-1alpha production was increased in both groups of Galpha(i) protein-depleted mice. LPS-induced production of KC and IL-1beta, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. These data suggest that G(i2) and G(i1/3) proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.

The phosphatidylinositol 3 kinase pathway regulates tolerance to lipopolysaccharide and priming responses to Staphylococcus aureus and lipopolysaccharide.

Our previous studies have demonstrated that although LPS and Staphylococcus aureus induce homologous tolerance, they induce priming to each other instead of cross-tolerance. The phosphatidylinositol 3 (PI3) kinase pathway has been implicated in microbial signaling and inflammatory gene expression regulation. We hypothesized that LPS or S. aureus induced tolerance and priming responses to each other are PI3 kinase pathway-dependent. CD1 mice received intraperitoneal injections of 1% Biogel and were treated intraperitoneally with vehicle, LPS, or S. aureus (5 mg/kg) 3 days later. Peritoneal macrophages (MØ) were harvested 24 h later and exposed to vehicle or the PI3 kinase inhibitors wortmannin (10 nmol/L) or LY294002 (10 nmol/L) 1 h before in vitro stimulation with LPS or S. aureus (10 microg/mL). Both LPS and S. aureus significantly induced tumor necrosis factor alpha and thromboxane B2 synthesis (P < 0.05, n = 3) in naive cells. LPS and S. aureus induced homologous tolerance were associated with suppressed tumor necrosis factor alpha and thromboxane B2 levels but augmented interleukin 10 production. However, LPS and S. aureus induced priming to each other, as shown by augmented mediator production. Wortmannin and LY294002 reversed LPS tolerance yet had no effect on S. aureus tolerance. PI3 kinase blockade attenuated the priming responses to both LPS and S. aureus. Mice pretreated with LPS and challenged with LPS were protected. In contrast, mice pretreated with LPS and wortmannin demonstrated LPS tolerance reversal. These data suggest that PI3 kinase is essential for LPS induced homologous tolerance and reciprocal LPS and S. aureus induced priming responses.

Prognostic and functional significance of thromboxane synthase gene overexpression in invasive bladder cancer.

Thromboxane synthase (TXAS) is one of the enzymes downstream from cyclooxygenase-2 and catalyzes the synthesis of thromboxane A(2) (TXA(2)). TXAS was among the genes we identified based on its overexpression in invasive bladder tumors. TXAS is overexpressed in common forms of bladder tumors: 69 of 97 (71.1%) transitional cell carcinoma (TCC), 38 of 53 (71.6%) squamous cell carcinoma, and 5 of 11 (45.5%) adenocarcinoma relative to nontumor tissue. Overall, 112 of 161 (69.5%) invasive tumors exhibited elevated expression. Significantly, patients with tumors having >4-fold levels of TXAS expression showed significant statistical evidence of lower overall survival expressed by the estimated hazard ratio of 2.74 with P = 0.009 in Cox's regression analysis. TXAS mRNA expression was found to be an independent prognostic marker for patients with bladder cancer. Treatment of bladder cancer cell lines (T24 and TCC-SUP) with TXAS inhibitors and TXA(2) (TP) receptor antagonists reduced cell growth, migration, and invasion, whereas TP agonists stimulated cell migration and invasion. The positive correlation between elevated TXAS expression and shorter patient survival supports a potential role for TXAS-regulated pathways in tumor invasion and metastases and suggests that modulation of the TXAS pathway may offer a novel therapeutic approach.