Hirahito Endo

Host Prostaglandin E2-EP3 Signaling Regulates Tumor-Associated Angiogenesis and Tumor Growth

Nonsteroidal antiinflammatories are known to suppress incidence and progression of malignancies including colorectal cancers. However, the precise mechanism of this action remains unknown. Using prostaglandin (PG) receptor knockout mice, we have evaluated a role of PGs in tumor-associated angiogenesis and tumor growth, and identified PG receptors involved. Sarcoma-180 cells implanted in wild-type (WT) mice formed a tumor with extensive angiogenesis, which was greatly suppressed by specific inhibitors for cyclooxygenase (COX)-2 but not for COX-1. Angiogenesis in sponge implantation model, which can mimic tumor-stromal angiogenesis, was markedly suppressed in mice lacking EP3 (EP3−/−) with reduced expression of vascular endothelial growth factor (VEGF) around the sponge implants. Further, implanted tumor growth (sarcoma-180, Lewis lung carcinoma) was markedly suppressed in EP3−/−, in which tumor-associated angiogenesis was also reduced. Immunohistochemical analysis revealed that major VEGF-expressing cells in the stroma were CD3/Mac-1 double-negative fibroblasts, and that VEGF-expression in the stroma was markedly reduced in EP3−/−, compared with WT. Application of an EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT, but not in EP3−/−. These results demonstrate significance of host stromal PGE2-EP3 receptor signaling in tumor development and angiogenesis. An EP3 receptor antagonist may be a candidate of chemopreventive agents effective for malignant tumors.

Selective recruitment of CCR6-expressing cells by increased production of MIP-3α in rheumatoid arthritis

Infiltration of various types of leucocytes has been shown to play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Macrophage inflammatory protein‐3α (MIP‐3α) is a recently identified chemokine which is a selective chemoattractant for leucocytes such as memory T cells, naïve B cells and immature dendritic cells. In this study, we investigated the expression of MIP‐3α and its specific receptor CCR6 in the inflamed joints of patients with RA. Increased amounts of MIP‐3α were found by ELISA in synovial fluids (SF) of patients with RA. MIP‐3α was apparently detected in all synovial tissue specimens of RA patients (n = 6), but it could not be detected in that of osteoarthritis (OA) patients (n = 4). Expression of MIP‐3α was detected especially in the sublining layer, and infiltrating mononuclear cells in RA synovial tissue. Gene expression of MIP‐3α was also found in six out of 11 RA‐synovial fluid cells by RT‐PCR. Cultured synovial fibroblasts derived from either RA or OA patients were capable of producing MIP‐3α in response to IL‐1β and TNFα in vitro. Furthermore, expression of CCR6 was found in infiltrating mononuclear cells in the cellular clusters and around the vessels of RA synovial tissue. These findings indicate that increased production of MIP‐3α may contribute to the selective recruitment of CCR6‐expressing cells in RA.

Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth.

We evaluated the significance of the host kallikrein-kinin system in tumor angiogenesis and tumor growth using two rodent models genetically deficient in a kallikrein-kinin system. Inoculation of Walker 256 carcinoma cells into the s.c. tissues of the back of normal Brown Norway Kitasato rats (BN-Ki rats) resulted in the rapid development of solid tumors with marked angiogenesis. By contrast, in kininogen-deficient Brown Norway Katholiek rats (BN-Ka rats), which cannot generate intrinsic bradykinin (BK), the weights of the tumors and the extent of angiogenesis were significantly less than those in BN-Ki rats. Daily administration of B2 receptor antagonists significantly reduced angiogenesis and tumor weights in BN-Ki rats to levels similar to those in BN-Ka rats but did not do so in BN-Ka rats. Angiogenesis and tumor growth were significantly suppressed in B2 receptor knockout mice bearing sarcoma 180 compared with their wild-type counterparts. Immunoreactive vascular endothelial growth factor (VEGF) was localized in Walker tumor stroma more extensively in BN-Ki rats than in BN-Ka rats, although immunoreactive B2 receptor also was detected in the stroma to the same extent in both types of rats. Cultured stromal fibroblasts isolated from BN-Ki rats and BN-Ka rats produced VEGF in response to BK (10−8-10−6 m), and this stimulatory effect of BK was abolished with a B2 receptor antagonist, Hoe140 (10−5 m). These results suggest that BK generated from kininogens supplied from the host may facilitate tumor-associated angiogenesis and tumor growth by stimulating stromal B2 signaling to up-regulate VEGF production mainly in fibroblasts.

Adiponectin stimulates prostaglandin E2 production in rheumatoid arthritis synovial fibroblasts

OBJECTIVE Adipokines may influence inflammatory and/or immune responses. This study was undertaken to examine whether adiponectin affects the production of prostaglandin E(2) (PGE(2)) by rheumatoid arthritis synovial fibroblasts (RASFs). METHODS Synovial tissue was obtained from patients with RA who were undergoing joint replacement surgery. Fibroblast-like cells from the third or fourth passage were used as RASFs. Expression of adiponectin receptor messenger RNA (mRNA) and protein was detected. PGE(2) (converted from arachidonic acid) was measured by enzyme-linked immunosorbent assay (ELISA). Expression of mRNA and protein for cyclooxygenase 2 (COX-2) and membrane-associated PGE synthase 1 (mPGES-1), key enzymes involved in PGE(2) synthesis, was detected in RASFs. The effects of RNA interference (RNAi) targeting the adiponectin receptor genes and the receptor signal inhibitors were examined. The influence of adiponectin on NF-kappaB activation in RASFs was measured with an ELISA kit. RESULTS Adiponectin receptors were detected in RASFs. Adiponectin increased both COX-2 and mPGES-1 mRNA and protein expression by RASFs in a time- and concentration-dependent manner. PGE(2) production by RASFs was also increased by the addition of adiponectin, and this increase was inhibited by RNAi for the adiponectin receptor gene, or coincubation with the receptor signal inhibitors. Enhancement of NF-kappaB activation by adiponectin as well as by interleukin-1beta was observed in RASFs. CONCLUSION Our findings indicate that adiponectin induces COX-2 and mPGES-1 expression, resulting in the enhancement of PGE(2) production by RASFs. Thus, adiponectin may play a role in the pathogenesis of synovitis in RA patients.

Experimental arthritis induced by continuous infusion of IL-8 into rabbit knee joints.

To determine the roles of IL‐8 in inflammatory synovitis. examination was made of the results of continuously injecting human recombinant IL‐8 into the knee joints of New Zealand white rabbits. Recombinant human lL‐8 was infused continuously into the joint cavity at 75 ng/h for 14 days by a polypropylene catheter connected to a mini‐osmotic pump implanted in each rabbit. Infiltration of inflammatory cells into joint cavity and histopathological changes in synovial tissue were examined at 7 and 14 days following the start of infusion. The continuous infusion of IL‐8 for 14days led to severe arthritis characterized by apparent erythema and joint pain, the accumulation of leucocytes, infiltration of mononuclear cells in synovial tissue, and marked hypervascularization in the synovial lining layer. IL‐8 may be a factor which can contribute to the inflammatory process of chronic arthritis by mediating leucocyte recruitment and hypervascularization in inflamed joints.

Up-regulation of prostaglandin E receptor EP2 and EP4 subtypes in rat synovial tissues with adjuvant arthritis

To evaluate the role of the prostaglandin E receptor (EP) subtypes in the development of inflammatory synovitis, we examined EP subtype mRNA distribution in the synovial tissue of rats with adjuvant arthritis and the effect of selective EP agonists on cytokine production by cultured rat synovial cells. We used reverse transcriptase‐polymerase chain reaction (RT‐PCR) and in situ hybridization to measure the level of EP subtype (EP1, EP2, EP3, and EP4) mRNA expression in synovial tissues and cultured synovial cells from the arthritic joints of rats. RT‐PCR and ELISA were used to analyse the effects of two selective EP agonists on IL‐6 production by cultured rat synovial cells. EP2 and EP4 mRNA expression in inflamed synovial tissues was up‐regulated. EP2 and EP4 mRNA were co‐expressed in synovial macrophages and fibroblasts in inflamed tissues. EP4 and EP2 agonists both inhibited IL‐1‐induced IL‐6 production. Our results suggest that prostaglandin E2 regulates the functions of synovial macrophages and fibroblasts through EP2 and EP4, which are induced by inflammatory stimuli in rats with adjuvant arthritis.

Predictors of Survival and Causes of Death in Japanese Patients with Systemic Sclerosis

Objective. To clarify the mortality rates, causes of death, and contributing clinical factors in Japanese patients with systemic sclerosis (SSc). Methods. A cohort of 405 patients with SSc, who attended our institution during the period 1973 to 2008, was retrospectively analyzed until the end of 2009. Clinical data were obtained from medical records or autopsy reports. Results. The 405 patients with SSc consisted of 310 (76.5%) survivors, 86 (21.2%) who died, and 9 who were lost to followup. Diffuse cutaneous SSc and involvement of organs other than the gastrointestinal tract were more frequent in patients who died, and were associated with a worse prognosis according to Kaplan-Meier analysis. Female sex, limited cutaneous SSc, anticentromere antibody (ACA), and overlap with Sjögren’s syndrome (SS) were factors favoring a better prognosis, while overlap with myositis contributed to a poor prognosis. The overall 10-year survival rate was 88%. The patients with SSc had a significantly higher mortality than the general population (standardized mortality ratio 2.76), but the patients with ACA or overlapping SS did not. The most common causes of death were unknown ones including sudden death, followed by malignancy and infection. In patients with pulmonary arterial hypertension, sudden death was the most common cause of mortality. Conclusion. The overall mortality rate of patients with SSc was higher than that of the general population, probably because of poor prognostic factors including organ involvement. These factors should be carefully monitored during followup.

Clinical usefulness of anti-RNA polymerase III antibody measurement by enzyme-linked immunosorbent assay

OBJECTIVE To evaluate the clinical usefulness of measuring anti-RNA polymerase (RNAP) III antibody with a commercially available ELISA in Japanese patients with SSc. METHODS This multicentre study involved 354 patients with SSc, 245 with non-SSc CTDs and 102 healthy controls. ELISAs were used to detect anti-RNAP III antibody, anti-topo I antibody and ACA. The presence of anti-RNAP III antibody in selected serum samples was confirmed by immunoprecipitation (IP) assay. RESULTS By ELISA, anti-RNAP III antibody was detected in 38 (10.7%) patients with SSc, 3 (1.2%) with non-SSc CTD and no healthy controls. The clinical specificity for SSc was excellent (98.8%), although a small number of false positives occurred. The sensitivity of the anti-topo I and ACA ELISAs for SSc was 59.9%, which increased to 68.2% without a reduction in specificity when the anti-RNAP III measurement was added. Clinical features associated with positivity for the anti-RNAP III antibody include dcSSc, a high total skin score and a trend towards high prevalence of renal crisis, consistent with previous studies that used an IP assay. Furthermore, on clinical severity scales, SSc patients with anti-RNAP III antibody scored highest for skin and renal involvement among patients subgrouped by the presence of individual SSc-related antibodies. CONCLUSIONS The measurement of anti-RNAP III antibody by ELISA is useful in routine clinical practice, because it helps diagnose SSc and identify a disease subset with severe skin and renal involvement.

Additive effects of IL-1 and TNF on induction of prostacyclin synthesis in human vascular endothelial cells.

The effect of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on the induction of prostacyclin (PGI2) synthesis in human vascular endothelial cells (EC) was investigated. Both IL-1 and TNF increased PGI2 production by EC in both a time- and dose-dependent manner, and a combination of the two cytokines additively enhanced PGI2 production. Metabolic inhibitors including indomethacin and cycloheximide abolished cytokine induced PGI2 synthesis. Since, the effect of TNF was not inhibited by neutralization with antibody to IL-1 alpha and IL-1 beta, it seems that the mechanism is not mediated by endogenous IL-1 induced by TNF.

Clinical features of 405 Japanese patients with systemic sclerosis.

We aimed to clarify the clinical features of Japanese patients with systemic sclerosis (SSc), especially with reference to organ involvement and autoantibodies. A cohort of 405 patients with SSc who attended our institution from 1973 to 2008 was identified retrospectively. Data on clinical features, including autoantibodies, organ involvement, and overlap of other connective tissue diseases, were obtained by following the medical records until 2009. The percentage of male patients during or after 1990 was greater than that before 1990 (3.9 vs. 10.6%, respectively). Limited cutaneous SSc (lSSc) was twice as frequent as diffuse cutaneous SSc (dSSc). About half of the patients had lung involvement (50.4%), while only 3.2% had scleroderma renal crisis. Male gender was associated with lung involvement, and dSSc was associated with most organ involvements except for pulmonary arterial hypertension (PAH). Anti-Scl-70 antibody was associated with lung or heart involvement, while anti-U1-RNP antibody was only associated with PAH. Conversely, patients with anti-centromere antibody had less organ involvement. SSc–Sjögren overlap syndrome was related to lSSc, further overlapping systemic lupus erythematosus (SLE), and less lung or heart involvement. In conclusion, these results not only confirmed previous reports but revealed several other findings, such as the increased proportion of male patients in recent years and the relationships between clinical features.