The role of thromboxane prostanoid receptor signaling in gastric ulcer healing.

The process of gastric ulcer healing includes cell migration, proliferation, angiogenesis and re-epithelialization. Platelets contain angiogenesis stimulating factors that induce angiogenesis. Thromboxane A2 (TXA2 ) not only induces platelet activity but also angiogenesis. This study investigated the role of TXA2 in gastric ulcer healing using TXA2 receptor knockout (TPKO) mice. Gastric ulcer healing was suppressed by treatment with the TXA2 synthase inhibitor OKY-046 and the TXA2 receptor antagonist S-1452 compared with vehicle-treated mice. TPKO showed delayed gastric ulcer healing compared with wild-type mice (WT). The number of microvessels and CD31 expression were lower in TPKO than in WT mice, and TPKO suppressed the expression of transforming growth factor beta (TGF-ß) and vascular endothelial growth factor A (VEGF-A) in areas around gastric ulcers. Immunofluorescence assays showed that TGF-ß and VEGF-A co-localized with platelets. Gastric ulcer healing was significantly reduced in WT mice transplanted with TPKO compared with WT bone marrow. These results suggested that TP signalling on platelets facilitates gastric ulcer healing through TGF-ß and VEGF-A.

Thromboxane A2 receptor signaling in endothelial cells attenuates monocrotaline-induced liver injury.

Sinusoidal obstruction syndrome (SOS) is a major complication of chemotherapy and hematopoietic stem cell transplantation. The early stage of SOS is characterized by liver sinusoidal endothelial cell (LSEC) injury accompanied by platelet aggregation. Thromboxane A2 (TxA2) induces platelet aggregation through the thromboxane prostanoid (TP) receptor. In this study, we explored the role of TP signaling in a monocrotaline (MCT)-induced mouse model of SOS. Relative to wild-type (WT) mice, TP-deficient (TP-/-) mice exhibited more severe MCT-liver injury, as indicated by elevated levels of alanine aminotransferase (ALT) and coagulative necrosis. Extensive accumulation of platelets in the liver was observed in both WT and TP-/- mice. TP expression co-localized with CD31-positive LSECs. MCT treatment caused LSEC destruction, concomitant with elevated expression of matrix metalloproteinases (MMPs) and adhesion molecules in WT mice, and LSEC damage was further exacerbated in TP-/- mice. Viability of isolated LSECs was lower in cells from TP-/- mice, whereas mRNA levels of MMPs and adhesion molecules were higher; U46619, a TxA2 agonist, reduced these levels in WT mice. These data suggest that TP signaling has no effect on platelet accumulation during MCT-induced liver injury, but instead prevents injury by suppressing LSEC damage.

The role of vascular endothelial growth factor receptor 1 tyrosine kinase signaling in bleomycin-induced pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with a poor prognosis. Fibroblast proliferation amplifies extracellular matrix deposition and increases angiogenesis. Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors. VEGF interacts with VEGF receptors (VEGFR1 and VEGFR2). A previous study showed that VEGFR1 tyrosine kinase (TK) signaling induced blood flow recovery mediated by bone marrow (BM)-derived stem cells. We hypothesized that VEGFR1-TK signaling might be related to pulmonary fibrosis. MATERIAL AND METHODS: Six-week-old male C57Bl/6 wild-type (WT) mice and VEGFR1 TK knockout mice (TKKO mice) were treated with a single intratracheal injection of bleomycin (BLM; 0.1 µg in 50 µl saline) or vehicle (saline; 50 µl). Lung fibrosis was evaluated by histology, real-time PCR and ELISA for pro-fibrotic factors, and assessment of lung mechanics. RESULTS: The fibrotic area in the lung and the lung elastance were significantly reduced in TKKO mice (P < 0.01). The expression of the fibrosis-related factors type I collagen, S100A4, and transforming growth factor (TGF)-ß was also significantly reduced in TKKO mice on day 21 after BLM injection. TKKO mice also had significantly lower levels of stromal cell-derived factor (SDF)-1 in the lungs and plasma on days 14 and 21 after BLM treatment (P < 0.05). Moreover, the expression of C-X-C chemokine receptor type 7 (CXCR7) and CXCR4, the receptors for SDF-1, was also suppressed in TKKO mice. Immunohistochemical analysis showed that treatment with a CXCR4 antibody decreased the accumulation of VEGFR1+ cells in the lung in WT mice but not in TKKO mice. CONCLUSION: These results suggest that VEGFR1 TK signaling promotes BLM-induced pulmonary fibrosis by activating the SDF-1/CXCR4 axis in infiltrating VEGFR1+ cells.

Vascular endothelial growth factor receptor 1 tyrosine kinase signaling facilitates healing of DSS-induced colitis by accumulation of Tregs in ulcer area.

BACKGROUND: Ulcerative Colitis (UC) is an inflammatory bowel disease that affects the colon. The development of UC is regulated by immune cells. Previously, we showed that vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase (TK) signaling induces healing of mucosal damage by recruiting VEGFR1+ cells appear to be lineage monocyte cells. Recent studies show that development of UC correlates with the number of regulatory T cells (Tregs). Here, we investigated whether VEGFR1-TK signaling induces healing of UC via accumulation of Tregs or not. METHOD: Acute colitis was induced in C57/Bl6N (wild-type [WT]) and VEGFR1 T K knockout (VEGFR1 T K-/-) mice by administration of 2.0% dextran sulfate sodium (DSS). RESULTS: Total colon length in VEGFR1 T K-/- mice was shorter than that in WT mice. The ulcer length and the disease activity index (DAI) score were significantly higher in VEGFR1 T K-/- mice than in WT mice, whereas CD31 mRNA and protein levels were significantly lower. Accumulation of forkhead box P3+ (Foxp3+) VEGFR1+ Tregs was lower in VEGFR1 T K-/- mice, as was expression of interleukin (IL)-10 and transforming growth factor (TGF)-ß. The survival rate of WT mice treated with an anti-folate receptor 4 (FR4) antibody was 40%, while that of WT mice treated with control IgG was 90%. Moreover, WT mice treated with a neutralizing antibody against C-X-C chemokine receptor type 4 (CXCR4) showed significantly shorter colon length than WT with control antibody. In VEGFR1 T K-/-, infiltration of Foxp3+ Tregs expressing VEGFR1 and CXCR4 into ulcerated areas was lower than that in WT mice. CONCLUSION: VEGFR1-TK signaling plays a critical role in UC healing and angiogenesis via accumulation of VEGFR1+CXCR4+Foxp3+ Tregs in ulcerated tissue. (264 words).

RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

The significance of the relationship between the nervous and immune systems with respect to disease course is increasingly apparent. Immune cells in the liver and spleen are responsible for the development of acute liver injury, yet the regulatory mechanisms of the interactions remain elusive. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, regulates innate immune activation via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. Here, we show that RAMP1 in Kupffer cells (KCs) plays a critical role in the etiology of immune-mediated hepatitis. RAMP1-deficient mice with concanavalin A (ConA)-mediated hepatitis, characterized by severe liver injury accompanied by infiltration of immune cells and increased secretion of pro-inflammatory cytokines by KCs and splenic T cells, showed poor survival. Removing KCs ameliorated liver damage, while depleting T cells or splenectomy led to partial amelioration. Adoptive transfer of splenic T cells from RAMP1-deficient mice led to a modest increase in liver injury. Co-culture of KCs with splenic T cells led to increased cytokine expression by both cells in a RAMP1-dependent manner. Thus, immune-mediated hepatitis develops via crosstalk between immune cells. RAMP1 in KCs is a key regulator of immune responses.

Angiotensin II subtype 1a receptor signaling in resident hepatic macrophages induces liver metastasis formation.

Liver metastases from colorectal cancer (CRC) are a clinically significant problem. The renin-angiotensin system is involved in tumor growth and metastases. This study was designed to evaluate the role of angiotensin II subtype receptor 1a (AT1a) in the formation of liver metastasis in CRC. A model of liver metastasis was developed by intrasplenic injection of mouse colon cancer (CMT-93) into AT1a knockout mice (AT1aKO) and wild-type (C57BL/6) mice (WT). Compared with WT mice, the liver weight and liver metastatic rate were significantly lower in AT1aKO. The mRNA levels of CD31, transforming growth factor- ß1 (TGF-ß1), and F4/80 were suppressed in AT1aKO compared with WT. Double immunofluorescence analysis showed that the number of accumulated F4/80+ cells expressing TGF-ß1 in metastatic areas was higher in WT than in AT1aKO. The AT1aKO bone marrow (BM) (AT1aKO-BM)→WT showed suppressed formation of liver metastasis compared with WT-BM→WT. However, the formation of metastasis was further suppressed in WT-BM→AT1aKO compared with AT1aKO-BM→WT. In addition, accumulated F4/80+ cells in the liver metastasis were not BM-derived F4/80+ cells, but mainly resident hepatic F4/80+ cells, and these resident hepatic F4/80+ cells were positive for TGF-ß1. Angiotensin II enhanced TGF-ß1 expression in Kupffer cells. Treatment of WT with clodronate liposomes suppressed liver metastasis by diminishing TGF-ß1+ F4/80+ cells accumulation. The formation of liver metastasis correlated with collagen deposition in the metastatic area, which was dependent on AT1a signaling. These results suggested that resident hepatic macrophages induced liver metastasis formation by induction of TGF-ß1 through AT1a signaling.