Kanako Hosono

COX-2 and prostaglandin EP3/EP4 signaling regulate the tumor stromal proangiogenic microenvironment via CXCL12-CXCR4 chemokine systems

Bone marrow (BM)-derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)-2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3-/- mice and EP4-/- mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins.

Roles of calcitonin gene-related peptide in facilitation of wound healing and angiogenesis.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide produced by tissue-specific alternative splicing of the primary transcript of the calcitonin/CGRP gene. CGRP is widely distributed in the central and peripheral neuronal systems and exhibits numerous biological activities in mammals. We examined in the present study whether or not endogenous CGRP released from neuronal systems facilitates neovascularization indispensable to wound healing. In CGRP knockout mice (CGRP-/-), wound-induced angiogenesis and wound closure were significantly suppressed compared with those in wild-type mice. The suppressed healing in CGRP-/- was accompanied by reduction in expressions of vascular endothelial growth factor (VEGF) in the wound granulation tissues. A CGRP antagonist, CGRP8-37 when infused with mini-osmotic pumps subcutaneously blocked the wound healing processes and reduced the expressions of CD31 and VEGF expression in the wound granulation tissues. Wound healing process was significantly delayed in neuropeptide-depleted mice pretreated with capsaicin, compared with vehicle-treated mice. These results indicate that CGRP derived from neuronal systems may facilitate wound healing and angiogenesis. Targeting of CGRP may be promising in controlling angiogenesis related to pathophysiological conditions.

Neuronal system-dependent facilitation of tumor angiogenesis and tumor growth by calcitonin gene-related peptide

A neuropeptide, calcitonin gene-related peptide (CGRP), is widely distributed in neuronal systems and exhibits numerous biological activities. Using CGRP-knockout mice (CGRP−/−), we examined whether or not endogenous CGRP facilitates angiogenesis indispensable to tumor growth. CGRP increased tube formation by endothelial cells in vitro and enhanced sponge-induced angiogenesis in vivo. Tumor growth and tumor-associated angiogenesis in CGRP−/− implanted with Lewis lung carcinoma (LLC) cells were significantly reduced compared with those in wild-type (WT) mice. A CGRP antagonist, CGRP8-37 or denervation of sciatic nerves (L1–5) suppressed LLC growth in the sites of denervation compared with vehicle infusion or sham operation. CGRP precursor mRNA levels in the dorsal root ganglion in LLC-bearing WT were increased compared with those in non-LLC-bearing mice. This increase was abolished by denervation. The expression of VEGF in tumor stroma was down-regulated in CGRP−/−. These results indicate that endogenous CGRP facilitates tumor-associated angiogenesis and tumor growth and suggest that relevant CGRP may be derived from neuronal systems including primary sensory neurons and may become a therapeutic target for cancers.

Roles of Prostaglandin E2–EP3/EP4 Receptor Signaling in the Enhancement of Lymphangiogenesis During Fibroblast Growth Factor-2–Induced Granulation Formation

Objective—One of the hallmarks of inflammation is lymphangiogesis that drains the interstitial fluids. During chronic inflammation, angiogenesis is induced by a variety of inflammatory mediators, such as prostaglandins (PGs). However, it remains unknown whether they enhance lymphangiogenesis. We examined the roles of cyclooxygenase-2 (COX-2) and PGE2 receptor signaling in enhancement of lymphangiogenesis during proliferative inflammation. Methods and Results—Lymphangiogenesis estimated by podoplanin/vascular endothelial growth factor (VEGF) receptor-3/LYVE-1 expression was upregulated during proliferative inflammation seen around and into subcutaneous Matrigel plugs containing fibroblast growth factor-2 (125 ng/site). A COX-2 inhibitor (celecoxib) significantly reduced lymphangiogenesis in a dose-dependent manner, whereas topical PGE2 enhanced lymphangiogenesis. Topical injection of fluorescein isothiocyanate–dextran into the Matrigel revealed that lymphatic flow from the Matrigels was COX-2 dependent. Lymphangiogenesis was suppressed in the granulation tissues of mice lacking either EP3 or EP4, suggesting that these molecules are receptors in response to endogenous PGE2. An EP3-selective agonist (ONO-AE-248) increased the expression of VEGF-C and VEGF-D in cultured macrophages, whereas an EP4-selective agonist (ONO-AE1-329) increased VEGF-C expression in cultured macrophages and increased VEGF-D expression in cultured fibroblasts. Conclusion—Our findings suggest that COX-2 and EP3/EP4 signaling contributes to lymphangiogenesis in proliferative inflammation, possibly via induction of VEGF-C and VEGF-D, and may become a therapeutic target for controlling lymphangiogenesis.

Calcitonin gene-related peptide facilitates revascularization during hindlimb ischemia in mice

It is known that the neural system plays a fundamental role in neovascularization. A neuropeptide, calcitonin gene-related peptide (CGRP), is widely distributed in the central and peripheral neuronal systems. However, it remains to be elucidated the role of CGRP in angiogenesis during ischemia. The present study examined whether endogenous CGRP released from neuronal systems facilitates revascularization in response to ischemia using CGRP knockout mice (CGRP-/-). CGRP-/- or their wild-type littermates (CGRP+/+) were subjected to unilateral hindlimb ischemia. CGRP-/- exhibited impaired blood flow recovery from ischemia and decreased capillary density expressed in terms of the number of CD-31-positive cells in the ischemic tissues compared with CGRP+/+. In vivo microscopic studies showed that the functional capillary density in CGRP-/- was reduced. Hindlimb ischemia increased the expression of pro-CGRP mRNA and of CGRP protein in the lumbar dorsal root ganglia. Lack of CGRP decreased mRNA expression of growth factors, including CD31, vascular endothelial growth factor-A, basic fibroblast growth factor, and transforming growth factor-β, in the ischemic limb tissue. The application of CGRP enhanced the mRNA expression of CD31 and VEGF-A in human umbilical vein endothelial cells (HUVECs) and fibroblasts. Subcutaneous infusion of CGRP8-37, a CGRP antagonist, using miniosmotic pumps delayed angiogenesis and reduced the expression of proangiogenic growth factors during hindlimb ischemia. These results indicate that endogenous CGRP facilitates angiogenesis in response to ischemia. Targeting CGRP may provide a promising approach for controlling angiogenesis related to pathophysiological conditions.

Vascular endothelial growth factor receptor-1 signaling promotes liver repair through restoration of liver microvasculature after acetaminophen hepatotoxicity

Vascular endothelial growth factor (VEGF) and its receptors promote liver regeneration. The objective of the present study was to examine the role of VEGF receptor 1 (VEGFR1) signaling in hepatic tissue repair after acetaminophen (N-acetyl-para-aminophenol) (APAP)-induced liver injury. To do this, we treated VEGFR1 tyrosine kinase knockout (VEGFR1 TK(-/-)) and wild-type (WT) mice with APAP (300 mg/kg, ip). In WT mice, serum levels of alanine aminotransferase (ALT) and the necrotic area peaked between 8 and 24 h and then declined. In VEGFR1 TK(-/-) mice, ALT levels remained high at 48 h and extensive hepatic necrosis and hemorrhage were observed, as well as high mortality. Downregulation of hepatic messenger RNA expression of VEGFR1 and VEGFR2 was also noted in VEGFR1 TK(-/-) mice. VEGFR1 TK(-/-) mice displayed lower expression of proliferating cell nuclear antigen and of growth factors including hepatocyte growth factor, CD31, and basic fibroblast growth factor than WT. The hepatic microvasculature in VEGFR1 TK(-/-) was compromised as evidenced by impaired sinusoidal perfusion, suppressed endocytosis in liver sinusoidal endothelial cells (LSECs), and the formation of large gaps in LSECs. In WT mice, immunofluorescence revealed that recruited VEGFR1(+) cells in the necrotic area were positive for CD11b. VEGFR1 TK(-/-) exhibited fewer VEGFR1(+) and VEGFR2(+) cells. These results suggest that VEGFR1 signaling facilitates liver recovery from APAP hepatotoxicity by preventing excessive hemorrhage and reconstituting the sinusoids through recruitment of VEGFR1-expressing macrophages to the injured area and also through affecting expression of genes including hepatotrophic and pro-angiogenic growth factors.

Prostanoid induces premetastatic niche in regional lymph nodes

The lymphatic system is an important route for cancer dissemination, and lymph node metastasis (LNM) serves as a critical prognostic determinant in cancer patients. We investigated the contribution of COX-2-derived prostaglandin E2 (PGE2) in the formation of a premetastatic niche and LNM. A murine model of Lewis lung carcinoma (LLC) cell metastasis revealed that COX-2 is expressed in DCs from the early stage in the lymph node subcapsular regions, and COX-2 inhibition markedly suppressed mediastinal LNM. Stromal cell-derived factor-1 (SDF-1) was elevated in DCs before LLC cell infiltration to the lymph nodes, and a COX-2 inhibitor, an SDF-1 antagonist, and a CXCR4 neutralizing antibody all reduced LNM. Moreover, LNM was reduced in mice lacking the PGE2 receptor EP3, and stimulation of cultured DCs with an EP3 agonist increased SDF-1 production. Compared with WT CD11c+ DCs, injection of EP3-deficient CD11c+ DCs dramatically reduced accumulation of SDF-1+CD11c+ DCs in regional LNs and LNM in LLC-injected mice. Accumulation of Tregs and lymph node lymphangiogenesis, which may influence the fate of metastasized tumor cells, was also COX-2/EP3-dependent. These results indicate that DCs induce a premetastatic niche during LNM via COX-2/EP3-dependent induction of SDF-1 and suggest that inhibition of this signaling axis may be an effective strategy to suppress premetastatic niche formation and LNM.

Host prostaglandin EP3 receptor signaling relevant to tumor-associated lymphangiogenesis

Prostaglandin E(2) (PGE(2)) and prostaglandin E (EP) receptor signaling pathways have been implicated in the promotion of tumor growth and angiogenesis. However, little is known about their roles in lymphangiogenesis during tumor development. The present study evaluates whether endogenous PGE(2) exhibits a critical role in tumor-associated lymphangiogenesis. Treatment of male C57BL/6 mice with a cyclooxygenase-2 inhibitor, celecoxib, for seven days resulted in a 52.4% reduction in tumor size induced by subcutaneous injection of murine Lewis lung cells. Celecoxib treatment down-regulated the expression of vascular endothelial growth factor receptor (VEGFR)-3 in stromal tissues by 73.9%, and attenuated expression of podoplanin, a marker for lymphatic endothelial cells. To examine the role of host PGE receptor signaling, we tested four kinds of EP receptor knockout mice. At Day 7 after tumor cell implantation, EP3 receptor knockout mice, but not EP receptor knockout mice lacking EP1, EP2, or EP4, exhibited a 53.3% reduction in tumor weight, which was associated with a 74.5% reduction in VEGFR-3 mRNA expression in tumor stromal tissues. At Day 14, VEGFR-3 expression in EP3-/- mice remained significantly lower than that of their wild-type (WT) counterparts. The expression of VEGF-C in the tumor stromal tissues in EP3-/- mice were also reduced by 22.1% (Day 7) and 44.1% (Day 14), respectively. In addition, the level of immunoreactive podoplanin in the tumor tissues from EP3-/- mice was less than that of WT. These results suggest that host EP3 receptor signaling regulates tumor-associated lymphangiogenesis by up-regulating expression of VEGF-C and its receptor, VEGFR-3, in tumor stromal tissues. Host EP3 blockade together with COX-2 inhibition may be a novel therapeutic strategy to suppress tumor-associated lymphangiogenesis.

Suppressed recruitment of alternatively activated macrophages reduces TGF-β1 and impairs wound healing in streptozotocin-induced diabetic mice.

BACKGROUND Diabetes mellitus inhibits wound-induced angiogenesis, impairing the wound healing process and leading to the development of chronic wounds. Impaired healing of diabetic wounds is caused by persistent pro-inflammatory macrophages recruited to the granulation tissue; however, little is known about the phenotype of the macrophages involved in diabetic wound healing. The present study was conducted to examine the involvement of macrophages in impaired wound healing using streptozotocin (STZ)-induced diabetic mice. METHODS Full-thickness skin wounds were created on the backs of mice treated with STZ or vehicle. RESULTS Compared with controls, wound healing and angiogenesis were suppressed in STZ-treated mice, with attenuated expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in wound granulation tissue. STZ-treated mice exhibited enhanced recruitment of classically activated macrophages (M1) expressing inducible nitric oxide synthase (iNOS) and suppressed recruitment of alternatively activated macrophages (M2) expressing transforming growth factor-beta-1 (TGF-β1). Treatment of diabetic mice with TGF-β1 restored wound healing and angiogenesis and normalized M1/M2 macrophage polarization in the granulation tissue. CONCLUSIONS These results suggest that an imbalance of macrophage phenotypes contributes to impaired wound healing in STZ-induced diabetic mice, and treatment with cytokines derived from M2 macrophages may be an effective therapeutic strategy to increase angiogenesis and promote healing of diabetic wounds.

Role of COX-2 in lymphangiogenesis and restoration of lymphatic flow in secondary lymphedema

The pathophysiology of secondary lymphedema remains poorly understood. To clarify the roles of cyclooxygenase (COX)-2 in enhancement of lymphangiogenesis during secondary lymphedema, we tested a mouse tail model and evaluated the recurrence of lymph flow. To induce lymphedema, a circumferential incision was made in the tail of anesthetized mice to sever the dermal lymphatic vessels. The maximum diameters of the tails were measured weekly. We found that the diameters of the tails around the wounds were markedly increased after surgery, and reached maximum size 2 weeks after wounding in mice without a COX-2 inhibitor, celecoxib (Celecoxib–). Expression of COX-2 in wound granulation tissues was markedly increased 1 week after surgery compared with unwounded naive control mice. In Celecoxib–, recurrence of lymphatic flow in the wound granulation tissues was detected 3 weeks after surgical treatment. In contrast, lymphatic flow was markedly suppressed in mice treated with celecoxib (Celecoxib+). Newly formed lymphatic structures were identified in the granulation tissues formed at wounded lesions in Celecoxib–, whereas those were markedly suppressed in Celecoxib+. Interstitial tissue pressures in the distal areas of the tail wounds were markedly increased in Celecoxib+ with reduced expression of vascular endothelial cell growth factor (VEGF)-C. F4/80-positive cells were accumulated to the wound granulation tissues in Celecoxib–, and the accumulation of these cells was suppressed in Celecoxib+. Prostaglandin E2 (PGE2) upregulated the expressions of VEGF-A and VEGF-C in cultured macrophages, but not human lymphatic microvascular endothelial cells. The present study therefore suggests that lymphangiogenesis, together with recurrence of lymph flow after surgical induction of lymphedema, is upregulated by COX-2 possibly via generation of PGs.