Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo.

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a ß-arrestin/protease fusion protein. Activated S1P1 recruits the ß-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

The role of interleukin 1 in growth and metastasis of human cancer xenografts.

BACKGROUND: Interleukin 1 (IL-1) is a pluripotent cytokine that promotes angiogenesis, tumor growth, and metastasis in experimental models; its presence in some human cancers is associated with aggressive tumor biology. The purpose of these studies was to characterize the role of IL-1 in human cancers and determine if inhibition of IL-1 via its receptor antagonist, IL-1Ra, alters tumor growth and metastatic potential. METHODS: IL-1 mRNA or protein levels were determined in clinical tumor samples, cancer cell lines, and xenografts using quantitative reverse transcription-PCR or ELISA. Biological activity of tumor-derived IL-1 protein was shown via induction of permeability across endothelial cell monolayers. The effects of recombinant IL-1Ra on tumor lines in culture (cell proliferation and IL-8 secretion) and in xenograft models (tumor growth, metastatic potential, and intratumoral levels of IL-8 and VEGF) were characterized. The effects of IL-1Ra-mediated regression of xenograft growth on angiogenic proteins (IL-8 and VEGF) were evaluated in an IL-1-producing melanoma (SMEL) xenograft model. RESULTS: IL-1 mRNA was highly expressed in more than half of all tested metastatic human tumor specimens including non-small-cell lung carcinoma, colorectal adenocarcinoma, and melanoma tumor samples. Constitutive IL-1 mRNA expression was identified in several cancer cell lines; tumor supernatant from these cell lines produced a significant increase in endothelial cell monolayer permeability, a hallmark event in early angiogenesis, in an IL-1-dependent manner. Moreover, systemic recombinant IL-1Ra resulted in significant inhibition of xenograft growth and neovessel density of IL-1-producing, but not non-IL-1-producing, tumor cell lines. Subsequent analysis of SMEL, a melanoma cell line with constitutive IL-1 production, showed that neither exogenous IL-1 nor IL-1Ra altered tumor cell proliferation rates in vitro. Gene expression analyses of IL-1Ra-treated SMEL xenografts showed a >3-fold down-regulation of 100 genes compared with control including a marked down-regulation of IL-8 and VEGF. CONCLUSIONS: These data show that the IL-1 gene is frequently expressed in metastases from patients with several types of human cancers. IL-1Ra inhibits xenograft growth in IL-1-producing tumors but has no direct antiproliferative effects in vitro; decreased tumor levels of IL-8 and VEGF may be an early surrogate of IL-1Ra-mediated antitumor activity. IL-1Ra may have a role alone or with other agents in the treatment of human cancers.

Interferon gamma-inducible protein 10 selectively inhibits proliferation and induces apoptosis in endothelial cells.

BACKGROUND: Interferon gamma-inducible protein 10 (IP-10) has antitumor effects in various murine models. The IP-10 receptor has two distinct splice variants, CXCR3A and CXCR3B, that have paradoxical effects after ligand-receptor interaction. METHODS: To characterize the putative antiangiogenic effects of IP-10, we measured proliferation rates and apoptosis in human umbilical vein endothelial cells (HUVECs), fibroblasts, and A375 melanoma or WIDR adenocarcinoma cell lines after exposure to the recombinant protein. CXCR3A (activating) and CXCR3B (inhibitory/proapoptotic) messenger RNA (mRNA) expression levels in fibroblasts, 2 human tumor cell lines, T lymphocytes, and HUVECs of varying cell densities were characterized. RESULTS: IP-10 resulted in dose-dependent and selective inhibition of proliferation and countered the proliferative effects of vascular endothelial growth factor in HUVECs but did not affect fibroblasts or 2 human tumor cell lines. In addition, IP-10 resulted in potent and selective induction of apoptosis in HUVECS but had no effect on fibroblasts or A375 melanoma. Confluent HUVECs had a predominance of mRNA for the CXCR3B splice variant by reverse transcriptase-polymerase chain reaction, and the ratio of CXCR3B to CXCR3A mRNA was >40 in HUVECs, compared with

Interleukin-1beta induced vascular permeability is dependent on induction of endothelial tissue factor (TF) activity.

IL-1beta is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1beta are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1beta induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1beta caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1beta induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.

Alterations in tumor necrosis factor-induced endothelial cell procoagulant activity by hyperthermia.

TNF is a cytokine with potent antitumor activity in murine models and when administered clinically via regional perfusion. There is substantial evidence that this antitumor activity depends in large part on TNF's procoagulant effect on tumor neovasculature, which is mediated by induction of endothelial cell tissue factor (TF), a component of the extrinsic clotting cascade. In regional perfusion of a cancer-bearing limb or organ, TNF is always administered under hyperthermic temperatures; however, little is known about the effect of hyperthermia on TNF-mediated procoagulant activity in endothelium. We examined the effects of hyperthermia on TNF-mediated procoagulant activity in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to TNF at normothermic (37 degrees C) and hyperthermic (41 degrees C) temperatures for 90 min, then assayed for clotting activity, TF protein production and mRNA production of TF and tissue factor pathway inhibitor-2 (TFPI-2), an endogenous inducible inhibitor of TF activity in HUVECs. TNF treatment at 41 degrees C significantly reduced clotting activity, TF protein and mRNA as well as TFPI-2 mRNA compared to treatment at 37 degrees C. These data show that hyperthermia significantly reduces the procoagulant effects of TNF on endothelial tissue compared to normothermia, which may have important clinical implications for the use of TNF in regional perfusion.

Parathyroid gland-specific deletion of the mouse Men1 gene results in parathyroid neoplasia and hypercalcemic hyperparathyroidism.

The inactivation of the MEN1 tumor suppressor gene in patients leads to a constellation of changes in endocrine tissues, including parathyroid neoplasia, pituitary adenomas, pancreatic neuroendocrine tumors, and carcinoids. To study the pathophysiological consequences of the deletion of the MEN1 gene, we set out to create a mouse model of hyperparathyroidism resulting from the deletion of the Men1 gene in parathyroid tissue. We introduced a Men1 gene flanked by loxP sites into the mouse germ line and then used a parathyroid cell-specific promoter to drive the expression of Cre recombinase, resulting in the deletion of the Men1 gene. Here, we show that loss of Men1 gene function in the parathyroid glands of mice results in histological changes consistent with parathyroid neoplasia as well as systemic hypercalcemia. This model provides a means for dissecting the molecular basis of this familial cancer syndrome and may allow for the development of new strategies to treat related forms of hypercalcemia.

Effect of interleukin 1 receptor antagonist gene transduction on human melanoma xenografts in nude mice.

Interleukin (IL)-1 is a pleiotropic inflammatory cytokine that promotes angiogenesis and enhances tumor growth and metastases. We evaluated the effects of IL-1 receptor antagonist (IL-1ra) on tumor growth and metastases in human melanoma xenografts. We selected two human melanoma lines (SMEL and PMEL) with differential (high versus low, respectively) constitutive production of IL-1 by ELISA. The IL-1ra gene was isolated from monocyte RNA by PCR and retrovirally transduced into SMEL and PMEL. In vitro cell proliferation was evaluated using a WST-1 assay. Athymic nude mice received s.c. or i.v. injection with parental, vector-transduced, or IL-1ra-transduced melanoma cells, and tumor growth, lung metastases, and histology were characterized. IL-1 was produced by SMEL in vitro and ex vivo (117 and 67 pg/ml/10(6) cells/24 h, respectively), but not by PMEL (15 and 0 pg/ml/10(6) cells/24 h, respectively). Neither made IL-1ra natively. Gene-transduced cell lines secreted >1000 pg/ml/10(6) cells/24 h of IL-1ra by ELISA. In vitro proliferation of each parental cell line was comparable to the proliferation rate of each transduced cell line. IL-1ra-transduced SMEL (SMEL/IL-1ra) showed significantly slower tumor growth compared with null-transduced and parental cell lines (P < 0.001, ANOVA-Bonferroni/Dunn). There was no difference in growth rates between PMEL and IL-1ra-transduced PMEL (PMEL/IL-1ra). A mixing study of SMEL and SMEL/IL-1ra showed significant inhibition of tumor growth at various ratios (P < 0.001, ANOVA-Bonferroni/Dunn). There were significantly fewer lung metastases with SMEL/IL-1ra versus SMEL (P < 0.002). IL-1ra decreases in vivo growth and metastatic potential of a human melanoma xenograft that constitutively secretes IL-1. This effect may be exploitable using clinically available IL-1ra for the treatment of human cancers.

Augmentation of endothelial cell monolayer permeability by hyperthermia but not tumor necrosis factor: evidence for disruption of vascular integrity via VE-cadherin down-regulation.

Following isolated limb perfusion (ILP) with hyperthermia (H T), TNF and melphalan, there is immediate tumor softening secondary to augmentation of capillary leak in the tumor neovasculature. TNF can induce vascular permeability but is always used with HT during ILP and the contribution of the latter on permeability is not known. This study characterizes the effects of HT and TNF on vascular permeability in vitro. Permeability across confluent human umbilical vein endothelial cells exposed to HT (40 degrees C) with or without 0.1-1000 ng/ml TNF was assessed by quantitating flux of albumin bound Evan's Blue dye from the upper to lower chamber. Immunofluorescent staining for VE-cadherin and F-actin was performed after human umbilical vein endothelial cells (hUVECs) were exposed to these conditions. HT induced a significant and reversible increase in permeability compared to untreated hUVECs (p<0.001) whereas barrier function was not altered by TNF. Untreated hUVECs had uniform cell surface staining for VE-cadherin, the primary endothelial intercellular adhesion molecule, with colocalization of F-actin cytoskeletal elements. HT resulted in a marked decrease in VE-cadherin staining and contraction of F-actin at sites of endothelial cell-cell separation. These data demonstrate that under conditions relevant to those used in ILP, HT but not TNF contributes to a rapid and reversible change in endothelial cell permeability in association with a down regulation of VE-cadherin. These data support the use of HT in isolation perfusion and demonstrate a novel mechanism for alterations in microvascular permeability by HT.

Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF.

Tumor necrosis factor (TNF) has marked effects on permeability and procoagulant activity on tumor-associated neovasculature when used in isolation perfusion, the latter effect primarily mediated via induction of cell surface expression of tissue factor (TF) on endothelial tissue. However, the cellular events that result in rapid alterations in endothelial cell (EC) permeability after intravascular TNF administration in isolation perfusion are not well characterized. We demonstrate that short exposure intervals to TNF induces TF expression on ECs but has no effect on permeability as assessed by flux of Evans blue-bound albumin across confluent EC monolayers using a 2-compartment model under basal culture conditions. However, a rapid and significant increase in EC permeability occurred with TNF in the presence of factor VIII-deficient plasma. Permeability was induced only with luminal versus abluminal TNF exposure and was blocked by antithrombin III, TF pathway inhibitor, or anti-TF antibody cotreatment. These data indicate that EC surface expression of TF and extrinsic clotting factors are critical in augmenting capillary leak following intravascular TNF administration. Alterations in permeability were associated with intercellular gap formation at sites of down-regulation of vascular endothelial (VE)-cadherin expression, the primary endothelial intercellular adhesion molecule, and intracellular contraction and alignment of F-actin cytoskeletal elements. Rapid induction of TF by TNF may be the primary EC response that results in alterations in permeability and procoagulant activity observed following intravascular TNF administration in isolation perfusion.

Retroviral gene transfer of interferon-inducible protein 10 inhibits growth of human melanoma xenografts.

Interferon-inducible protein 10 (IP-10) is an immunomodulatory chemokine recently recognized to have potent antiangiogenic activity in vivo. Due to difficulties in the stability, manufacture and chronic administration of recombinant forms of endogenous antiangiogenic proteins, antiangiogenic gene therapy has emerged as a promising new form of cancer treatment. We retrovirally transduced A375 human melanoma cells with the human IP-10 gene and injected cells subcutaneously into nude mice. IP-10-transduced cells also were mixed with null-transduced cells in varying proportions before injection. In vivo growth of IP-10-transduced melanoma cells was markedly diminished compared to parental or null-transduced cells (p = 0.0002, Kruskal-Wallis test). This growth inhibition was associated with a marked reduction in microvessel density. The degree of growth inhibition of tumors following injection of a mixed population of null- and IP-10-transduced cells was directly associated with the fraction of IP-10-transduced cells present. We conclude that retroviral transduction of human melanoma cells with the IP-10 gene leads to sufficient protein secretion to inhibit angiogenesis and tumor growth. These findings suggest that IP-10 gene therapy might be an effective therapy in patients with cancer.