Antigen presentation, autoantibody production, and therapeutic targets in autoimmune liver disease.

The liver is an important immunological organ that controls systemic tolerance. The liver harbors professional and unconventional antigen-presenting cells that are crucial for tolerance induction and maintenance. Orchestrating the immune response in homeostasis depends on a healthy and well-toned immunological liver microenvironment, which is maintained by the crosstalk of liver-resident antigen-presenting cells and intrahepatic and liver-infiltrating leukocytes. In response to pathogens or autoantigens, tolerance is disrupted by unknown mechanisms. Intrahepatic parenchymal and nonparenchymal cells exhibit unique antigen-presenting properties. The presentation of microbial and endogenous lipid-, metabolite- and peptide-derived antigens from the gut via conventional and nonconventional mechanisms can educate intrahepatic immune cells and elicit effector responses or tolerance. Perturbation of this balance results in autoimmune liver diseases, such as autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis. Although the exact etiologies of these autoimmune liver diseases are unknown, it is thought that the disruption of tolerance towards self-antigens and microbial metabolites and lipids, as well as alterations in bile acid composition, may result in changes in effector cell activation and polarization and may reduce or impair protective anti-inflammatory regulatory T and B cell responses. Additionally, the canonical and noncanonical transmission of antigens and antigen:MHC complexes via trogocytosis or extracellular vesicles between different (non) immune cells in the liver may play a role in the induction of hepatic inflammation and tolerance. Here, we summarize emerging aspects of antigen presentation, autoantibody production, and the application of novel therapeutic approaches in the characterization and treatment of autoimmune liver diseases.

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium.

BACKGROUND: Brain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC. METHODS: Immunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice. RESULTS: ALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors. CONCLUSIONS: Our findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM. KEY POINTS: 1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC.2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium.3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.

Contribution of Macrophage Efferocytosis to Liver Homeostasis and Disease.

The clearance of apoptotic cells is pivotal for both maintaining tissue homeostasis and returning to homeostasis after tissue injury as part of the regenerative resolution response. The liver is known for its capacity to remove aged and damaged cells from the circulation and can serve as a graveyard for effector T cells. In particular Kupffer cells are active phagocytic cells, but during hepatic inflammatory responses incoming neutrophils and monocytes may contribute to pro-inflammatory damage. To stimulate resolution of such inflammation, myeloid cell function can change, via sensing of environmental changes in the inflammatory milieu. Also, the removal of apoptotic cells via efferocytosis and the signaling pathways that are activated in macrophages/phagocytes upon their engulfment of apoptotic cells are important for a return to tissue homeostasis. Here, we will discuss, how efferocytosis mechanisms in hepatic macrophages/phagocytes may regulate tissue homeostasis and be involved in tissue regeneration in liver disease.

CEACAM1 in Liver Injury, Metabolic and Immune Regulation.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein that is expressed on epithelial, endothelial and immune cells. CEACAM1 is a differentiation antigen involved in the maintenance of epithelial polarity that is induced during hepatocyte differentiation and liver regeneration. CEACAM1 regulates insulin sensitivity by promoting hepatic insulin clearance, and controls liver tolerance and mucosal immunity. Obese insulin-resistant humans with non-alcoholic fatty liver disease manifest loss of hepatic CEACAM1. In mice, deletion or functional inactivation of CEACAM1 impairs insulin clearance and compromises metabolic homeostasis which initiates the development of obesity and hepatic steatosis and fibrosis with other features of non-alcoholic steatohepatitis, and adipogenesis in white adipose depot. This is followed by inflammation and endothelial and cardiovascular dysfunctions. In obstructive and inflammatory liver diseases, soluble CEACAM1 is shed into human bile where it can serve as an indicator of liver disease. On immune cells, CEACAM1 acts as an immune checkpoint regulator, and deletion of Ceacam1 gene in mice causes exacerbation of inflammation and hyperactivation of myeloid cells and lymphocytes. Hence, hepatic CEACAM1 resides at the central hub of immune and metabolic homeostasis in both humans and mice. This review focuses on the regulatory role of CEACAM1 in liver and biliary tract architecture in health and disease, and on its metabolic role and function as an immune checkpoint regulator of hepatic inflammation.

Carcinoembryonic antigen-related cell adhesion molecule 1 controls IL-2-dependent regulatory T-cell induction in immune-mediated hepatitis in mice.

A dysbalance between effector T cells (Tconv) and regulatory T cells (Tregs) and impaired Treg function can cause autoimmune liver disease. Therefore, it is important to identify molecular mechanisms that control Treg homeostasis. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a) is an immune coreceptor with dichotomous roles in T-cell regulation: its short isoform (CEACAM1S) can activate T cells and induce Tregs, whereas its long isoform (CEACAM1L), containing two intracellular immune receptor tyrosine-based inhibitory motifs, can inhibit activated T-cell function. In the liver, CEACAM1 has antifibrotic effects in models of nonalcoholic steatohepatitis. However, its role in immune-mediated hepatitis is unknown. In the mouse model of concanavalin A-induced CD4+ T-cell-dependent liver injury, liver damage was aggravated and persisted in Ceacam1-/- mice. Concomitantly, we observed hyperexpansion of Tconv, but reduction of interleukin (IL)-2 production and hepatic forkhead box protein P3+ (Foxp3+ )CD4+ Treg numbers. CEACAM1-/- CD4+ T cells showed impaired IL-2-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation, which correlated with a failure of naïve CEACAM1-/- CD4+ T cells to convert into Tregs in vitro. Furthermore, CEACAM1-/- Tregs expressed reduced levels of Foxp3, CD25, and B-cell lymphoma 2. Adoptive transfer experiments demonstrated that hepatic Treg expansion and suppressive activity required CEACAM1 expression on both CD4+ T cells and Tregs. We identified predominant CEACAM1S expression on hepatic CD4+ T cells and Tregs from mice with acute liver injury and expression of both isoforms in liver-derived CD4+ T-cell clones from patients with liver injury. CONCLUSION: Our data suggest that CEACAM1S expression in CD4+ T cells augments IL-2 production and STAT5 phosphorylation leading to enhanced Treg induction and stability, which, ultimately, confers protection from T-cell-mediated liver injury. (Hepatology 2018;68:200-214).

CEACAM1 controls the EMT switch in murine mammary carcinoma in vitro and in vivo.

We analyzed the molecular basis for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-controlled inhibition of epithelial-mesenchymal transition (EMT) in a mouse model for mammary adenocarcinoma (WAP-T mice). We demonstrate that silencing of CEACAM1 in WAP-T tumor-derived G-2 cells induces epithelial-mesenchymal plasticity (EMP), as evidenced by typical changes of gene expression, morphology and increased invasion. In contrast, reintroduction of CEACAM1 into G-2 cells reversed up-regulation of genes imposing mesenchymal transition, as well as cellular invasion. We identified the Wnt-pathway as target for CEACAM1-mediated repression of EMT. Importantly, ß-catenin phosphorylation status and transcriptional activity strongly depend on CEACAM1 expression: CEACAM1high G-2 cells displayed enhanced phosphorylation of ß-catenin at S33/S37/T41 and decreased phosphorylation at Y86, thereby inhibiting canonical Wnt/ß-catenin signaling. We identified Src-homology 2 domain-containing phosphatase 2 (SHP-2) as a critical binding partner of CEACAM1 that could modulate ß-catenin Y86 phosphorylation. Hence, CEACAM1 serves as a scaffold that controls membrane proximal ß-catenin signaling. In vivo, mammary tumors of WAP-T/CEACAM1null mice displayed increased nuclear translocation of ß-catenin and a dramatically enhanced metastasis rate compared to WAP-T mice. Hence, CEACAM1 controls EMT in vitro and in vivo by site-specific regulation of ß-catenin phosphorylation. Survival analyses of human mammary carcinoma patients corroborated these data, indicating that CEACAM1 is a prognostic marker for breast cancer survival.

Modulation of liver tolerance by conventional and nonconventional antigen-presenting cells and regulatory immune cells.

The liver is a tolerogenic organ with exquisite mechanisms of immune regulation that ensure upkeep of local and systemic immune tolerance to self and foreign antigens, but that is also able to mount effective immune responses against pathogens. The immune privilege of liver allografts was recognized first in pigs in spite of major histo-compatibility complex mismatch, and termed the "liver tolerance effect". Furthermore, liver transplants are spontaneously accepted with only low-dose immunosuppression, and induce tolerance for non-hepatic co-transplanted allografts of the same donor. Although this immunotolerogenic environment is favorable in the setting of organ transplantation, it is detrimental in chronic infectious liver diseases like hepatitis B or C, malaria, schistosomiasis or tumorigenesis, leading to pathogen persistence and weak anti-tumor effects. The liver is a primary site of T-cell activation, but it elicits poor or incomplete activation of T cells, leading to their abortive activation, exhaustion, suppression of their effector function and early death. This is exploited by pathogens and can impair pathogen control and clearance or allow tumor growth. Hepatic priming of T cells is mediated by a number of local conventional and nonconventional antigen-presenting cells (APCs), which promote tolerance by immune deviation, induction of T-cell anergy or apoptosis, and generating and expanding regulatory T cells. This review will focus on the communication between classical and nonclassical APCs and lymphocytes in the liver in tolerance induction and will discuss recent insights into the role of innate lymphocytes in this process.

Progesterone and HMOX-1 promote fetal growth by CD8+ T cell modulation.

Intrauterine growth restriction (IUGR) affects up to 10% of pregnancies in Western societies. IUGR is a strong predictor of reduced short-term neonatal survival and impairs long-term health in children. Placental insufficiency is often associated with IUGR; however, the molecular mechanisms involved in the pathogenesis of placental insufficiency and IUGR are largely unknown. Here, we developed a mouse model of fetal-growth restriction and placental insufficiency that is induced by a midgestational stress challenge. Compared with control animals, pregnant dams subjected to gestational stress exhibited reduced progesterone levels and placental heme oxygenase 1 (Hmox1) expression and increased methylation at distinct regions of the placental Hmox1 promoter. These stress-triggered changes were accompanied by an altered CD8+ T cell response, as evidenced by a reduction of tolerogenic CD8+CD122+ T cells and an increase of cytotoxic CD8+ T cells. Using progesterone receptor- or Hmox1-deficient mice, we identified progesterone as an upstream modulator of placental Hmox1 expression. Supplementation of progesterone or depletion of CD8+ T cells revealed that progesterone suppresses CD8+ T cell cytotoxicity, whereas the generation of CD8+CD122+ T cells is supported by Hmox1 and ameliorates fetal-growth restriction in Hmox1 deficiency. These observations in mice could promote the identification of pregnancies at risk for IUGR and the generation of clinical interventional strategies.

Increased osteoclastogenesis in mice lacking the carcinoembryonic antigen-related cell adhesion molecule 1.

Alterations in bone remodeling are a major public health issue, as therapeutic options for widespread bone disorders such as osteoporosis and tumor-induced osteolysis are still limited. Therefore, a detailed understanding of the regulatory mechanism governing bone cell differentiation in health and disease are of utmost clinical importance. Here we report a novel function of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a member of the immunoglobulin superfamily involved in inflammation and tumorigenesis, in the physiologic regulation of bone remodeling. Assessing the expression of all members of the murine Ceacam family in bone tissue and marrow, we found CEACAM1 and CEACAM10 to be differentially expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts. While Ceacam10-deficient mice displayed no alteration in structural bone parameters, static histomorphometry demonstrated a reduced trabecular bone mass in mice lacking CEACAM1. Furthermore, cellular and dynamic histomorphometry revealed an increased osteoclast formation in Ceacam1-deficient mice, while osteoblast parameters and the bone formation rate remained unchanged. In line with these findings, we detected accelerated osteoclastogenesis in Ceacam1-deficient bone marrow cells, while osteoblast differentiation, as determined by mineralization and alkaline phosphatase assays, was not affected. Therefore, our results provide in vivo and in vitro evidence for a physiologic role of CEACAM1 in the regulation of osteoclastogenesis.

CEACAM1 confers resistance toward oxygen-induced vessel damage in a mouse model of retinopathy of prematurity.

PURPOSE: To determine a functional role for the carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1) in retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). METHODS: In a 21/75/21% OIR mouse model, retinal neovascularization was compared in wild-type and CEACAM1-deficient mice. Animals were housed under normoxic conditions until postnatal day 7, followed by exposure to 75% oxygen for 5 days, and further housing under normoxic conditions. Retinal vascular anatomy, vaso-obliteration, neovascularization, and tuft formation were characterized and quantified in retinal flat-mounts from untreated mice and from experimental mice during and at different time points after exposure to high oxygen levels. The vascular network was stained with fluorescently labeled isolectin B4. RESULTS: Mice deficient in CEACAM1 did not present any apparent abnormalities in their postnatal retinal vascular development under normoxic housing conditions. However, after hyperoxia and under relative hypoxic conditions, retinal neovascularization and tuft formation were aggravated in the mutant. Congruently, revascularization and vessel maturation were delayed in CEACAM1-deficient mice whereas in wild-type mice, tuft regression and vascular remodeling occurred efficiently after exposure to high oxygen levels. CONCLUSIONS: Our report describes a functional role for CEACAM1 in retinal neovascularization in a mouse model of OIR. This is the first study demonstrating that CEACAM1 enhances vascular remodeling and tuft regression by increasing endothelial resistance to alterations in oxygen tension, thus accelerating vascular recovery after systemic hypoxia.

CEA a thrombus CAM: CEACAM2, a twin of CEACAM1?

In this issue of Blood, Alshahrani et al demonstrate that carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) is expressed on platelets and negatively regulates the collagen receptor glycoprotein (GP)VI-FcRγ chain and C-type lectin-like receptor 2 (CLEC-2)-mediated platelet activation.

Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits MMP-9-mediated blood-brain-barrier breakdown in a mouse model for ischemic stroke.

RATIONALE: Blood-brain-barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated. OBJECTIVE: We sought to identify and characterize the relevance of CEACAM1-expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1(-/-) and wild-type mice. METHODS AND RESULTS: Focal ischemia was induced by temporary occlusion of the middle cerebral artery with a microfilament. Using MRI and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion, and brain atrophy in Ceacam1(-/-) mice. This translated into poor performance in neurological scoring and high poststroke-associated mortality. Elevated neutrophil influx, hyperproduction, and release of neutrophil-related matrix metalloproteinase-9 in Ceacam1(-/-) mice were confirmed by immune fluorescence, flow cytometry, zymography, and stimulation of neutrophils. Importantly, neutralization of matrix metalloproteinase-9 activity in Ceacam1(-/-) mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human poststroke autoptic brains congruently identified abundance of CEACAM1(+)matrix metalloproteinase-9(+) neutrophils in the ischemic hemispheres. CONCLUSIONS: CEACAM1 controls matrix metalloproteinase-9 secretion by neutrophils in postischemic inflammation at the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia.

Acceleration of collateral development by carcinoembryonic antigen-related cell adhesion molecule 1 expression on CD11b/⁺Gr-1⁺ myeloid cells--brief report.

OBJECTIVE: Previously, we demonstrated the relevance for endothelial carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression in collateral formation. However, a proarteriogenic role for CEACAM1(+) myeloid cells is unknown. Here, we investigated the contribution of CEACAM1(+) myeloid cells on collateral formation. METHODS AND RESULTS: Collateral growth and vascular remodeling were analyzed in CEACAM1-competent and CEACAM1 null mice after femoral artery ligation in hindlimb ischemia. Reperfusion of the adductor muscles was evaluated by Laser Doppler measurements and microcomputed tomography imaging. In CEACAM1 null mice, poor reperfusion and reduced collateral formation were observed, accompanied by reduction in arterial diameters. Using flow cytometry, we identified an increase of the muscle-resident CD11b(+)/granulocyte receptor-1+ (Gr-1+) population in CEACAM1 null mice only, pointing toward a CEACAM1-dependent functional deviation. Direct and reciprocal bone marrow transplantations between CEACAM1-competent and CEACAM1 null mice, and antibody-mediated depletion of the CD11b(+)/Gr-1(+) population, confirmed the requirement of CEACAM1 expression on the CD11b(+)/Gr-1(+) population for reestablishment of perfusion after arterial occlusion. CONCLUSIONS: CEACAM1 expression on CD11b(+)/Gr-1(+) myeloid cells is a prerequisite for adequate collateral formation.

XMRV induces cell migration, cytokine expression and tumor angiogenesis: are 22Rv1 cells a suitable prostate cancer model?

22Rv1 is a common prostate cancer cell line used in xenograft mouse experiments as well as in vitro cell culture assays to study aspects of prostate cancer tumorigenesis. Recently, this cell line was shown to harbor multiple copies of a gammaretrovirus, called XMRV, integrated in its genome. While the original prostate cancer xenograft CWR22 is free of any retrovirus, subsequently generated cell lines 22Rv1 and CWR-R1, carry this virus and additionally shed infectious gammaretroviral particles in their supernatant. Although XMRV most likely was generated by recombination events in cell culture this virus has been demonstrated to infect human cells in vitro and 22Rv1 as well as CWR-R1 cells are now considered biosafety 2 reagents. Here, we demonstrate that 22Rv1 cells with reduced retroviral transcription show reduced tumor angiogenesis and increased necrosis of the primary tumor derived from xenografted cells in scid mice when compared to the parental cell line. The presence of XMRV transcripts significantly increases secretion of osteopontin (OPN), CXCL14, IL13 and TIMP2 in 22Rv1 cells. Furthermore, these data are supported by in vitro cell invasion and differentiation assays. Collectively, our data suggest that the presence of XMRV transcripts at least partially contributes to 22Rv1 characteristics observed in vitro and in vivo with regard to migration, invasion and tumor angiogenesis. We propose that data received with 22Rv1 cells or equivalent cells carrying xenotropic gammaretroviruses should be carefully controlled including other prostate cancer cell lines tested for viral sequences.

Transgenic over-expression of interleukin-33 in osteoblasts results in decreased osteoclastogenesis.

Interleukin-33 (IL-33) is the most recently identified member of the IL-1 family of cytokines, which is primarily known for its proinflammatory functions. We have previously reported that IL-33 is expressed by bone-forming osteoblasts, and that administration of recombinant IL-33 to bone marrow cultures inhibits their differentiation into bone-resorbing osteoclasts. Likewise, while the inhibitory effect of IL-33 on osteoclast differentiation was fully abolished in cultures lacking the IL-33 receptor ST2, mice lacking ST2 displayed low bone mass caused by increased osteoclastogenesis. Although these data suggested a physiological role of IL-33 as an inhibitor of bone resorption, direct in vivo evidence supporting such a function was still missing. Here we describe the generation and bone histomorphometric analysis of a transgenic mouse model (Col1a1-Il33) over-expressing IL-33 specifically in osteoblasts. While we did not observe differences in osteoblast number and bone formation between wildtype and Col1a1-Il33 mice, the number of osteoclasts was significantly reduced compared to wildtype littermates in two independent transgenic lines. Since we did not observe quantitative differences in the populations of eosinophils, neutrophils, basophils or M2-macrophages from the bone marrow of wildtype and Col1a1-Il33 mice, our data demonstrate that an inhibition of osteoclastogenesis is one of the major physiological functions of IL-33, at least in mice.

Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells.

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL-1) and IL-18, two cytokines of the IL-1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL-33, another IL-1 family member, has not been addressed yet. Since we observed that the expression of IL-33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL-33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1-deficient mice, which lack the IL-33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL-33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL-33 completely abolished the generation of TRACP(+) multinucleated osteoclasts, even in the presence of RANKL and macrophage colony-stimulating factor (M-CSF). Although our molecular studies revealed that IL-33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL-33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL-33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL-33 as an inhibitor of bone resorption.

TAE226-mediated inhibition of focal adhesion kinase interferes with tumor angiogenesis and vasculogenesis.

Neoangiogenesis plays an important role in tumor growth and metastasis. Evaluation of new anti-angiogenic targets may broaden the armament for future therapeutic concepts. Focal adhesion kinase (FAK), expressed in endothelial and tumor cells, is essential for adhesion and mobility of adherent cells. In the current study we analyzed the anti-angiogenic properties of the FAK inhibitor TAE226 on the proliferation of blood outgrowth endothelial cell (OEC) and differentiation of endothelial progenitor cells (EPC), derived from peripheral blood CD133(+) cells, tube formation and on neovascularization in a HT29 xenotransplant model. The effects of TAE226 were compared to those of the rapamycin analogue RAD001. The combination of both drugs was also studied. We showed that HT29 tumor cells and OEC were most sensitive to the action of TAE226 compared to EPC in vitro. In contrast, RAD001 affected the proliferation of both types of endothelial cells stronger than that of HT29 cells. Furthermore we could show that TAE226 inhibited tube formation in a dose dependent manner. In a HT29 subcutaneous tumor model TAE226 and RAD001 diminished MVD at commonly employed doses to a similar degree. Combination of both compounds did not show synergy in vitro or in vivo. Since TAE226 has been shown to inhibit the PI3 kinase, Akt kinase, mTor pathway, addition of RAD001 may not increase this effect. In conclusion, we have shown that treatment with TAE leads to a reduction of neoangiogenesis in vitro and in a mouse model. The effects are mediated by inhibition of angiogenesis and vasculogenic OEC and EPC.

Carcinoembryonic antigen-related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1-/- mice, the Ceacam1 gene was deleted systemically, and in CEACAM1(endo+) mice, CEACAM1 was overexpressed under the control of the endothelial cell-specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1-/- mice failed to establish new capillaries whereas in CEACAM1(endo+) mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1-/- mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1(endo+) mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease.