Lymphatic Vessel Endothelial Receptor Research Articles

Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model

IntroductionTumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors.Methods4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted.ResultsThe co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45+ leukocytes into tumor tissues. The proportions of Ki-67+ proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone.ConclusionThe crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis.

Abstract 606: Inhibitory effects of piceatannol on tumor angiogenesis and lung metastasis in the 4T1 mammary cancer model

Abstract Piceatannol (trans-3,4,3′,5′-tetrahydroxystilbene) is a polyphenol found in Rheum undulatum, grapes, red wine, and berries and has been shown to exert anticarcinogenic effects. In the present study, we determined whether oral administration of piceatannol inhibits solid tumor growth, angiogenesis, and lung metastasis of mammary cancer cells. 4T1 cells (5 x 104 cells) were injected into the mammary fat pad of syngeneic, 6-week old, female BALB/c mice. One day after, the mice were subjected to gavage for 30 days with piceatannol (0, 10, or 20 mg/kg body weight/day). Oral piceatannol treatment induced a significant reduction in the growth of solid tumors. Immunohistochemical staining of tumor tissues revealed that the expression of Ki67 (prototypic cell cycle related nuclear antigen), cyclin D1, cyclin A, cyclin-dependent kinase (CDK)4, CDK2, hypoxia-inducible factor 1, vascular endothelial growth factor, CD31 (platelet endothelial cell adhesion molecule-1) and LYVE-1 (lymphatic vessel endothelial receptor 1) was significantly decreased in the mice received 20 mg/kg piceatannol as compared to mice received vehicle. Piceatannol significantly increased the numbers of TUNEL-positive, apoptotic cells in tumor tissues, which was accompanied by increased Bax and cleaved caspase-3 expression and reduced Bcl-2 expression. Additionally, piceatannol feeding reduced the number and volume of pulmonary tumor nodules, which was accompanied by reduced expression of matrix metalloproteinase-9 in lung tissues. Piceatannol induced a significant reduction in the blood levels of chemokine ligand (CXCL) 16, CXCL13 (BCA-1), interleukin-16, macrophage colony-stimulating factor, monocyte chemoattractant protein-1, triggering receptor expressed on myeloid cells-1, stromal cell-derived factor-1, and plasminogen activator inhibitor-1 compared to the vehicle-fed mice. The present results indicate that piceatannol has an ability to inhibit breast cancer development and metastasis and this effect is attributable to its ability to inhibit tumor angiogenesis, lymphangiogenesis and reduce the blood levels of a broad variety of cytokines and chemokines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 606. doi:1538-7445.AM2012-606

Abstract 589: Benzyl isothiocyanate effectively inhibits solid tumor growth and lung metastasis of 4T1 mammary carcinoma cells in BALB/c mice fed on a high-fat diet

Abstract Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables. We previously observed that chronic consumption of a high-fat diet increased the breast cancer progression and lung metastasis in the 4T1 orthotopic BALB/c mouse model. In the present study, we determined whether BITC inhibits high-fat diet-induced acceleration of breast cancer growth and metastasis in the 4T1 model. 4-week old, female BALB/c mice were fed on a high-fat diet (HFD, 60 kcal% fat) or control diet (CD, 10 kcal% fat) and BITC was administered daily with oral gavage (0, 5, or 10 mg/kg body weight/day). After 16 weeks, 4T1 cells (5 × 104 cells) were injected into the mammary fat pads of syngeneic, female, BALB/c mice and the mice were continuously fed on the same diets and BITC. 26 day after the cancer cell injection, animals were killed. The weights of tumors and lungs were significantly increased in the HFD group as compared to the CD group, and the increases were suppressed by BITC treatment. Additionally, chronic consumption of the HFD markedly increased the number and volume of tumor nodules in the lung; the increases were markedly suppressed by BITC treatment. Immunohistochemical staining of tumor tissues revealed that the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31, an angiogenesis marker), vascular endothelial growth factor (VEGF)-A, LYVE-1 (lymphatic vessel endothelial receptor-1), VEGF-D, Ki67, cyclin-dependent kinase 2, cyclin A, cyclin D1, and hypoxia-inducible factor 1α (HIF-1α) was markedly increased in the HFD group as compared to the CD group; the increases were blocked by BITC treatment. Chronic consumption of the HFD reduced TUNEL-positive apoptotic cells, which was prevented by BITC treatment. The present results demonstrate that BITC suppressed HFD-stimulated breast cancer progression, which may have been mediated via the suppression of cell cycle progression, tumor angiogenesis and lymph angiogenesis, and induction of apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 589. doi:1538-7445.AM2012-589

Abstract 591: Licochalcone E inhibits solid tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells in the BALB/c mouse orthotopic model

Abstract Licochalcone E a phenolic constituent of licorice that is reported to exert excellent anti-cancer effects. In the present study, to investigate the effect of licochalcone E oral administration on the solid tumor growth and metastasis of breast cancer cells, 4T1 cells were injected into the inguinal mammary fat pads of syngeneic female BALB/c mice. One week after the injection the mice were subjected to oral gavage for 25 days with licochalcone E (0, 7, or 14 mg/kg body weight/day). Licochalcone E treatment induced a significant reduction in solid tumor growth, which was accompanied by 1) reduced Ki67 (prototypic cell cycle related nuclear antigen), cyclin-dependent kinase (CDK)2, CDK4, cyclin A, and cyclin D1 and 2) increased numbers of TUNEL-positive apoptotic cells with increased Bax and cleaved caspase-3 expression but reduced Bcl-2 expression in tumor tissues. Licochalcone E also reduced the expression of hypoxia inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), VEGF-R2, CD31 (platelet endothelial cell adhesion molecule-1), VEGF-C and LYVE-1 (lymphatic vessel endothelial receptor-) in tumor tissues. In addition to tumor growth, oral licochalcone E administration markedly reduced the number of pulmonary tumor nodules. In lung tissues, licochalcone E induced a significant reduction in the levels of cytokines and angiogenesis-related proteins including DPPIV (CD26), endoglin (CD105), MMP-9, C5a, interleukin (IL)-1ra, IL-16, MIG (CXCL9). In vitro culture studies showed that licochalcone E dose-dependently inhibited the migration of MDA-MB-231 human breast cancer cells at concentrations of 0-20 μmol/L without significant changes in cell viability. Licochalcone E significantly reduced secretion of matrix metalloproteinase-9, urokinase-type plasminogen activator and VEGF in concentration-dependent manners in MDA-MB-231 cells. These results demonstrated that licochalcone E inhibits solid tumor growth and metastasis of breast cancer cells and these effects were mediated via the reduction of a wide variety of cytokines and chemokines, which subsequently inhibits tumor angiogenesis, lymphangiogenesis, proliferation, and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 591. doi:1538-7445.AM2012-591

Blockade of Prolymphangiogenic Vascular Endothelial Growth Factor C in Dry Eye Disease

To determine whether blocking prolymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C) would suppress alloimmunity in dry eye disease using a murine model. The effects of intraperitoneal injections of 400 μg of anti-VEGF-C antibody (treated group) and intraperitoneal normal saline (untreated group) were studied in murine dry eyes induced by exposing mice to high-flow desiccated air in a controlled-environment chamber. Growth of lymphatic vessels and infiltration of macrophages were evaluated by immunohistochemistry using CD31 (panendothelial marker), lymphatic vessel endothelial receptor 1 (lymphatic endothelial marker), and CD11b (monocyte and macrophage marker). Real-time polymerase chain reaction was performed to quantify expression of different inflammatory cytokine transcripts in the conjunctiva and lymph nodes as well as vascular endothelial growth factors and their receptors (VEGF-A, VEGF-C, VEGF-D, VEGFR-2, and VEGFR-3) in the cornea. Blocking VEGF-C led to significant reductions in lymphatic caliber (P = .02) and lymphatic area (P = .006) in the corneas of mice with dry eye disease. In addition to significantly decreasing CD11b(+) cells (P = .005), anti-VEGF-C treatment significantly decreased transcript levels of VEGF-C (P = .002), VEGF-D (P = .01), and VEGFR-3 (P = .02) in the corneas of the treated group. A significant decrease in expression of inflammatory cytokines in the conjunctiva (interleukin 1α, P = .003; interleukin 1β, P = .02; interleukin 6, P = .005) and lymph nodes (interferon γ, P = .008; interleukin 17, P = .003) was also seen with anti-VEGF-C treatment. Treatment with anti-VEGF-C led to significant improvement in dry eye disease, reflected by a decrease in inflammation at the clinical, molecular, and cellular levels. Targeting prolymphangiogenic growth factors or their receptors could inhibit the trafficking of antigen-presenting cells to the draining lymph nodes and hence prove to be a potential therapeutic target for dry eye disease.

The relationship between corneal lymphangiogenesis and inflammation index after corneal alkali injury

To discuss the relationship between corneal lymphangiogenesis and inflammation index (IF) in alkali burned corneas. Experimental research. Rat corneal hemangiogenesis and lymphangiogenesis were examined by 5'-nase-alkaline phosphatase (5'-NA-ALP) double enzyme-histochemistry and whole mount immunofluorescence at 1 day, 3 days, and 1, 2, 3, 4, 5, 6, 7, 8 weeks after alkaline burns, and the blood vessel counting (BVC) and the lymphatic vessel counting (LVC) were recorded. The state of corneal inflammation was observed under the slit lamp and evaluated by inflammation index (IF) grading at the same time. Then, the association of LVC with IF was examined. In addition, eleven human alkali burned corneas were obtained from 11 patients undergoing corneal transplantation in Zhongshan Ophthalmic Center from January 2005 to June 2008. Corneal lymphangiogenesis was examined by lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry. The significance of the differences in IF, inflammatory cells counting, burn history, and age between two groups was analyzed by using paired student's t-test. New lymphatic vessels were present in rat alkali burned corneas. Corneal lymphangiogenesis developed 3 days after alkaline burns, reached the top 2 weeks after the injury, then decreased gradually, and disappeared at the end of the 5th week. Corneal lymphatics occurred behind corneal inflammation, but disappeared before corneal inflammation and hemangiogenesis. LVC was strongly and positively correlated with IF (r = 0.572, P < 0.01) after corneal alkaline burns. Among eleven human alkali burned corneas, corneal lymphatic vessels were present in 3 corneas. Compared with the other 8 cases without corneal lymphangiogenesis, the scores of IF was significantly higher (t = 3.28, P < 0.05), the inflammatory cells counting dramatically increased (t = 2.42, P < 0.05), but the age decreased significantly (t = 2.62, P < 0.05). However, the difference in burn history between two groups was not significant (t = 1.28, P > 0.05). Corneal lymphangiogenesis develops after alkaline-burns and correlates closely with inflammation index.

Novel Discovery of LYVE-1 Expression in the Hyaloid Vascular System

The hyaloid vascular system (HVS) is a transient network nourishing developing eyes and has been widely used as a natural model to study blood vessel regression. Failure of its regression in humans leads to several blinding diseases. Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a recently defined lymphatic marker that is also expressed by a subpopulation of macrophages. To date, there is no report on its expression in the HVS. This study was conducted to investigate whether LYVE-1 is expressed in the HVS and how it is associated with the vascular structure and macrophage phenotype. Normal C57BL/6 mouse eyeballs were sampled from embryonic day (E) 10.5 to postnatal (P) and adult stages for immunofluorescent microscopic studies with antibodies against LYVE-1, CD31 (panendothelial cell marker), and F4/80 (macrophage marker). Additionally, Angiopoietin-2 (Ang-2) knockout mice with abnormally persistent HVS were examined. The LYVE-1 expression was detected on normal HVS between E12.5 and P14. The LYVE-1(+) cells were F4/80(+) but CD31(-), indicating a macrophage lineage. Additionally, LYVE-1(+) cells bud on CD31(+) vessels and constitute an integral part of the network in both normal developing and Ang-2 knockout mice. This study provides the first evidence that the HVS contains a LYVE-1(+) cellular component in both physiological and pathologic conditions. This novel finding not only provides a new concept in defining the embryogenesis and pathogenesis of the HVS, it also leads to a completely natural model in which to study the functions of the LYVE-1 pathway, an important topic for lymphatic research as well.

Lymphatic Vessels Correlate Closely with Inflammation Index in Alkali Burned Cornea

Purpose: To study the relationship between corneal lymphangiogenesis and inflammation in alkali burned corneas.Methods: Rat corneal lymphatic and blood vessels were labeled and distinguished by whole mount immunofluorescence and 5'-nase-alkaline phosphatase (5'-NA-ALP) double enzyme-histochemistry. Then, lymphatic vessel areas (LVA) and lymphatic vessel counting (LVC) were examined. Corneal inflammation was evaluated by inflammation index (IF) grading, histopathology, electron microscope, and polymorphonuclear leukocyte (PMN) infiltration. The relationship between LVC, LVA, IF, and PMN was examined, respectively. In addition, corneal lymphatic vessels of eleven human alkali burned corneas were examined by lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry.Results: Corneal lymphangiogenesis occurred on Day 3, reached the peak at the end of two weeks, and disappeared five weeks after alkaline burns. Both LVA and LVC were strongly and positively correlated with IF after corneal alkaline burns. However, the relationship between LVC and PMN, between LVA and PMN were significant but converse. Among eleven human alkali burned corneas, corneal lymphangiogenesis was present in three corneas.Conclusions: Corneal lymphagiogenesis develops after alkaline burns and correlates closely with corneal inflammation.

Distinctive Properties of the Hyaluronan-binding Domain in the Lymphatic Endothelial Receptor Lyve-1 and Their Implications for Receptor Function

The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.

Corneal lymphangiogenesis correlates closely with hemangiogenesis after keratoplasty.

To examine the relationship between corneal lymphangiogenesis and hemangiogenesis after keratoplasty. Nineteen human corneas were obtained from 19 patients undergoing a second corneal transplantation in Zhongshan Ophthalmic Center in 2005. Blood and lymphatic vessels in human transplanted corneas were identified by lymphatic vessel endothelial receptor (LYVE-1) and platelet endothelial cell adhesion modecule-1 (PECAM-1) immunohistochemistry, and double enzyme-histochemistry; then the association of corneal blood vessel counting (BVC) with lymphatic vessel counting (LVC) was examined. Corneal hemangiogenesis was present in 12 cases (63%), and lymphangiogenesis occurred in 5 cases (26%) human transplanted corneas. In addition, corneal lymphangiogenesis was only present in vascularized corneas. LVC was strongly and positively correlated with BVC (r=0.725, P<0.01). Corneal lymphangiogenesis develops after keratoplasty and strongly associates with hemangiogenesis.

TGF-β1is a negative regulator of lymphatic regeneration during wound repair

Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -beta1 (TGF-beta1) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-beta1 on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-beta1 expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-beta1 on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-beta1, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-beta1 to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-beta1 inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-beta1 caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-beta1 during wound repair resulted in lymphatic fibrosis and the coexpression of alpha-smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-beta1 expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-beta1 expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-beta1 expression may be useful in promoting more rapid lymphatic regeneration.

The Normal Human Choroid Is Endowed with a Significant Number of Lymphatic Vessel Endothelial Hyaluronate Receptor 1 (LYVE-1)–Positive Macrophages

Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a newly discovered lymphatic endothelium-specific marker that is also expressed by a subpopulation of macrophages. To date, there is no report on its expression in the posterior human uvea. The purpose of this study was to investigate the expression of LYVE-1 in normal human choroids. Eyes of body/cornea donors (55-89 years of age; 4-9 hours postmortem) were obtained. Choroids were dissected and prepared for cryosections followed by immunohistochemistry with anti-human LYVE-1 antiserum and immunogold labeling. In addition, anti-human antibodies against macrophage markers (CD68, MHC class II) and lymphatic (podoplanin) and blood vascular endothelium (CD31, vWF) were used. For documentation, light-, fluorescence-, confocal laser scanning-, and electron-microscopy were used. The normal human choroidal stroma contained 274 +/- 86 LYVE-1 positive cells/mm(2). The cells displayed irregular shapes with a relatively uniform diameter of 32 mum. Costaining with CD68 and negativity for CD31, podoplanin, and melan-A/HMB45, as well as electron microscopic features, suggest these LYVE-1(+) cells to be macrophages. Besides that, no classic LYVE-1(+)/podoplanin(+) lymphatic vessels were detected within the normal adult human choroid. The normal adult human choroid does not contain typical lymph vessels, but is endowed with a significant number of LYVE-1 positive macrophages. These cells may be involved in choroidal hyaluronic acid metabolism or contribute to temporary formation of lymphatic channels under inflammatory conditions.

Clinical and Experimental Research of Corneal Lymphangiogenesis after Keratoplasty

The objective of this study is to provide further evidence that corneal lymphangiogenesis occurs after keratoplasty, and to explore the association of corneal hemangiogenesis, corneal inflammation and transplantation history with corneal lymphangiogenesis. Rat corneal lymphangiogenesis was examined by electron microscopy, lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry, and whole-mount immunofluorescence at 1, 3, 7, 10 and 14 days after corneal transplantation. Blood and lymphatic vessels in human transplanted corneas were identified by LYVE-1 and CD₃₁ immunohistochemistry, then the association between corneal blood vessel counting, inflammatory index and transplantation history with the lymphatic vessel counting was examined.The results showed that corneal lymphangiogenesis was present in all rat corneas and 26% of human transplanted corneas. Lymphatic vessel counting was significantly associated with blood vessel counting, inflammatory index and transplantation history (all p values <0.0001). We conclude that corneal lymphangiogenesis develops after keratoplasty, and is strongly associated with hemangiogenesis, inflammation and the history of transplantation.

Modification of the Primary Tumor Microenvironment by Transforming Growth Factor α-Epidermal Growth Factor Receptor Signaling Promotes Metastasis in an Orthotopic Colon Cancer Model

The transforming growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) signaling pathway appears to play a critical role in colon cancer progression, but the cellular and molecular mechanisms that contribute to metastasis remain unknown. KM12C colon cancer cell clones expressing high (C9) or negligible (C10) levels of TGFalpha were implanted into the cecal walls of nude mice. C9 tumors formed autocrine and paracrine EGFR networks, whereas C10 tumors were unable to signal through EGFR. The tumor microenvironment of C9, but not C10, contained cells enriched in vascular endothelial growth factor (VEGF) A, interleukin-8, and matrix metalloproteinases-2 and -9 and had a high vascular surface area. C9 tumors recruited a macrophage population that co-expressed F4/80 and lymphatic vessel endothelial hyaluronic acid receptor and produced VEGFC. The mean lymphatic density of C9 tumors was threefold higher than that of C10 tumors. C9, but not C10, tumor cells metastasized to regional lymph nodes in all mice and to the liver in 5 of 10 mice. Forced expression of TGFalpha in C10 tumor cells led to the generation of autocrine and paracrine EGFR signaling, macrophage recruitment, enhanced blood and lymphatic vascular surface areas, and increased lymphatic metastasis. Collectively, these data show that activation of TGFalpha-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFalpha-positive colon cancers.

Mapping of the Neonatal Fc Receptor in the Rodent Eye

The neonatal Fc receptor (FcRn) has been known to modulate IgG transport and protect against IgG catabolism, resulting in extension of the serum half-life of IgG. The goal of this study was to localize FcRn receptor expression in the rat's eye. The cornea, retina, conjunctiva, ciliary body and iris, retinal pigment epithelium and choroid, and lens were dissected from each rat's eye, and total RNA was purified. The first-strand cDNAs were synthesized and subjected to PCR reaction. For control samples, reverse transcriptase was omitted. A monoclonal antibody against the FcRn heavy chain was used to localize the distribution of the FcRn receptor in ocular tissues. Lymphatic vessels and blood vessels were stained with a rabbit anti-mouse lymphatic vessel endothelial receptor-1 polyclonal antibody and a rabbit anti-human von Willebrand factor polyclonal antibody, respectively. RT-PCR demonstrated expression of FcRn RNA in cornea, retina, conjunctiva, ciliary body and iris, and lens but absence of expression in the retinal pigment epithelium and choroid. Immunohistochemistry and double staining confirmed the expression of FcRn receptor to the conjunctival lymphatic vessels but not in the conjunctival blood vessels. In the ciliary body, the FcRn receptor was found to be expressed in both the nonpigmented ciliary epithelium and the ciliary blood vessels. The expression of FcRn receptor was confirmed in the retinal blood vessels, iris blood vessels, optic nerve vascular structures, corneal epithelium and endothelium, and lens epithelium. The FcRn receptor is expressed in multiple ocular tissues. The blood-ocular barrier showed FcRn receptor expression, indicating that IgG transport from ocular tissues to the blood system may use this receptor. The role of the FcRn receptor in the anterior segment and the conjunctiva remains unclear.

Pulmonary Lymphangioma

We report on the very rare case of a 49-year-old man with a large solitary pulmonary lymphangioma. Rapid growth of the tumor led to dyspnea and pain. A chest roentgenogram and computed tomography scan revealed a large 18 x 12-cm space-occupying cystic lesion in the posterior mediastinum. The tumor was resected by lateral thoracotomy. Histopathology revealed a pulmonary cystic lymphangioma.

Molecular epidemiologic features of inflammatory breast cancer: a comparison between Egyptian and US patients

Inflammatory breast cancer (IBC) is a lethal form of breast cancer with unknown etiology. A higher frequency of IBC and a more aggressive IBC phenotype was reported in Egypt than in the United States. This difference in disease frequency and presentation might be related to molecular epidemiologic factors. We used tumor blocks and demographic, epidemiologic, and clinical data of 48 IBC patients from Egypt and 12 patients from the United States. We counted tumor emboli in tumors before and after immunohistochemical staining with lymphatic vessel endothelial receptor-1 (LYVE-1), and measured the expression of RhoC GTPase protein in the two groups. Erythema, edema, and peau d'orange were found in 77% of the Egyptian patients as compared with 29% found in the US patients (P=0.02). The number of tumor emboli was significantly higher in tumors from Egypt (mean+/-SD, 14.1+/-14.0) than in the tumors from the United States (5.0+/-4.0, P=0.01). The number of tumor emboli in LYVE-1 positive vessels was higher in tumors from Egypt (3.5+/-2.8) than tumors from the United States (1.6+/-0.5, P=0.15). We detected a high level of RhoC in 87% of the tumors from Egypt and 14% of the tumors from the United States (P=0.0003). Patients from Egypt have a more aggressive form of IBC than those in the United States. Our analysis of IBC patients shows that distinct molecular phenotypes can be found when these two study populations are compared. Future studies should explore the epidemiologic and environmental exposures and the genetic factors that might lead to the different clinical and molecular features of IBC in patients from these two countries.

VEGF-D in Association With VEGFR-3 Promotes Nodal Metastasis in Human Invasive Lobular Breast Cancer

We assessed the expression of vascular endothelial growth factors (VEGF-C and VEGF-D) in breast cancer cells and the density of lymph vessels and VEGF receptor-3 (VEGFR-3)-positive vessels in and around the tumor in invasive lobular breast cancer. We found significant correlation between peritumoral lymph vessel density and presence of lymph node metastases (P=.001) and the number of metastatic lymph nodes (P<.001). A significant correlation was detected between tumor cell VEGF-D expression and lymph node status (P=.001) and density of lymphatic vessel endothelial receptor (LYVE)-1-positive vessels (P=.035). VEGFR-3+/VEGF-D+ and VEGFR-3+/VEGF-C+ tumors had a significantly higher number of metastatic lymph nodes than tumors with other staining patterns (P<.001). Tumors positive for neither VEGF-D nor VEGFR-3 had a lower density of LYVE-1+ vessels than tumors with other staining patterns (P=.033). Our results indicate that peritumoral lymph vessel density is associated with lymph node metastases in invasive lobular breast cancer and that invasive lobular cancer producing VEGF-D, surrounded by VEGFR-3+ vessels, has a significantly higher peritumoral lymph vessel density and a higher number of metastatic lymph nodes.

Radiogenic Lymphangiogenesis in the Skin

The time course of microvascular changes in the environment of irradiated tumors was studied in a standardized human protocol. Eighty skin biopsies from 40 patients with previously treated primary breast cancer were taken from irradiated skin and corresponding contralateral unirradiated control areas 2 to 8 weeks, 11 to 14 months, or 17+ months after radiotherapy (skin equivalent dose 30 to 40 Gy). Twenty-two biopsies of 11 melanoma patients who had undergone lymph node dissection were used for unirradiated control. We found an increase of total podoplanin(+) lymphatic microvessel density resulting mainly from a duplication of the density of smallest lymphatic vessels (diameter <10 microm) in the samples taken 1 year after radiation. Our findings implicate radiogenic lymphangiogenesis during the 1st year after therapy. The numbers of CD68(+) and vascular endothelial growth factor-C(+) cells were highly elevated in irradiated skin in the samples taken 2 to 8 weeks after radiotherapy. Thus, our results indicate that vascular endothelial growth factor-C expression by invading macrophages could be a pathogenetic route of induction of radiogenic lymphangiogenesis.

Angiogenesis and lymphangiogenesis in thyroid proliferative lesions: relationship to type and tumour behaviour

The role of angiogenesis and lymphangiogenesis in thyroid cancer pathogenesis has not been elucidated. Patterns for tumour behaviour and metastasic spread vary according to tumour type and whether differences in the angiogenic or lymphangiogenic phenotype influence the route for tumour metastases or determine a more aggressive behaviour has not been fully explored. The angiogenic and lymphangiogenic phenotypes of a large cohort of thyroid proliferative lesions (n=191) were studied. Using immunohistochemistry for CD34, lymphatic vessel endothelial receptor-1 (LYVE-1) (specific markers for vascular and lymphatic endothelium respectively), vascular endothelial growth factor (VEGF-A), VEGF-C and fibroblast growth factor-2 (FGF-2), this study analyses microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic factors in normal thyroid (NT; n=19), multinodular goitre (n=25), toxic multinodular goitre (n=8), Graves' hyperplasia (n=22), follicular adenoma (n=54), papillary carcinoma (PC; n=27), incidental papillary microcarcinoma (PMC; n=8), follicular carcinoma (FC; n=20) and medullary carcinoma (MC; n=8). MVD was decreased in proliferative lesions, benign and malignant, compared with NT (P<0.0001). In contrast, VEGF-A expression was increased in thyroid carcinomas (PC, FC and MC) when compared with PMC, benign lesions and NT (P<0.0001). LVD was higher in PC and PMC (P=0.001), and VEGF-C expression was increased in PC (P<0.0001). Despite higher LVD and increased expression of VEGF-A and VEGF-C in thyroid cancers, these markers were not related to poor prognosis in terms of tumour size, multifocality and/or presence of lymphatic or distant metastases. In conclusion, angiogenesis is reduced in thyroid proliferative lesions compared with NT tissue. However, VEGF-A expression is upregulated in thyroid cancers. Lymphangiogenesis and VEGF-C expression are increased in thyroid tumours prone to lymphatic metastases. This may be an important mechanism underlying the differences in metastatic behaviour between papillary and follicular thyroid cancer.