Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis

Lymphangiogenesis is a novel prognostic parameter for several cancers that is preferentially quantified by immunohistochemistry of the lymphatic endothelium-specific hyaluronan receptor LYVE-1. Recently, the specificity of LYVE-1 was challenged by serendipitous observations of LYVE-1 expression in rare tissue macrophages. As expression of the hyaluronan receptor-like molecule stabilin-1 is shared by sinusoidal endothelium and macrophages, a thorough analysis of LYVE-1 expression was performed using macrophage-specific markers in vivo and in vitro. In murine tumour models and excisional wound healing, LYVE-1 expression occurred in a subset of CD11b(+), F4/80(+) tissue macrophages that preferentially co-expressed stabilin-1. Upon comparison of single- and double-labelling immunofluorescence, it became apparent that LYVE-1(+) macrophages mimic sprouting and collapsed lymphatic vessels. In vitro, LYVE-1 expression was induced in 25-40% of murine bone marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium and IL-4/dexamethasone. By FACS analysis, 11.5% of bone marrow-derived macrophages were LYVE-1(+), stabilin-1(+) double-positive, while 9.9% were LYVE-1(+), stabilin-1(-) and 33.5% were LYVE-1(-), stabilin-1(+). Northern and western analyses confirmed expression of LYVE-1 mRNA and protein in bone marrow-derived macrophages. In the light of the current debate about true endothelial trans-differentiation versus endothelial mimicry of monocytes/macrophages, LYVE-1(+), stabilin-1(+) non-continuous endothelial-like macrophages will require further developmental and functional analyses. In conclusion, the findings imply that LYVE-1 staining must be supplemented by double labelling with macrophage markers in order to differentiate clearly between LYVE-1(+) lymphatics and LYVE-1(+) tumour-infiltrating macrophages. This improved approach will help to clarify the prognostic significance of lymphangiogenesis in malignant tumours.     

Expression of vascular endothelial growth factor receptor 3 in blood and lymphatic vessels of lung adenocarcinoma

Vascular endothelial growth factor receptor 3 (VEGFR-3) has been proposed as a marker for lymphatic endothelial cells. This study investigated the expression of VEGFR-3 in the tumour vessels of lung adenocarcinoma and evaluated whether VEGFR-3 staining was useful for identifying lymphatic vessels within the tumour stroma. It also explored whether active growth of lymphatic vessels occurred in lung adenocarcinoma. Formalin-fixed, paraffin-embedded specimens obtained from 60 cases of lung adenocarcinoma, including five cases of pure bronchiolo-alveolar carcinoma (BAC) without stromal, vascular, and pleural invasion, were examined. No VEGFR-3-positive vessels were observed in pure BAC, but varying numbers of VEGFR-3-positive vessels were found in 39 of 55 (70.9%) invasive adenocarcinomas. A comparison of serial sections stained for VEGFR-3, CD31, and laminin-1 showed that most of the VEGFR-3-positive vessels appeared to be blood vessels (CD31-positive, laminin-1-positive), but some had the characteristics of lymphatic vessels (variable staining for CD31, little or no staining for laminin-1). VEGFR-3 staining highlighted lymphatic invasion by cancer cells; this invasion could not be detected by CD31 or haematoxylin and eosin (H&E) staining. Active growth of lymphatic vessels (as indicated by nuclear Ki-67 labelling of the endothelium) was observed in five tumours, four of which showed a high level of lymphatic invasion by cancer cells. It was concluded that VEGFR-3 immunostaining did not discriminate clearly between vascular and lymphatic endothelial cells, since expression of VEGFR-3 can be up-regulated in tumour blood vessels. However, VEGFR-3 staining combined with laminin-1 and CD31 staining would be useful for identifying lymphatic vessels and their invasion by tumour cells in a more objective way. Finally, proliferation of lymphatic endothelial cells may occur in association with lymphatic invasion by cancer cells.     

Lymphatic vascular endothelial hyaluronan receptor (lyve)-1- and ccl21-positive lymphatic compartments in the diabetic thymus

To explore the biological significance of the lymphatics in the autoimmune process, the thymus from non-obese diabetic (NOD) mice was evaluated by histochemistry and western blot analysis. Thymic lymphatic endothelial cells showed suggestive expression patterns of the functional molecules lymphatic vascular endothelial hyaluronan receptor (LYVE)-1, CCL21, CD31 and podoplanin. With increasing age, the expression of CCL21 was reduced in the medullary epithelial cells and lymphatics. Of note, LYVE-1-expressing lymphatics, filled with a cluster of thymocytes, increased in number and size and extended from the corticomedullary boundary into the medulla as the insulitis progressed. The development of lymphatic compartments was occasionally accompanied by a regional disappearance between the cortex and medulla. The CD4- and CD8-positive T cells frequently penetrated through the slender lymphatic walls. The epithelial reticular cell layer lining the perivascular spaces was extensively stained with cytokeratin, but the expression of cytokeratin showed an age-dependent decrease. These findings indicate that the occurrence of LYVE-1-expressing lymphatic compartments and the alteration of CCL21 expression in the lymphatics may be involved in defective thymocyte differentiation and migration, and play a significant role in insulitic and diabetic processes.     

胃癌组织淋巴管LYVE-1、钙粘蛋白的表达及其与癌转移的关系

目的：探讨胃癌组织钙粘蛋白的表达与癌淋巴道转移的相关性。方法：取胃癌标本14例，正常胃组织标本2例。通过E-cadherin、β-catenin和LYVE-1免疫组化方法，观察肿瘤淋巴管的表达。结果：LYVE-1在正常和癌组织淋巴管阳性表达，正常淋巴管E．cadherin、β-catenin表达阴性，β-catenin在胃癌淋巴管表达弱阳性，β-catenin在淋巴管的表达与肿瘤的分化程度及有无转移有负相关性。结论：LYVE-1在淋巴管特异性表达，E-cadherin／β-catenin复合物在淋巴道转移过程中，对肿瘤细胞与淋巴管内皮细胞的粘附起一定的作用，但不是主要作用。     

Immunohistochemical detection of human small lymphatic vessels under normal and pathological conditions using the LYVE-1 antibody

The spread of tumor cells via lymphatic vessels to the lymph nodes is an important indicator of malignancy. However, previous markers used to identify lymphatic endothelium gave ambiguous results in immunohistochemical analyses with paraffin-embedded tissues. In this study, we attempted to prepare a polyclonal antibody against human lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) for detecting lymphatic vessels using immunohistochemistry. The antibody was raised against a region near the transmembrane anchor of LYVE-1 in New Zealand white rabbits. Immunostainings with anti-LYVE-1 and von Willebrand factor antibodies were performed in various normal and pathological tissues. LYVE-1 expression was confined to the endothelial surface of lymphatic vessels but was not found in the endothelium of blood vessels, which were positive for von Willebrand factor. Our LYVE-1 polyclonal antibody was useful for the identification of small lymphatic vessels in normal human tissues. In addition, the immunostaining enabled us to distinguish lymphatic invasion by malignant tumor cells from blood vessel invasion using paraffin-embedded sections. In conclusion, our polyclonal antibody against the transmembrane anchor of the peptide can be used to detect human lymphatic vessels under various conditions.     

High expression of insulin receptor on tumour‐associated blood vessels in invasive bladder cancer predicts poor overall and progression‐free survival

Bladder cancer is a frequently recurring disease with a very poor prognosis once progressed to invasive stages, and tumour‐associated blood vessels play a crucial role in this process. In order to identify novel biomarkers associated with progression, we isolated blood vascular endothelial cells (BECs) from human invasive bladder cancers and matched normal bladder tissue, and found that tumour‐associated BECs greatly up‐regulated the expression of insulin receptor (INSR). High expression of INSR on BECs of invasive bladder cancers was significantly associated with shorter progression‐free and overall survival. Furthermore, increased expression of the INSR ligand IGF‐2 in invasive bladder cancers was associated with reduced overall survival. INSR may therefore represent a novel biomarker to predict cancer progression. Mechanistically, we observed pronounced hypoxia in human bladder cancer tissue, and found a positive correlation between the expression of the hypoxia marker gene GLUT1 and vascular INSR expression, indicating that hypoxia drives INSR expression in tumour‐associated blood vessels. In line with this, exposure of cultured BECs and human bladder cancer cell lines to hypoxia led to increased expression of INSR and IGF‐2, respectively, and IGF‐2 increased BEC migration through the activation of INSR in vitro. Taken together, we identified vascular INSR expression as a potential biomarker for progression in bladder cancer. Furthermore, our data suggest that IGF‐2/INSR mediated paracrine crosstalk between bladder cancer cells and endothelial cells is functionally involved in tumour angiogenesis and may thus represent a new therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The effect of neoadjuvant chemotherapy on the correlation of tumor-associated macrophages with CD31 and LYVE-1

Angiogenesis and lymphangiogenesis play a crucial role in tumor growth, invasion and metastasis. Tumor-associated macrophages (TAM) induce both angiogenesis and lymphangiogenesis in mouse breast cancer models and positively correlate with these processes in human breast cancer patients. Neoadjuvant chemotherapy (NAC) is a widely used therapeutic option for cancer treatment. However, the effect of NAC on the distribution of TAM within intratumoral compartments and their correlation with angiogenesis and lymphangiogenesis remained unknown. In the present study we analyzed the effect of NAC on the distribution of CD68+ and stabilin-1+ TAM in five functionally distinct areas of human breast cancer and their correlations with microvessel density (MVD) and lymphatic microvessel density (LMVD), identified by CD31 and LYVE1, respectively. We found that NAC enhances blood vessel density in soft fibrous stroma and in coarse fibrous stroma. Without NAC the amount of CD68+ TAM in gaps of ductal tumor structures positively correlate with CD31+ microvessel density in soft fibrous stroma. NAC had enhancing effect on the amount of CD68+ TAM but not stabilin-1+ TAM in soft fibrous stroma. However, no correlation between TAM and CD31+ microvessel density was identified after NAC. NAC did not enhance the lymphatic microvessel density. But after NAC stabilin-1 expressing subpopulation of TAM positively correlated with expression of LYVE-1. We hypothesized that CD68+ TAM can support tumor angiogenesis primarily before NAC, while stabilin-1+ TAM can contribute to the maintenance of lymphatic microvessel density after NAC.     

Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors,Flk‐1 and Flt‐1

The biological effects of vascular endothelial growth factor A (VEGF‐A) are mediated by fetal liver kinase‐1 (Flk‐1) and fms‐like tyrosine kinase‐1 (Flt‐1). In lung tissue, VEGF‐A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk‐1 and Flt‐1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk‐1 and Flt‐1 of embryonic mouse lung tissue. When Flk‐1 was predominantly expressed (embryonic day [E] 9.5–E13.5), then vascular endothelial cells actively proliferated. When Flt‐1 was enhanced (E14.5–E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk‐1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt‐1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt‐1 promoted Flk‐1 expression. These results suggest that the two VEGF‐A receptors regulate pulmonary vascular development by modulating the VEGF‐A signaling. Anat Rec, 290:958–973, 2007. © 2007 Wiley‐Liss, Inc.     

Cellular phenotypes and spatio‐temporal patterns of lymphatic vessel development in embryonic mouse hearts

Background: The origin of cardiac lymphatics from venous endothelial cells or from scattered lymphangioblasts has been discussed in the literature. We aimed to establish the stage when lymphatic vessels appear in the developing mouse heart, the location of the first lymphatics, and to define cellular phenotypes of growing lymphatics. Results: We found that scattered Lyve‐1‐positive cells located in the subepicardial area of developing heart expressed CD45, CD68, F4/80, or CD11b but not CD31. Prox‐1⁺/Lyve‐1⁺ cellular cords or vessels were found to invade 12.5–13.5‐dpc hearts via two routes: from the venous pole, i.e., dorsal atrioventricular sulcus, or on the dorsal atrial surface from mediastinum and from the arterial pole, i.e., along the great arteries. The Prox‐1⁺/Lyve‐1⁺ vessels were located among the Prox‐1⁺/Lyve‐1^? cords and among the scattered Prox‐1^?/Lyve‐1⁺ cells. The Prox‐1⁺/Lyve‐1^? cellular cords/tubules dominate initially at the arterial pole whereas Lyve‐1⁺/Prox‐1^? cellular cords/tubules dominate initially on the venous pole, i.e., dorsal atrioventricular sulcus. The Lyve‐1⁺/CD45⁺, Lyve‐1⁺/CD11b⁺, Lyve‐1⁺/F4/80⁺ and Lyve‐1⁺/CD68⁺ cells were subsequently found to be co‐opted to the wall of the developing lymphatic vessels while gaining Flk‐1. Conclusions: Lymphatic primordia exhibit different cellular phenotypes and different spatiotemporal pattern on the venous pole as compared with the arterial pole of the heart. Developmental Dynamics 241:1473–1486, 2012. © 2012 Wiley Periodicals, Inc.     

淋巴管内皮细胞透明质酸受体-1在发育小鼠肾内的表达

目的:探讨淋巴管内皮细胞透明质酸受体-1(LYVE-1)在发育小鼠肾内的表达.方法:胚胎期第13～18天(E13～18)的胎鼠和出生后第4、 14和21天(P4～21)及成年小鼠的肾,行LYVE-1免疫组织化学显色.结果:LYVE-1免疫阳性淋巴管丛最早在胚胎期第14天的小鼠肾检测到,主要位于肾皮质的动脉周围.在胚胎期第15天的肾小球中最早检测到LYVE-1免疫阳性细胞,但LYVE-1免疫阳性细胞在肾小球中的数量和位置不定.结论:LYVE-1免疫阳性细胞主要位于肾动脉周围淋巴管,在肾小球中也有LYVE-1表达.     

The role of high‐mobility group box protein 1 in collagen antibody‐induced arthritis is dependent on vascular endothelial growth factor

High‐mobility group box 1 (HMGB1) has been implicated in angiogenesis and rheumatoid arthritis (RA). The aim of this study was to define more clearly the role of HMGB1 in the synovial angiogenesis and pathogenesis of an immune model of arthritis. BALB/c mice were injected with monoclonal anti‐collagen antibody cocktail followed by lipopolysaccharide to induce arthritis. HMGB1 and vascular endothelial growth factor (VEGF) were over‐expressed in the areas of the synovium where more inflammation and neoangiogenesis were present. The selective blockade of HMGB1 or VEGF resulted alternatively in a lower severity of arthritis evaluated by the arthritis index. Furthermore, exogenous HMGB1 administration caused a worsening of arthritis, associated with VEGF up‐regulation and increased synovial angiogenesis. The selective inhibition of VEGF also resulted in no induction of arthritis in mice receiving exogenous HMGB1. Cytokine enzyme‐linked immunosorbent assay (ELISA) analyses performed on peripheral blood and synovial fluid demonstrated a significant reduction of interleukin (IL)?1β, IL‐6 and tumour necrosis factor (TNF)‐α in mice where HMGB1 and VEGF pathways were blocked. Interestingly, the selective blockade of HMGB1 and VEGF resulted in an increase of the peripheral IL‐17A concentration. The development of arthritis mediated by HMGB1 and the synovial angiogenesis can be blocked by inhibiting the VEGF activity. The proinflammatory and proangiogenic cytokine IL‐17A was increased when HMGB1 is inhibited, but the synovial angiogenesis was nevertheless reduced in this model of arthritis. Taken together, these findings shed new light on the role of this nuclear protein in the pathogenesis of arthritis in an RA‐like model.     

Organ‐specific lymphangiectasia,arrested lymphatic sprouting,and maturation defects resulting from gene‐targeting of the PI3K regulatory isoforms p85α, p55α, and p50α

The phosphoinositide 3‐kinase (PI3K) family has multiple vascular functions, but the specific regulatory isoform supporting lymphangiogenesis remains unidentified. Here, we report that deletion of the Pik3r1 gene, encoding the regulatory subunits p85α, p55α, and p50α impairs lymphatic sprouting and maturation, and causes abnormal lymphatic morphology, without major impact on blood vessels. Pik3r1 deletion had the most severe consequences among gut and diaphragm lymphatics, which share the retroperitoneal anlage, initially suggesting that the Pik3r1 role in this vasculature is anlage‐dependent. However, whereas lymphatic sprouting toward the diaphragm was arrested, lymphatics invaded the gut, where remodeling and valve formation were impaired. Thus, cell‐origin fails to explain the phenotype. Only the gut showed lymphangiectasia, lymphatic up‐regulation of the transforming growth factor‐β co‐receptor endoglin, and reduced levels of mature vascular endothelial growth factor‐C protein. Our data suggest that Pik3r1 isoforms are required for distinct steps of embryonic lymphangiogenesis in different organ microenvironments, whereas they are largely dispensable for hemangiogenesis. Developmental Dynamics 238:2670–2679, 2009. © 2009 Wiley‐Liss, Inc.     

Immune receptor expressed on myeloid cells 1 (IREM-1) inhibits B cell activation factor (BAFF)-mediated inflammatory regulation of THP-1 cells through modulation of the activities of extracellular regulated kinase (ERK)

The immune receptor expressed on myeloid cells 1 (IREM‐1) has been known to regulate the activities of myeloid cells through its immunoreceptor tyrosine‐based inhibition motifs (ITIMs) in its intracellular region. In order to investigate its effect on macrophage activation, a human macrophage cell line (THP‐1) was tested after stimulation of its membrane‐bound form of B cell activation factor (BAFF), which has been shown to modulate inflammatory activities through induction of proinflammatory mediator expression and suppression of phagocytosis. IREM‐1‐specific monoclonal antibodies detected the expression of high levels of IREM‐1 in THP‐1 cells. Cross‐linking of IREM‐1 with these antibodies resulted in the blockage of the BAFF‐mediated expression of interleukin (IL)‐8 and matrix metalloproteinase (MMP)‐9 through inhibition of the activation of extracellular regulated kinase (ERK) and phosphorylation/degradation of IκB. Furthermore, cross‐linking of IREM‐1 also reversed the BAFF‐mediated inhibition of phagocytosis. In order to demonstrate the role of ITIM in the IREM‐1‐mediated suppression of BAFF signalling, a decapeptide containing YADL (an ITIM in IREM‐1) was fused with HIV–TAT_48–57 which was required for the internalization of the synthetic polypeptide (TAT–YADL). TAT–YADL, but not control peptides, recapitulated the effect of the anti‐IREM‐1 monoclonal antibody. These observations indicate that IREM‐1 exerted its inhibitory effect on BAFF‐medicated signalling through ITIM‐mediated regulation of ERK activities in THP‐1 cells.     

Tissue‐specific venous expression of the EPH family receptor EphB1 in the skin vasculature

Background : The major arteries and veins are formed early during development. The molecular tools to identify arterial and venous endothelial cells improve our understanding of arterial–venous differentiation and branching morphogenesis. Compared with arterial differentiation, relatively little is known about what controls venous development, due to lack of definitive molecular markers for venous endothelial cells. Results: Here we report that the antibody against EphB1, an EphB class receptor, makes it possible to establish a reliable whole‐mount immunohistochemical analysis of venous identity with greater resolution than previously possible in embryonic and adult skin vasculature models. EphB1 expression is restricted to the entire venous vasculature throughout embryonic development to adulthood, whereas the previously established venous marker EphB4 is also detectable in lymphatic vasculature. This venous‐restricted expression of EphB1 is established after the vascular remodeling of the primary capillary plexus has occurred. Compared with its venous‐specific expression in the skin, however, EphB1 is not restricted to the venous vasculature in yolk sac, trunk and lung. Conclusions: These studies introduce EphB1 as a new venous‐restricted marker in a tissue‐specific and time‐dependent manner. Developmental Dynamics 242:976–988, 2013. © 2013 Wiley Periodicals, Inc.     

Increased FXYD1 and PGC‐1α mRNA after blood flow‐restricted running is related to fibre type‐specific AMPK signalling and oxidative stress in human muscle

Aim

This study explored the effects of blood flow restriction (BFR) on mRNA responses of PGC‐1α (total, 1α1, and 1α4) and Na⁺,K⁺‐ATPase isoforms (NKA; α_1‐3, β_1‐3, and FXYD1) to an interval running session and determined whether these effects were related to increased oxidative stress, hypoxia, and fibre type‐specific AMPK and CaMKII signalling, in human skeletal muscle.

Methods

In a randomized, crossover fashion, 8 healthy men (26 ± 5 year and 57.4 ± 6.3 mL kg^?1 min^?1) completed 3 exercise sessions: without (CON) or with blood flow restriction (BFR), or in systemic hypoxia (HYP, ~3250 m). A muscle sample was collected before (Pre) and after exercise (+0 hour, +3 hours) to quantify mRNA, indicators of oxidative stress (HSP27 protein in type I and II fibres, and catalase and HSP70 mRNA), metabolites, and α‐AMPK Thr¹⁷²/α‐AMPK, ACC Ser²²¹/ACC, CaMKII Thr²⁸⁷/CaMKII, and PLBSer¹⁶/PLB ratios in type I and II fibres.

Results

Muscle hypoxia (assessed by near‐infrared spectroscopy) was matched between BFR and HYP, which was higher than CON (~90% vs ~70%; P < .05). The mRNA levels of FXYD1 and PGC‐1α isoforms (1α1 and 1α4) increased in BFR only (P < .05) and were associated with increases in indicators of oxidative stress and type I fibre ACC Ser²²¹/ACC ratio, but dissociated from muscle hypoxia, lactate, and CaMKII signalling.

Conclusion

Blood flow restriction augmented exercise‐induced increases in muscle FXYD1 and PGC‐1α mRNA in men. This effect was related to increased oxidative stress and fibre type‐dependent AMPK signalling, but unrelated to the severity of muscle hypoxia, lactate accumulation, and modulation of fibre type‐specific CaMKII signalling.

     

Estrogen receptor alpha is expressed on the cell-surface of embryonic hypothalamic neurons

Although the biological activity of estrogen is generally mediated through nuclear estrogen receptors, a large body of evidence indicates that estrogen may also affect target cells upon binding to putative membrane estrogen receptors (mER) coupled to intracellular signaling cascades; however, no agreement has been reached on the nature and precise location of the putative estrogen receptor (ER) responsible for these rapid effects. In the present report we show that the expression of ERalpha is associated with the plasma membrane fraction of rat hypothalamic tissue at embryonic day 16. Moreover, our experiments extend these results to rat hypothalamic neurons in vitro showing that ERalpha can be detected from the cell exterior as a biotinylated cell-surface protein. We have also shown that the mERalpha is under regulation of estradiol, and the ERalpha agonist, 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol, induced extracellular-signal-regulated kinase signaling in a dose-dependent manner and in a time-course not compatible with genomic actions, supporting the notion of a membrane-initiated phenomenon.