Expression Of LYVE-1 Research Articles

Three unique interstitial macrophages in the murine lung at steady state

Abstract Rationale The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin such as microglia in the brain, while other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs such as the skin, there are both embryonic and postnatal-derived macrophages. Objectives In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages (AMs), dendritic cells and few extravascular monocytes. Methods We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover and lack of survival dependency on CX3CR1. Measurements and Main Results Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature. All IMs expressed MerTK+CD64+ CD11b+ CX CR1+ and were furthermore distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2 and MHCII, along with the absence of Ly6C, Ly6G, and Siglec F. Ex vivo analysis revealed that all three IMs were highly phagocytic compared to AMs. Finally, similar phenotypic populations of IMs were present in other non-lymphoid organs, suggesting that these IMs may not be unique to the lung. Conclusions These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

Severe damage to the placental fetal capillary network causes mid- to late fetal lethality and reduction in placental size in Peg11/Rtl1 KO mice.

Paternally expressed 11/Retrotransposon-like 1 (Peg11/Rtl1) knockout (KO) mice show mid- to late fetal lethality or late fetal growth retardation associated with frequent neonatal lethality. The lethal phenotype is largely dependent on genetic background and becomes more severe with each succeeding generation in the course of backcross experiments to C57BL/6 (B6). We previously suggested that these lethal and growth phenotypes in the fetal stages were due to severe defects in placental fetal capillaries in the labyrinth layer. In this study, we re-examined KO fetuses and placentas and confirmed that the severe clogging of fetal capillaries was associated with KO fetuses showing mid-fetal lethality with internal bleeding. Importantly, the basal region of the fetal capillary network was specifically damaged, also leading to poor expansion of the labyrinth layer and placental size reduction in the later stage. An apparent down-regulation of transmembrane protein 100 (Tmem100), mesenchyme homeobox 2 (Meox2) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) expression were observed in earlier stage placentas even before apparent size reduction became, suggesting that these genes are involved in the maintenance of fetal capillaries associated with Peg11/Rtl1 during development.

Effects of Melatonin, Aluminum Oxide, and Polymethylsiloxane Complex on the Expression of LYVE-1 in the Liver of Mice with Obesity and Type 2 Diabetes Mellitus

The effects of melatonin, aluminum oxide, and polymethylsiloxane complex on the expression of LYVE-1 (lymphatic vessel endothelial hyaluronan receptor) in the liver were studied in db/db mice with experimental obesity and type 2 diabetes mellitus. The complex or placebo was administered daily by gavage from week 8 to week 16 of life. The animals receiving the complex exhibited enhanced, in comparison with the placebo group, immunohistochemical LYVE-1+ staining of endothelial cells in sinusoids. Enhanced expression of LYVE-1 was associated with less pronounced dilatation of interlobular arteries, veins, and lymphatic vessels. Thee findings suggest a protective effect of the complex towards structural changes in the liver of mice with obesity and type 2 diabetes.

Lymphatic vascular endothelial hyaluronan receptor-1 immunoexpression in placenta of HIV infected pre-eclamptic women

IntroductionLymphangiogenesis is the formation of new vessels from pre-existing lymphatic vessels. Data on lymphangiogenesis in the placenta of HIV-infected pre-eclamptics are sparse and the findings are conflicting. The aim of this novel study was to evaluate LYVE-1 immunoexpression in the placenta of HIV infected normotensive versus pre-eclamptic women. MethodsPlacental tissue was obtained from normotensive and pre-eclamptic women stratified according to their HIV status. The pre-eclamptic group was divided into early (<34 weeks) and late (>34 weeks) onset. Immunohistochemistry utilized mouse anti-human LYVE-1 antibody and was morphometrically evaluated. ResultsLYVE-1 immunostaining was localized within endothelium of the arterial supply and venous drainage of both conducting and exchange villi as well as within medial cells of arteries. LYVE-1 immunostained macrophage-like cells were observed within the fetal and maternal circulation. LYVE-1 immunoexpression was higher (p=0.0001) in HIV positive cohort, regardless of pregnancy and villous type. Irrespective of HIV status and pregnancy type, LYVE-1 immunoexpression was significantly elevated in the conducting compared to the exchange villi (p=0.01). LYVE-1 immunoexpression was higher in N and LOPE compared to EOPE groups for both conducting and exchange villi types respectively (p=0.0001 and p=0.006). There is a decrease of LYVE-1 expression in EOPE+ (conducting villi) and EOPE− (exchange villi) compared to N and LOPE subgroups. ConclusionThis study provides a novel insight into an up-regulation of LYVE-1 expression in the fetal circulation of conducting and exchange villi of HIV-infected pre-eclamptics.

Abstract 1460: Bone marrow-derived CD11b+/Podoplanin+ cells are lymphatic progenitors directly responsible for breast cancer lymphatic formation

Abstract Introduction: Lymphatic metastasis, a key factor for poor outcome of breast cancer (BC), strongly depends on lymphatic vessel (LV) formation. Recent studies suggest that lymphangiogenesis is strongly promoted by bone marrow (BM)-derived CD11b+ monocytes that differentiate into Lymphatic Endothelial Cell Progenitors dubbed here M-LECP. While BC are known to recruit BM monocytes, a specific BM subset responsible for tumor lymphatic formation has not been identified. We recently discovered that podoplanin (Pdpn) is the highest upregulated lymphatic marker that signifies transdifferentiation of either human or mouse immature myeloid cells into M-LECP. We hypothesized that BM-derived M-LECP are represented by a subset expressing podoplanin in CD11b+ myeloid cells that are capable of inducing tumor lymphatics. Methods: Tumor lymphatic vessels expressing myeloid markers were detected immunohistochemically by triple-staining in clinical specimens and experimental breast tumors. Expression of Vegfr-3, Lyve-1, Sca-1, and other markers in BM-derived CD11b+/Pdpn+ and Pdpn-negative subsets was determined by FACS. Expression of lymphatic-specific markers in CD11b+/Pdpn+ and Pdpn− myeloid cells isolated from MDA-MB-231 tumors was determined by RT-qPCR. CD11b+/Pdpn+ and Pdpn-negative subsets from BM of GFP-expressing mice were adoptively transferred to mice with orthotopic breast tumors followed by quantifying mobilized GFP+/CD11b+/Pdpn+ cells and the density of tumor LV. Results: M-LECP were absent in normal human breast tissues but highly present in tumors. Analysis of clinical BC specimens showed significant correlations between tumor-mobilized M-LECP, density of LV and metastasis to regional nodes. All examined BC models exhibited very high densities (60-90%) of double-positive macrophages expressing lymphatic markers, and lymphatic vessels expressing myeloid proteins. At least a half of CD11b+ cells isolated from tumors were positive for podoplanin and nearly 90% of CD11b+/Pdpn+ subset expressed markers of progenitor cells. Only CD11b+/Pdpn+ subset (but not other myeloid cells) expressed a broad panel of proteins specific to the lymphatic endothelial lineage. Adoptive transfer of BM-derived CD11b+/Pdpn+ or Pdpn-negative fractions into tumor-bearing mice showed that only Pdpn+ BM myeloid cells integrated into preexisting LV and caused a 10-fold increase in peritumoral and intratumoral LV density. Conclusion: We show for the first time in clinical BC samples that tumor-recruited M-LECP significantly correlate with LN metastasis. We also identified a phenotypically distinct BM fraction of CD11b+/Pdpn+ cells that directly promote generation of new tumor lymphatic vessels. Our data suggest that tumors induce myeloid BM cells to undergo differentiation into M-LECP that subsequently promote cancer lymphatics thereby promoting tumor spread. Citation Format: Caitlin Griggs, Kelly Hall, Lisa Volk-Draper, Kathy Robinson, Sophia Ran. Bone marrow-derived CD11b+/Podoplanin+ cells are lymphatic progenitors directly responsible for breast cancer lymphatic formation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1460.

Abstract 2499: Sox2 enhances breast tumor angiogenesis by promoting the transition of cancer cell to CD31+ and LYVE-1+ cells

Abstract Angiogenesis plays an important role during the development of tumor. Sox2 is one of the transcription factors involved in tumorigenesis and metastasis. However, its function during the process of tumor angiogenesis and lymphogenesis is still poorly understood. Here, we demonstrated that down-regulation of Sox2 significantly reduced the tumor angiogenesis and lymphogenesis as reflected by attenuated immunofluorescence staining of CD31 and LYVE-1 and improved prognosis in xenograft models of breast tumor. Furthermore, Sox2 also promoted the recruitment of bone marrow derived endothelial progenitor cells (MDEPC) to tumor microenvironment. Most importantly, we demonstrated that some of CD31+ and LYVE-1+ cells were derived from Sox2+ cancer cells and silencing of Sox2 could inhibit this transition. We revealed that Sox2 promoted the expression of LYVE-1, VEGFs and enhanced the phosphorylation of VEGFR2 in breast cancer. ChIP-seq and dual-luciferase assay further revealed the binding of Sox2 protein on proximate promoter region of VEGFB and LYVE1 to regulate their transcriptional activities, disclosing the underlying molecular mechanism on the regulatory effects of Sox2 on angiogenesis and lymphogenesis. Our study indicated that Sox2 improved tumor angiogenesis and lymphogenesis through regulating the related genes and unveiled a novel function of Sox2 in tumor progression. Citation Format: Rongrong Wang, Xuefei Li, Yingxi Xu, Tongchao Sun, Weijun Su, Chenhua Yu, Xiaoli Dong, Rong Xiang, Na Li. Sox2 enhances breast tumor angiogenesis by promoting the transition of cancer cell to CD31+ and LYVE-1+ cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2499.

Licoricidin, an Active Compound in the Hexane/Ethanol Extract of Glycyrrhiza uralensis, Inhibits Lung Metastasis of 4T1 Murine Mammary Carcinoma Cells.

Licorice extracts containing glycyrrhizin exhibit anti-carcinogenic properties. Because glycyrrhizin induces severe hypokalemia and hypertension, we prepared a hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) that lacks glycyrrhizin, and showed that HEGU induces apoptosis and G1 cell cycle arrest and inhibits migration of DU145 human prostate cancer cells. Our previous in vitro studies identified two active components in HEGU: isoangustone A, which induces apoptosis and G1 cycle arrest, and licoricidin, which inhibits metastasis. This study examined whether HEGU and licoricidin inhibit metastasis using the 4T1 mammary cancer model. Both HEGU and licoricidin treatment reduced pulmonary metastasis and the expression of CD45, CD31, HIF-1α, iNOS, COX-2, and VEGF-A in tumor tissues. Additionally, a decrease in protein expression of VEGF-R2, VEGF-C, VEGF-R3, and LYVE-1 was noted in tumor tissues of licoricidin-treated mice. Furthermore, the blood concentrations of MMP-9, ICAM-1, VCAM-1, and VEGF-A were decreased in HEGU-treated mice. In vitro 4T1 cell culture results showed that both HEGU and licoricidin inhibited cell migration, MMP-9 secretion, and VCAM expression. The present study demonstrates that the licoricidin in HEGU inhibits lung metastasis of 4T1 mammary carcinoma cells, which may be mediated via inhibition of cancer cell migration, tumor angiogenesis, and lymphangiogenesis.

Absence of Lymphatic Vessels in PCNSL May Contribute to Confinement of Tumor Cells to the Central Nervous System.

Primary central nervous system (CNS) lymphoma (PCNSL) is a mature lymphoma of the diffuse large B-cell lymphoma (DLBCL) type confined to the CNS. Despite cytomorphological similarities between PCNSL and systemic DLBCL, molecular differences between both entities have been identified. The exclusively topographical restriction of PCNSL to the CNS is an unexplained mystery. To address the question of whether the unique lymphatic drainage system of the CNS, which differs from that of other organs, may play a role for this peculiar behavior, we investigated a series of 20 PCNSLs for the presence of lymphatic vessels by immunohistochemistry for Lyve-1, podoplanin, and Prox-1 expression. All PCNSLs lacked lymphatic vessels and, in this regard, were similar to 20 glioblastoma multiforme samples. In contrast to these tumors, all of which were located in the deep brain parenchyma, dural and meningeal DLBCL harbored lymphatic vessels that expressed Lyve-1 (3/8 tumors), podoplanin (5/8 tumors), and Prox-1 (5/8 tumors) in areas where the tumors had invaded the fibrous tissue of the dura. These data indicate that local topographical characteristics of the specific lymphatic drainage system may contribute to confinement of the tumor cells in PCNSL and malignant gliomas.

Expression of Lymphatic Markers in the Adult Rat Spinal Cord.

Under physiological conditions, lymphatic vessels are thought to be absent from the central nervous system (CNS), although they are widely distributed within the rest of the body. Recent work in the eye, i.e., another organ regarded as alymphatic, revealed numerous cells expressing lymphatic markers. As the latter can be involved in the response to pathological conditions, we addressed the presence of cells expressing lymphatic markers within the spinal cord by immunohistochemistry. Spinal cord of young adult Fisher rats was scrutinized for the co-expression of the lymphatic markers PROX1 and LYVE-1 with the cell type markers Iba1, CD68, PGP9.5, OLIG2. Rat skin served as positive control for the lymphatic markers. PROX1-immunoreactivity was detected in many nuclei throughout the spinal cord white and gray matter. These nuclei showed no association with LYVE-1. Expression of LYVE-1 could only be detected in cells at the spinal cord surface and in cells closely associated with blood vessels. These cells were found to co-express Iba1, a macrophage and microglia marker. Further, double labeling experiments using CD68, another marker found in microglia and macrophages, also displayed co-localization in the Iba1+ cells located at the spinal cord surface and those apposed to blood vessels. On the other hand, PROX1-expressing cells found in the parenchyma were lacking Iba1 or PGP9.5, but a significant fraction of those cells showed co-expression of the oligodendrocyte lineage marker OLIG2. Intriguingly, following spinal cord injury, LYVE-1-expressing cells assembled and reorganized into putative pre-vessel structures. As expected, the rat skin used as positive controls revealed classical lymphatic vessels, displaying PROX1+ nuclei surrounded by LYVE-1-immunoreactivity. Classical lymphatics were not detected in adult rat spinal cord. Nevertheless, numerous cells expressing either LYVE-1 or PROX1 were identified. Based on their localization and overlapping expression with Iba1, the LYVE-1+ cell population likely represents a macrophage subpopulation, while a significant fraction of PROX1+ cells belong to the oligodendrocytic lineage based on their distribution and the expression of OLIG2. The response of these LYVE-1+ and PROX1+ cell subpopulations to pathological conditions, especially in spinal cord inflammatory conditions, needs to be further elucidated.

The selective distribution of LYVE-1-expressing endothelial cells and reticular cells in the reticulo-endothelial system(RES).

LYVE-1, a receptor molecule for hyaluronan, is expressed in the lymphatic endothelium, blood sinus endothelium, and certain macrophage lineages. The present immunohistochemical study revealed a broader distribution of LYVE-1 in vascular endothelial cells of the murine lung, adrenal gland, and heart as well as the liver and spleen. In addition, sinus reticular cells-including sinuslining cells-in the medulla of the lymph node also intensely expressed LYVE-1. Ultrastructurally, immuno-gold particles for LYVE-1 were localized on the entire length of plasma membrane in all cell types. Most of these LYVE-1-expressing cells had previously been classified as the reticuloendothelial system (RES) specialized for eliminating foreign particles. An LPS stimulation decreased the LYVE-1 expression in macrophages but elevated the expression at mRNA and protein levels in the liver and lung, major organs for the elimination of blood-born waste substances. LYVE-1-expressing endothelial cells in these organs participated in the endocytosis of exogenous particles, and the uptake ability was conspicuously enhanced by the LPS challenge. Although the expression of the degrading enzyme, hyaluronidase, was generally low in the LYVE-1-expressing cells, they were topographically associated with a dense distribution of macrophages possessing hyaluronidase activities in each tissue. These findings suggest that the LYVE-1-expressing cells might be involved in the uptake of hyaluronan and other waste products as well as foreign particles circulating in the blood and lymph while participating in the subsequent degradation in relay with adjacent macrophage populations.

A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 “multi-points priming” mechanisms in vitro and in vivo

BackgroundTumor lymphangiogenesis plays an important role in promoting growth and metastasis of tumors, but no antilymphangiogenic agent is used clinically. Based on the effect of norcantharidin (NCTD) on lymphangiogenesis of human lymphatic endothelial cells (LECs), we firstly investigated the antilymphangiogenic activity of NCTD as a tumor lymphangiogenic inhibitor for human colonic adenocarcinomas (HCACs).MethodsIn vivo and in vitro experiments to determine the effects of NCTD on tumor growth and lymphangiogenesis of the in-situ colonic xenografts in nude mice, and lymphatic tube formation of the three-dimensional (3-D) of the co-culture system of HCAC HT-29 cells and LECs were done. Proliferation, apoptosis, migration, invasion, Ki-67, Bcl-2 and cell cycle of LECs and the co-culture system in vitro were respectively determined. Streparidin-peroxidase staining, SABC, western blotting and RT-PCR were respectively used to examine the expression of LYVE-1, D2-40, CK20 (including their LMVD), and VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in vitro and in vivo.ResultsNCTD inhibited tumor growth and lymphangiogenesis of the in-situ colonic xenografts in vivo, and these observations were confirmed by facts that lymphatic tube formation, proliferation, apoptosis, migration, invasion, S-phase cell cycle, and Ki-67 and Bcl-2 expression in vitro, and LYVE-1, D2-40, CK20 expression and their LMVD in vitro and in vivo were inhibited and affected. Furthermore, the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 at protein/mRNA levels in the process of lymphatic tube formation in vitro and tumor lymphangiogenesis in vivo was downregulated; NCTD in combination with mF4-31C1 or Sorafenib enhanced these effects.ConclusionsNCTD inhibits tumor growth and lymphangiogenesis of HCACs through “multi-points priming” mechanisms i.e. affecting related malignant phenotypes, inhibiting Ki-67 and Bcl-2 expression, inducing S-phase cell cycle arrest, and directly or indirectly downregulating VEGF-A,-C,-D/VEGFR-2,-3 signaling pathways. The present finding strongly suggests that NCTD could serve as a potential antilymphangiogenic agent for tumor lymphangiogenesis and is of importance to explore NCTD is used for antitumor metastatic comprehensive therapy for HCACs.

The role of CCL21/CCR7 chemokine axis in breast cancer-induced lymphangiogenesis.

BackgroundTumor-induced lymphangiogenesis facilitates breast cancer progression by generating new lymphatic vessels that serve as conduits for tumor dissemination to lymph nodes and beyond. Given the recent evidence suggesting the implication of C-C chemokine ligand 21/chemokine receptor 7 (CCL21/CCR7) in lymph node metastasis, the aim of our study was to define the role of this chemokine pair in breast cancer-associated lymphangiogenesis.MethodsThe expression analysis of CCL21/CCR7 pair and lymphatic endothelial cell (LEC) markers in breast cancer specimens was performed by means of quantitative real-time PCR. By utilizing CCR7 and CCL21 gene manipulated breast cancer cell implants into orthotopic sites of nude mice, lymphatic vessel formation was assessed through quantitative real-time PCR, immunohistochemistry and immunofluorescence assays. Finally, the lymphangiogenic potential of CCL21/CCR7 was assessed in vitro with primary LECs through separate functional assays, each attempting to mimic different stages of the lymphangiogenic process.ResultsWe found that CCR7 mRNA expression in human breast cancer tissues positively correlates with the expression of lymphatic endothelial markers LYVE-1, podoplanin, Prox-1, and vascular endothelial growth factor-C (VEGF-C). We demonstrated that the expression of CCL21/CCR7 by breast cancer cells has the ability to promote tumor-induced lymph-vascular recruitment in vivo. In vitro, CCL21/CCR7 chemokine axis regulates the expression and secretion of lymphangiogenic factor VEGF-C and thereby promotes proliferation, migration, as well as tube formation of the primary human LECs. Finally, we showed that protein kinase B (AKT) signaling pathway is the intracellular mechanism of CCR7-mediated VEGF-C secretion by human breast cancer cells.ConclusionsThese results reveal that CCR7 and VEGF-C display a significant crosstalk and suggest a novel role of the CCL21/CCR7 chemokine axis in the promotion of breast cancer-induced lymphangiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0306-4) contains supplementary material, which is available to authorized users.

Warthin's tumor: an unknown pathogenesis: a neoplasm or a reactive hyperplasia?

To examine the probable role of angiogenesis and lymphangiogenesis in the pathogenesis of Warthin's tumor. Sixty-one patients with Warthin's tumor (n = 40), branchial cysts (n = 6), thymic cysts (n = 3), or tonsillar lymphoepithelial cysts (n = 12) were included. Forty Warthin's tumors were used as the lesion group, and 21 lymphoepithelial cysts were used as a control group. 29 lymph nodes around the Warthin's tumor, four of which showed salivary duct inclusions, were also evaluated. Blood vessel density was defined as an indicator of angiogenesis by examining CD31 and FVIII Ag expression, and lymphatic vascular density was defined as an indicator of lymphangiogenesis by evaluating LYVE-1 and podoplanin expression by immunohistochemical analysis. Data are expressed with descriptive statistics. Comparative analysis was performed using Shapiro-Wilks, Mann-Whitney U, and Kruskal-Wallis tests. A P < 0.005 was considered to indicate statistical significance. Statistical analysis was performed using the MedCalc ® v.10.3.0 software. The lesion group had higher mean values of age (58 vs. 11 years, P = 0.001), smoking rate (92.3% vs. 19%, P < 0.001), stromal degeneration (100% vs. 42.9%, P < 0.001), lymph node involvement around the lesion (87.9% vs. 12.1%, P < 0.001), salivary duct inclusion (25% vs. 0%, P = 0.0001), than those of lymphoepithelial cysts. Blood vessel density (51.92 ± 25.64 vs. 8 ± 5.35, number/5 high power fields (HPF), P < 0.001) and lymphatic vascular density (68.95 ± 21.32 vs. 21.10 ± 4.05 number/5 HPF, P < 0.001) were higher in Warthin's tumors than lymphoepithelial cysts. Warthin's tumors, and lymph nodes with inclusions had similar levels of blood and lymphatic vascular density, which was higher than those of lymph nodes (P < 0.0001). Warthin's tumor is a true neoplastic epithelial proliferation associated with increased angiogenesis and lymphangiogenesis and induces reactive lymph node hyperplasia.

ETS Transcription Factor Etsrp/Etv2 Is Required for Lymphangiogenesis in Zebrafish and Is a Potential Therapeutic Target in Lymphatic Disease

Lymphangiogenesis is of great clinical interest because of its essential role in such pathologic settings as inflammation, vascular malformations, and tumor metastasis. Previous observational hurdles are now overcome by use of the optically clear and genetically modifiable zebrafish embryo, which provides a vertebrate model for understanding lymphangiogenesis and for revealing putative therapeutic targets for metastatic disease and vascular/lymphatic malformations that currently have no cure. We and others have previously demonstrated that an evolutionarily conserved ETS transcription factor Etv2 / Etsrp functions as a master regulator during embryonic vascular development. However, its role in lymphatic development is not defined. Here we performed conditional knockdown of Etv2 function using photoactivatable morpholinos (MO) in a zebrafish model system to test Etv2 requirement during lymphangiogenesis. Our data show that conditional Etv2 inhibition at 1-day-post-fertilization (dpf) resulted in specific defects in lymphatic development, while blood vessel formation was not affected. The major lymphatic vessel, the thoracic duct, was absent or discontinuous in Etv2-depleted embryos (Figure 1) and the lymphatic progenitors failed to migrate (Figure 2) in absence of Etv2 function. Expression of the lymphatic markers lyve1, prox1a and the major VegfC receptor VegfR3 was strongly down regulated in the conditional Etv2 MO knockdown embryos. We further show that Etv2 expression is enriched in the posterior cardinal vein where lymphangioblasts (lymphatic endothelial cell precursors) originate during lymphangiogenesis and that overexpression of Etv2 throughout blood vasculature perturbs lymphangiogenesis. We further show that in the absence of Etv2 function, lymphatic progenitors fail to respond to VegfC signaling. Our results suggest that Etv2 initiates lymphangiogenesis in part by regulating Vegf3 expression. These results argue that Etv2 is a novel critical factor during lymphatic development, and it may represent a new target for chemical modulation in multiple lymphatic disorders. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Temporal expression of hyaluronic acid and hyaluronic acid receptors in a porcine small intestinal submucosa-augmented rat bladder regeneration model

Hyaluronic acid (HA), a non-sulfated glycosaminoglycan, is an essential component of the extracellular matrix (ECM). Since HA is involved in many phases of wound healing and may play a key role in tissue repair and regeneration, this study was intended to understand temporal and spatial expression of HA and HA receptors (HARs) during the course of bladder regeneration in rats. Sprague-Dawley rats were subjected to partial cystectomy followed by augmentation with porcine small intestinal submucosal (SIS) prepared from distal sections of the small intestine. SIS-augmented bladders were harvested between postoperative days 2 and 56. Bladder regeneration proceeded without complications. All augmented bladders had complete urothelial lining and smooth muscle bundles by day 56 post-augmentation. Temporal and spatial distributions of HA and HARs were studied by immunohistochemistry in regenerating bladders. The strongest HA immunoreactivity was observed in the ECM on postoperative days 28 and 56. Cluster of differentiation 44 (CD44) immunoreactivity was detected in the cytoplasm of urothelial cells on day 56; and LYVE-1 immunoreactivity was exclusively limited to lymphatic vessels on days 28 and 56. We demonstrated that HA was synthesized throughout the course of bladder wound healing and regeneration; and HA deposition coincided with urothelial differentiation. Expression of CD44 and LYVE-1 followed the same temporal pattern as HA deposition. Therapeutic modalities through local delivery of exogenous HA to improve the outcome of SIS-mediated bladder regeneration might need to be coordinated with HAR expression in order to achieve maximal regenerative responses as opposed to fibrosis.

Endothelial cell-derived signals in liver development and regeneration

The liver harbors a specialized capillary bed. It is characterized by Lyve-1 and VEGFR3 expression, low CD31 and vWF expression and lack of CD34 expression [1], Moreover, the basement membrane is discontinuous, and liver sinusoidal endothelial cells are adjacent to a discontinuous basement membrane. Finally, the blood flow through liver sinusoidal capillaries is slow compared to many other organs. Besides the role of hepatic capillary beds in exchange of substances and gases between liver and blood, the endothelium of the liver is required for both liver development [2,3] and liver regeneration [4]. Interestingly, liver regeneration seems to be similar to liver development in that endothelial signals seem to be required during both processes. In the regenerating liver, it was shown that so-called angiocrine signals are needed and involved in discriminating between liver regeneration and liver fibrosis [4,5]. As a model for liver regeneration in mice, a 2/3rd partial hepatectomy is normally performed. Importantly, the liver starts to divide and grow, and within a week the original liver cell mass is restored. Therefore, signals are needed to turn on and off liver regeneration. Previously, Michalopoulos and colleagues have proposed that after PHx three times more blood has to pass the liver when it is reduced to one-third of its original mass based on simple mathematics [6]. To this end, we propose that this increase in blood volume relative to liver mass might turn on angiocrine signals via mechanotransduction (Buschmann, Axnick & Lammert, unpublished data). To this end, we perform different measurements to determine changes in the endothelium, blood flow and blood volume during the course of liver regeneration.

Mo1726 Failure of Bilirubin-Mediated Immunosuppression Can Be Linked to Increased Efflux From Cells Driven by Heightened Activation of MDR1 Glycoprotein on T-Cells in Crohn's Disease

Background: Lymphocyte infiltration into the intestinal mucosa is an important pathogenic feature of inflammatory bowel diseases (IBD). In case of dermatitis, lymphatics play a role as drainage route of lymphocytes from skin and an inhibition of lymphangiogenesis keeps lymphocytes remaining in the local area leading to exacerbate inflammation. However, the role of lymphatics in enterocolitis is not well understood. In this study, we examined changes in lymphatic densities and expression of lymphangiogenic factors in patients with IBD and in animal model of colitis, and investigated a role of lymphangiogenic using inhibitor of VEGF (vascular endothelial growth factor)-R3. Method: Colonic biopsies were obtained from 40 patients with ulcerative colitis (UC), 17 patients with Crohn's disease (CD), and controls. Messenger RNA expressions of lymphangiogenic factors, VEGFC & D, VEGFR3, podoplanin, Prox-1, and LYVE1 were determined using real time RT-PCR. Expression of CD34 and LYVE1 was studied immunohistochemically. In dextran sulfate sodium (DSS)-induced mice colitis, a VEGF-R3 kinase inhibitor (10mg/kg) was administered every day and the effects of lymphangiogenetic inhibition on activity of colitis and lymphatic density were determined in the colonic mucosa. Result: In the colonic mucosa of patients with UC and CD, the expressions of VEGFC, VEGFR3, podoplanin, and LYVE1 were significantly increased than the mucosa of control patients. The expression levels of podoplanin and LYVE1 were well correlated to endoscopic Matt's score in UC patients. By immunohistochemistry, the lymphatic densities in the colonic mucosa of IBD patients also increased compared to control patients. However, interestingly those densities were not significantly increased in the severely inflamed mucosa of IBD patients compared with those in the quiescent mucosa, suggesting that lymphangiogenesis and lymphatic drainage are not well organized in the severely inflamed mucosa. In murine colitis model, VEGF-R3 inhibition group showed significant aggravation of colitis such as shortening of colonic length and increase in the lymphocyte infiltration in the submucosa compared with DSS-treatment alone. Lymphatic densities decreased in the submucosa of VEGF-R3 inhibition group. Conclusions: The results of our study revealed that lymphangiogenic factors and lymphatic density are increased in both UC and CD patients. However, the density of lymphatic did not increase as inflammation advanced. Aggravation of colitis by lymphangiogenic receptor inhibition suggests that lymphatic networks may play some protective roles as exit of immune cells from intestinal mucosa, and that some mechanisms which block lympangiogenesis play aggravating role in severe IBD patients. In the severely inflamed conditions lymphatic drainage through lymphangiogenesis is not well functioning in IBD.

Mo1727 Impaired Expression of the Aryl Hydrocarbon Receptor and CD39 Is Associated With Heightened Pathogenicity of T Helper Type 17 Cells in Crohn's Disease

Background: Lymphocyte infiltration into the intestinal mucosa is an important pathogenic feature of inflammatory bowel diseases (IBD). In case of dermatitis, lymphatics play a role as drainage route of lymphocytes from skin and an inhibition of lymphangiogenesis keeps lymphocytes remaining in the local area leading to exacerbate inflammation. However, the role of lymphatics in enterocolitis is not well understood. In this study, we examined changes in lymphatic densities and expression of lymphangiogenic factors in patients with IBD and in animal model of colitis, and investigated a role of lymphangiogenic using inhibitor of VEGF (vascular endothelial growth factor)-R3. Method: Colonic biopsies were obtained from 40 patients with ulcerative colitis (UC), 17 patients with Crohn's disease (CD), and controls. Messenger RNA expressions of lymphangiogenic factors, VEGFC & D, VEGFR3, podoplanin, Prox-1, and LYVE1 were determined using real time RT-PCR. Expression of CD34 and LYVE1 was studied immunohistochemically. In dextran sulfate sodium (DSS)-induced mice colitis, a VEGF-R3 kinase inhibitor (10mg/kg) was administered every day and the effects of lymphangiogenetic inhibition on activity of colitis and lymphatic density were determined in the colonic mucosa. Result: In the colonic mucosa of patients with UC and CD, the expressions of VEGFC, VEGFR3, podoplanin, and LYVE1 were significantly increased than the mucosa of control patients. The expression levels of podoplanin and LYVE1 were well correlated to endoscopic Matt's score in UC patients. By immunohistochemistry, the lymphatic densities in the colonic mucosa of IBD patients also increased compared to control patients. However, interestingly those densities were not significantly increased in the severely inflamed mucosa of IBD patients compared with those in the quiescent mucosa, suggesting that lymphangiogenesis and lymphatic drainage are not well organized in the severely inflamed mucosa. In murine colitis model, VEGF-R3 inhibition group showed significant aggravation of colitis such as shortening of colonic length and increase in the lymphocyte infiltration in the submucosa compared with DSS-treatment alone. Lymphatic densities decreased in the submucosa of VEGF-R3 inhibition group. Conclusions: The results of our study revealed that lymphangiogenic factors and lymphatic density are increased in both UC and CD patients. However, the density of lymphatic did not increase as inflammation advanced. Aggravation of colitis by lymphangiogenic receptor inhibition suggests that lymphatic networks may play some protective roles as exit of immune cells from intestinal mucosa, and that some mechanisms which block lympangiogenesis play aggravating role in severe IBD patients. In the severely inflamed conditions lymphatic drainage through lymphangiogenesis is not well functioning in IBD.

Mo1725 Possible Protective Role of Lymphangiogenesis in the Inflamed Colonic Mucosa of Inflammatory Bowel Diseases

Background: Lymphocyte infiltration into the intestinal mucosa is an important pathogenic feature of inflammatory bowel diseases (IBD). In case of dermatitis, lymphatics play a role as drainage route of lymphocytes from skin and an inhibition of lymphangiogenesis keeps lymphocytes remaining in the local area leading to exacerbate inflammation. However, the role of lymphatics in enterocolitis is not well understood. In this study, we examined changes in lymphatic densities and expression of lymphangiogenic factors in patients with IBD and in animal model of colitis, and investigated a role of lymphangiogenic using inhibitor of VEGF (vascular endothelial growth factor)-R3. Method: Colonic biopsies were obtained from 40 patients with ulcerative colitis (UC), 17 patients with Crohn's disease (CD), and controls. Messenger RNA expressions of lymphangiogenic factors, VEGFC & D, VEGFR3, podoplanin, Prox-1, and LYVE1 were determined using real time RT-PCR. Expression of CD34 and LYVE1 was studied immunohistochemically. In dextran sulfate sodium (DSS)-induced mice colitis, a VEGF-R3 kinase inhibitor (10mg/kg) was administered every day and the effects of lymphangiogenetic inhibition on activity of colitis and lymphatic density were determined in the colonic mucosa. Result: In the colonic mucosa of patients with UC and CD, the expressions of VEGFC, VEGFR3, podoplanin, and LYVE1 were significantly increased than the mucosa of control patients. The expression levels of podoplanin and LYVE1 were well correlated to endoscopic Matt's score in UC patients. By immunohistochemistry, the lymphatic densities in the colonic mucosa of IBD patients also increased compared to control patients. However, interestingly those densities were not significantly increased in the severely inflamed mucosa of IBD patients compared with those in the quiescent mucosa, suggesting that lymphangiogenesis and lymphatic drainage are not well organized in the severely inflamed mucosa. In murine colitis model, VEGF-R3 inhibition group showed significant aggravation of colitis such as shortening of colonic length and increase in the lymphocyte infiltration in the submucosa compared with DSS-treatment alone. Lymphatic densities decreased in the submucosa of VEGF-R3 inhibition group. Conclusions: The results of our study revealed that lymphangiogenic factors and lymphatic density are increased in both UC and CD patients. However, the density of lymphatic did not increase as inflammation advanced. Aggravation of colitis by lymphangiogenic receptor inhibition suggests that lymphatic networks may play some protective roles as exit of immune cells from intestinal mucosa, and that some mechanisms which block lympangiogenesis play aggravating role in severe IBD patients. In the severely inflamed conditions lymphatic drainage through lymphangiogenesis is not well functioning in IBD.

The expression of vascular endothelial growth factor-C correlates with lymphatic microvessel density and lymph node metastasis in prostate carcinoma: An immunohistochemical study.

Aim:To evaluate the expression of two different lymphatic vascular density (LVD) markers (D2-40 and LYVE-1) and a lymphangiogenic cytokine (Vascular Endothelial Growth Factor-C, [VEGF-C]) in prostate carcinoma and to investigate their relationship with the lymph node status.Settings and Design:Archival material study of 92 non-consecutive radical prostatectomy specimens.Materials and Methods:The mean LVD was assessed immunohistochemically in 24 prostate carcinoma specimens from patients with clinically localized disease, who were found to have nodal metastasis (pN1), and was compared with 68 pN0 cases. Furthermore, the mean LVD, VEGF-C expression, and lymphatic invasion were examined in relation to lymph node involvement.Results:Peritumoral (but not intratumoral) mean LVD assessed by D2-40 was higher in pN1 tumors (P = 0.015). LYVE-1 expression was limited and not associated with lymph node status. The VEGF-C expression was higher in the N1 cases and also correlated with the increased mean LVD in both the peri- and intratumoral compartments. Lymphatic invasion was strongly associated with nodal metastasis and higher VEGF-C expression.Conclusions:Our results indicate that increased peritumoral (but not intratumoral) LVD in the tumor specimen is associated with lymph node metastasis. Increased expression of VEGF-C is associated with higher LVD (in both intratumoral and peritumoral compartments) and with positive lymph node status, indicating a possible dual role in both lymphangiogenesis and lymphatic vessel invasion.