Dermal Endothelial Cells Research Articles

CIAP2 promotes gallbladder cancer invasion and lymphangiogenesis by activating the NF-κB pathway.

Several studies have produced contradictory findings about the prognostic implications for inhibitor of apoptosis proteins (IAP) in different types of cancer. Cellular inhibitor of apoptosis 2 (cIAP2/BIRC) is one of the most extensively characterized human IAP. To date, no studies have focused on the expression level of cIAP2 in human gallbladder cancer (GBC), and the mechanism of cIAP2 in GBC invasion and lymphangiogenesis remains unclear. Therefore, in the present study, cIAP2 expression in GBC was detected using quantitative real‐time polymerase chain reaction and immunohistochemistry, and the relationship between cIAP2 levels in cancer tissues and the clinicopathological characteristics of patients was analyzed. The biological effect of cIAP2 in GBC cells was tested using the Cell Counting Kit‐8 Assay, Transwell assays and the ability of human dermal lymphatic endothelial cells (HDLEC) to undergo tube formation. The role of cIAP2 in activating the NF‐κB pathway was determined using a dual‐luciferase reporter assay, immunofluorescence staining, western blotting and ELISA. Finally, an animal model was used to further confirm the role of cIAP2 in lymphangiogenesis. We showed that cIAP2 expression was elevated in human GBC tissues and correlated with a negative prognosis for patients. Moreover, cIAP2 was identified as a lymphangiogenic factor of GBC cells and, thus, promoted lymph node metastasis in GBC cells. Our study is the first to suggest that cIAP2 can promote GBC invasion and lymphangiogenesis by activating the NF‐κB pathway.

A heparin conjugate, LHbisD4, inhibits lymphangiogenesis and attenuates lymph node metastasis by blocking VEGF-C signaling pathway.

Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in invitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway.

Fli1 Deficiency Induces CXCL6 Expression in Dermal Fibroblasts and Endothelial Cells, Contributing to the Development of Fibrosis and Vasculopathy in Systemic Sclerosis.

CXCL6, a chemokine with proangiogenic property, is reported to be involved in vasculopathy associated with systemic sclerosis (SSc). We investigated the contribution of CXCL6 to SSc development by focusing on the association of friend leukemia virus integration 1 (Fli1) deficiency, a potential predisposing factor of SSc, with CXCL6 expression and clinical correlation of serum CXCL6 levels. mRNA levels of target genes and the binding of Fli1 to the CXCL6 promoter were evaluated by quantitative reverse transcription-PCR and chromatin immunoprecipitation, respectively. Serum CXCL6 levels were determined by ELISA. FLI1 siRNA significantly enhanced CXCL6 mRNA expression in human dermal fibroblasts and human dermal microvascular endothelial cells, while Fli1 haploinsufficiency significantly suppressed CXCL6 mRNA expression in murine peritoneal macrophages stimulated with lipopolysaccharide. Supporting a critical role of Fli1 deficiency to induce SSc-like phenotypes, CXCL6 mRNA expression was higher in SSc dermal fibroblasts than in normal dermal fibroblasts. Importantly, Fli1 bound to the CXCL6 promoter in dermal fibroblasts, endothelial cells, and THP-1 cells. In patients with SSc, serum CXCL6 levels correlated positively with the severity of dermal and pulmonary fibrosis and were elevated in association with cardiac and pulmonary vascular involvement and cutaneous vascular symptoms, including Raynaud phenomenon, digital ulcers (DU)/pitting scars, and telangiectasia. Especially, serum CXCL6 levels were associated with DU/pitting scars and heart involvement by multiple regression analysis. CXCL6 expression is upregulated by Fli1 deficiency in fibroblasts and endothelial cells, potentially contributing to the development of fibrosis and vasculopathy in the skin, lung, and heart of SSc.

Abstract P30: The Mechanical Environment Modulates Lymphatic Tube Formation In Vitro

PURPOSE: Clinical lymphedema can be tempered by exogenous administration of pro-lymphangiogenic molecules such as VEGF-C or retinoic acids at the time of lymphatic injury. In this study, we sought to explore the effect of three-dimensional mechanical environment on VEGF induced lymphangiogenesis. Improved understanding of growth factor induced lymphangiogenesis will aid clinical translation of novel therapies against lymphedema. METHODS: Human dermal lymphatic endothelial cells (LEC) were isolated from human foreskin. The cells were expanded in traditional 2D culture in EBM in LEC media with 15% fetal bovine serum supplement and 1% Ampicillin/Gentamycin. Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) were prepared with a 12% gelatin stock gel washed with PBS, and further diluted for various gel concentrations. Mechanical tests were carried out with an indentation test, and the deformation distance of the gel construct was used as an indicator of relative gel stiffness. A total of 5 different concentrations were used with relative stiffness including: <1, 5, 9, 18 and 25 kPa. The 3D gels were cultured as a single droplet on a 48-well suspension cell culture plate with exchange of fresh media and 100ng/mL of VEGF-A/C every 2–3 days. The gels were directly observed daily under the light microscope and recorded. CONCLUSIONS: The 3D hydrogel served as the interstitial substrate to support LEC tube formation under different mechanical properties. Our experimental results showed a high density of tube formation in LECs cultured in ~18 kPa Col-Tgels beginning as early as 48 hours after plating. Shorter and early stages of tubule structure formation was also visible in the ~9 kPa gel. Col-Tgels with stiffness lesser than 9 kPA or greater than 18 kPa were not conducive to lymphatic tube formation. Lymphatic tube formation was similarly seen in the 18 kPa gels cultured with LEC media alone without soluble cytokines, but only after a 72–96 hour lag compared to the gels with cytokine treatment (images not shown). Our results show that the 3D environment plays an integral role in both cell-cell and cell-extracellular matrix (ECM) interactions. LECs respond not only to soluble factors associated with the ECM, but also the biomechanical cues for tubulogenesis.

IL‐36γ has proinflammatory effects on human endothelial cells

Interleukin-36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL-36 is now a recognised characteristic of psoriatic skin inflammation. IL-36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL-36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated by IL-36γ stimulation, and this is reversed by the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. These results suggest a role for IL-36γ in the dermal vascular compartment, and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leucocyte recruitment.

Psoriasis and Pro-angiogenetic Factor CD93: Gene Expression and Association with Gene Polymorphism Suggests a Role in Disease Pathogenesis

CD93 is involved in angiogenesis and inflammation, both of which are key processes in the pathogenesis of psoriasis. CD93 was studied in serum, peripheral blood mononuclear cells and skin of patients with psoriasis and controls. Furthermore, allele frequencies for CD93 single-nucleotide polymorphisms rs2749812 and rs2749817 were assessed in patients with psoriasis compared with controls and the effect of narrowband ultraviolet B (NB-UVB) treatment on CD93 gene expression was evaluated in the skin of patients with psoriasis. CD93 gene expression was significantly increased in lesional and non-lesional skin from patients with psoriasis compared with controls. Immunohistochemistry revealed CD93 staining in dermal endothelial cells in lesional skin, and psoriasis was significantly associated with rs2749817 CD93 gene polymorphism. NB-UVB treatment of patients with psoriasis did not alter skin CD93 gene expression. Increased protein expression of CD93 psoriatic skin and association with the rs2749817 polymorphism suggests that CD93 plays a role in psoriasis disease pathogenesis.

Efficacy of AdipoDren® in Reducing Interleukin-1-Induced Lymphatic Endothelial Hyperpermeability

Lymphatic leakage can be seen as a detrimental phenomenon associated with fluid retention and deposition as well as gain of weight. Moreover, lymphatic dysfunction is associated with an inflammatory environment and can be a substrate for other health conditions. A number of treatments can ameliorate lymphatic vasculature: natural substances have been used as treatment options particularly suitable for their consolidated effectiveness and safety profile. Here we report the protective effect of AdipoDren®, an association of a series of plant-derived natural complexes, on lymphatic endothelium permeability promoted by interleukin-1 beta (IL-1β) and the associated molecular mechanisms. AdipoDren® demonstrated a protective effect on dermal lymphatic endothelial cell permeability increased by IL-1β. Reduced permeability was due to the maintenance of tight junctions and cell-cell localisation of occludin and zonula occludens-1 (ZO-1). Moreover, AdipoDren® reduced the expression of the inflammatory key element cyclooxygenase-2 (COX-2), while not altering the levels of endothelial and inducible nitric oxide synthases (eNOS and iNOS). The upregulation of antioxidant enzymatic systems (catalase and superoxide dismutase-1, SOD-1) and the downregulation of pro-oxidant markers (p22 phox subunit of NADPH oxidase) were also evident. In conclusion, AdipoDren® would be useful to ameliorate conditions of altered lymphatic vasculature and to support the physiological functionality of the lymphatic endothelium.

Psoriasin (S100A7) promotes stress-induced angiogenesis.

Vascular modifications occur early in the development of psoriasis, and angiogenesis is one of the key features in the pathogenesis of the disease. To identify the role of the S100 protein psoriasin in psoriasis-associated angiogenesis. The role of psoriasin in mediating angiogenesis was investigated by silencing psoriasin with small interfering RNA (siRNA) and measuring psoriasis-associated angiogenic factors in human epidermal keratinocytes. The secretion of psoriasin and the effect of psoriasin on general regulators of angiogenesis in keratinocytes, and on endothelial cell migration, proliferation, tube formation and production of angiogenic mediators, was evaluated. Reactive oxygen species (ROS) and hypoxia induced the expression of psoriasin. Downregulation of psoriasin in keratinocytes using siRNA altered the ROS-induced expression of the psoriasis-associated angiogenic factors vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor-like growth factor, matrix metalloproteinase 1 and thrombospondin 1. Overexpression of psoriasin altered several regulators of angiogenesis and led to the secretion of psoriasin. Treatment with extracellular psoriasin induced proliferation, migration and tube formation in dermal-derived endothelial cells to a similar extent as VEGF and interleukin-17, and induced the expression and release of proangiogenic mediators. These effects were suggested to be mediated by the PI3K and nuclear factor kappa B pathways. These findings suggest that psoriasin expression is promoted by oxidative stress in keratinocytes and amplifies the ROS-induced expression of angiogenic factors relevant to psoriasis. Moreover, extracellularly secreted psoriasin may act on dermal endothelial cells to contribute to key features angiogenesis.

ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection.

Lipopolysaccharide increases the release of VEGF-C that enhances cell motility and promotes lymphangiogenesis and lymphatic metastasis through the TLR4- NF-κB/JNK pathways in colorectal cancer.

Lipopolysaccharide (LPS) exists in the outer membrane of Gram-negative bacteria. Colorectal normal epithelium and colorectal cancer cells in situ are continuously exposed to LPS from intestinal bacteria, while little is known about the influence of LPS on colorectal cancer progression and metastasis. In this study, we investigated the potential role of LPS on colorectal cancer progression and metastasis as well as the underlying mechanisms. We measured higher LPS concentration in colorectal cancer tissues and even higher LPS concentration in colorectal cancer tissues with lymph node metastasis. LPS significantly enhanced cancer cell motility and promoted human dermal lymphatic endothelial cells' (HDLECs') capacity of tube-like formation in vitro, as well as accelerates lymphangiogenesis and lymph node metastasis in nude mice. Furthermore, we demonstrated LPS notably increased the expression of VEGF-C in a time-dependent and concentration-dependent manner. VEGF-C is a key regulator for lymphangiogenesis and lymph node metastasis. By constructing lentivirus-mediated shVEGF-C cells, VEGF-C down-regulation suppressed LPS' promotive effect on cancer cell motility and HDLEC tube-like formation capacity. In addition, we found TLR4- NF-κB/JNK signal pathways were important for LPS to increase VEGF-C expression. All these result suggested a critical role for LPS in migration, invasion, lymphangiogenesis and lymph node metastasis of colorectal cancer, providing evidence that LPS increased VEGF-C secretion to promote cell motility and lymphangiogenesis via TLR4- NF-κB/JNK signaling.

Myeloid Fli1 deficiency induces tissue fibrosis, vasculopathy, and immune abnormalities recapitulating systemic sclerosis

Systemic sclerosis (SSc) is a multisystem chronic disease characterized by immune abnormalities, vasculopathy, and tissue fibrosis. In SSc lesional skin, transcription factor Friend leukemia virus integration 1 (Fli1) is constitutively down-regulated in various cell types, especially by an epigenetic mechanism in dermal fibroblasts, and Fli1 deficiency in dermal fibroblasts and endothelial cells induces an SSc-like phenotype in each cell type. However, it has still remained to be elucidated whether Fli1 deficiency in immune cells has an impact on the pathogenesis of SSc. As a part of studies to address this issue, we focused our interests on myeloid cells in this study. We generated myeloid cell-specific Fli1 deficient (Fli1 Mye-KO) mice (LysM-Cre+/−Fli1flox/flox) and examined the phenotypic changes in these mice. Fli1 Mye-KO mice showed increased dermal thickness and collagen production through activation of dermal fibroblasts. As for vasculopathy, Fli1 Mye-KO mice showed stenosis, dilatation, and regression of dermal blood vessels as visualized by FITC-conjugated dextran injection. Increased permeability of dermal blood vessels was also confirmed by leakage of Evans blue dye into interstitial spaces in Fli1 Mye-KO mice. Histopathological analysis of the skin of Fli1 Mye-KO mice showed decreased pericyte coverage in dermal blood vessels, leading to endothelial hyperplasia, vascular dilatation, and decreased stability of blood vessels. Regarding immune dysfunction, Fli1 Mye-KO mice showed production of anti-nuclear antibodies. Thus, Fli1 deficiency in myeloid cells may contribute to the induction of three cardinal pathological features of SSc, including dermal fibrosis, vascular injury, and immune abnormalities.

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion

ABSTRACTEndothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo. Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-β-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

Abstract LB-269: Engineering a vascularized 3D in vitro model of cancer progression

Abstract Microenvironmental cues regulate the neo-vascularisation and angiogenic processes within a tumour. The hallmark of cancer is the ability of cancer cells to disseminate and invade to distant sites. The presence of a ‘leaky’ vasculature in the tumour microenvironment facilitates this migration of cancer cells. Using tissue-engineering strategies, we have developed a 3D in vitro cancer model (tumouroid), where we have engineered specific compartments for both the cancer mass and the surrounding stroma. We have utilized a biomimetic matrix for the cancer mass the stroma engineered of collagen I and laminin, and used plastic compression to increase the cell and matrix density by removal of interstitial fluid in collagen hydrogels. We created artificial cancer masses (ACMs) containing colorectal cancer cells (HT29 and HCT116) and nested them within collagen/laminin stromal surrounds (SS) (both ∼10% w/v collagen) containing human dermal fibroblasts (HDFs) and endothelial cells (HUVECs). We have delineated cell specific mechanisms to describe changes in the invasion pattern of cancer cells dependent upon the stromal composition and density. HT29 cells invaded as spherical aggregates of cells while HCT116 cells invaded as contiguous cell sheets. Multicellular cultures were maintained for up to 21 days and endothelial cell morphology was investigated using immunofluorescence of CD31. In HDF-HUVEC co-cultures, HUVECs formed highly branched (29.25 ± 3.86 branches) end-to-end networks with an average length and width of 197.4μm ± 87.5μm and 19μm ± 8.1μm respectively. In the presence of HT29 cancer cells, HUVEC network formation was considerably altered with HUVECs forming much longer and wider networks (456.8μm ± 136μm and 24.2μm ± 7μm) but significantly less interconnected (13.5 ± 5.1 branches). The loss of CK20 expression by the invading cancer epithelial cell sheet was also confirmed by immunofluorescence. The loss of CK20 expression has been known to correspond with high levels of microsatellite instability and overall poor prognosis1. These findings signify the importance of a biomimetic environment in which to culture cancer cells, to mimic the native physiological response of these cells. The tumouroid model is an important tool to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo. 1. Kim, J. H., Rhee, Y., Bae, J. M., Cho, N. &amp; Kang, G. H. Loss of CDX2/CK20 expression is associated with poorly differentiated carcinoma, the CpG island methylator phenotype, and adverse prognosis in microsatellite-unstable colorectal cancer. Am. J. Surg. Pathol. 37, 1532-41 (2013). Citation Format: Tarig Magdeldin, Mark Emberton, Marilena Loizidou, Umber Cheema. Engineering a vascularized 3D in vitro model of cancer progression. [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 LB-269.

S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells.

MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery.

Molecular basis of retinol anti-ageing properties in naturally aged human skin in vivo.

Retinoic acid has been shown to improve the aged-appearing skin. However, less is known about the anti-ageing effects of retinol (ROL, vitamin A), a precursor of retinoic acid, in aged human skin in vivo. This study aimed to investigate the molecular basis of ROL anti-ageing properties in naturally aged human skin in vivo. Sun-protected buttock skin (76 ± 6 years old, n = 12) was topically treated with 0.4% ROL and its vehicle for 7 days. The effects of topical ROL on skin epidermis and dermis were evaluated by immunohistochemistry, in situ hybridization, Northern analysis, real-time RT-PCR and Western analysis. Collagen fibrils nanoscale structure and surface topology were analysed by atomic force microscopy. Topical ROL shows remarkable anti-ageing effects through three major types of skin cells: epidermal keratinocytes, dermal endothelial cells and fibroblasts. Topical ROL significantly increased epidermal thickness by stimulating keratinocytes proliferation and upregulation of c-Jun transcription factor. In addition to epidermal changes, topical ROL significantly improved dermal extracellular matrix (ECM) microenvironment; increasing dermal vascularity by stimulating endothelial cells proliferation and ECM production (type I collagen, fibronectin and elastin) by activating dermal fibroblasts. Topical ROL also stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, and thus enriched the deposition of ECM in aged human skin in vivo. 0.4% topical ROL achieved similar results as seen with topical retinoic acid, the biologically active form of ROL, without causing noticeable signs of retinoid side effects. 0.4% topical ROL shows remarkable anti-ageing effects through improvement of the homeostasis of epidermis and dermis by stimulating the proliferation of keratinocytes and endothelial cells, and activating dermal fibroblasts. These data provide evidence that 0.4% topical ROL is a promising and safe treatment to improve the naturally aged human skin.

Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation.

Leptin is a hormone produced by adipose tissue that regulates various physiological processes. Recent studies have shown that the level of circulating leptin is elevated in obese patients and have suggested a relationship between obesity and postoperative lymphedema. However, the mechanisms by which postoperative lymphedema develops in obese patients and the mechanisms by which leptin regulates lymphatic endothelial cell homeostasis such as tube formation and cell proliferation remain unknown. Here we report that leptin regulates tube formation and cell proliferation in human dermal lymphatic endothelial cells (HDLECs) by activation of the signal transducer and activator of transcription 3 pathway, which is downstream signaling of the leptin receptor. Additionally, we found that upregulation of suppressor of cytokine signaling 3 underlies the mechanisms by which a high dose of leptin inhibits cell proliferation and tube formation. Leptin also enhanced expression of the proinflammatory cytokine IL-6 in HDLECs. Interestingly, IL-6 rescues the compromised cell proliferation and tube formation caused by treatment with a high dose of leptin in an autocrine or paracrine manner. Taken together, our findings reveal a novel mechanism by which compromised HDLECs maintain their homeostasis during inflammation mediated by leptin and IL-6. Thus, regulating the level of leptin or IL-6 may be a viable strategy to reduce the incidence of postoperative lymphedema.

465 Autocrine IFN-κ maintains baseline type I interferon responses in keratinocytes in a Tyk2 dependent manner

Interferon κ (IFNK) is a type I interferon (IFN) whose biology is poorly understood. Using RNA-Seq and qRT-PCR in 2D and 3D primary human skin culture systems, we found that IFNK expression was concentrated in undifferentiated keratinocytes (KCs), decreasing as epidermal differentiation progressed (n=3, FC= 0.054, P<0.001), and was not present in dermal endothelial cells or fibroblasts (n=3, P<0.001). Intriguingly, while suppressed in psoriatic skin (FC = 0.78, P<0.001), IFNK was increased in cutaneous lupus (n=23, n=7 control, FC=1.63, P<0.05). Since type I IFN signaling is known to be dependent on Tyk2, which is a genetic risk factor for both psoriasis and lupus, we performed a knockout (KO) of the TYK2 gene using non-homologous end joining (NHEJ) repair via CRISPR/Cas9 to address the role of IFN-k in KCs. KO was validated by Western-blot for the Tyk2 protein. Notably, TYK2 KO suppressed basal expression of IFN response genes in unstimulated KCs (P=6.2E-06), including IFNK mRNA (n=3, FC=0.38, P<2.00E-15). Moreover, TYK2 KO suppressed IFN-a induction of IFNK mRNA (n=3, FC=0.24, P<3.00E-36). To address the contribution of IFN-k to baseline IFN responses in KCs, we treated with a neutralizing anti-IFN-k antibody (10mg/ml). This led to significant suppression of IFN inducible genes in both unstimulated (AIM2, FC=0.39; IRF7, FC=0.61; n=3, P<0.001 for all) as well as IFN-a treated KCs (50ng/ml, AIM2, FC=0.32; IRF7, FC= 0.63; n=3, p<0.001 for all), suggesting that IFN-k contributes to baseline type I IFN responses in KCs and further amplifies IFN-a responses. In summary, we demonstrate that IFN-k is an endogenous, KC-derived, type I IFN that maintains baseline IFN responses in KCs in a Tyk2 and differentiation dependent manner. Furthermore, our data indicate that this system is activated in cutaneous lupus and may contribute to its pathogenesis.

ACKR4 on Stromal Cells Scavenges CCL19 To Enable CCR7-Dependent Trafficking of APCs from Inflamed Skin to Lymph Nodes.

Dermal dendritic cells and epidermal Langerhans cells are APCs that migrate from skin to draining lymph nodes (LN) to drive peripheral tolerance and adaptive immunity. Their migration requires the chemokine receptor CCR7, which directs egress from the skin via dermal lymphatic vessels and extravasation into the LN parenchyma from lymph in the subcapsular sinus. CCR7 is activated by two chemokines: CCL19 and CCL21. CCL21 alone is sufficient for the migration of APCs from skin to LN. CCL19 and CCL21 also bind atypical chemokine receptor (ACKR) 4. ACKR4-mediated CCL21 scavenging by lymphatic endothelial cells lining the subcapsular sinus ceiling stabilizes interfollicular CCL21 gradients that direct lymph-borne CCR7(+)APCs into the parenchyma of mouse LN. In this study, we show that ACKR4 also aids APC egress from mouse skin under steady-state and inflammatory conditions. ACKR4 plays a particularly prominent role during cutaneous inflammation when it facilitates Langerhans cell egress from skin and enables the accumulation of dermal dendritic cells in skin-draining LN. Stromal cells in mouse skin, predominantly keratinocytes and a subset of dermal lymphatic endothelial cells, express ACKR4 and are capable of ACKR4-dependent chemokine scavenging in situ. ACKR4-mediated scavenging of dermal-derived CCL19, rather than CCL21, is critical during inflammation, because the aberrant trafficking of skin-derived APCs inAckr4-deficient mice is completely rescued by genetic deletion ofCcl19 Thus, ACKR4 on stromal cells aids the egress of APCs from mouse skin, and, during inflammation, facilitates CCR7-dependent cell trafficking by scavenging CCL19.

TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway.

BackgroundTumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined.MethodsThe expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment.ResultsTNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo.ConclusionTaken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.

Abstract B36: Orally active low molecular weight heparin conjugates as a tumoral lymphangiogenesis inhibitor

Abstract Heparin has been reported as not only an anticoagulant drug but also as a tumoral angiogenesis inhibitor by blocking tumor growth factors including VEGF, bFGF. Low molecular weight heparin conjugated with 4 pairs of deoxycholic acid (LHbisD4) was newly synthesized to enhance the oral absorption and efficacy. As the tumoral lymphangiogenesis is being considered to play the most important role in tumor metastasis, the purpose of this study is to evaluate whether LHbisD4 could suppress the tumoral lymphangiogenesis as well as the incidence of metastasis. To evaluate the inhibition effect of LHbisD4 on VEGF-C induced lymphangiogenesis, several in vitro experiments using human dermal lymphatic endothelial cell(HDLEC) was carried out. The inhibition effect of LHbisD4 on VEGF-C induced VEGFR-3 phosphorylation was evaluated by western blot. Proliferation assay, tubular formation assay, spheroid based sprouting assay were also done to evaluate the efficacy of LHbisD4 on suppressing lymphagiogenesis using HDLEC. For in vivo test, orthotopic breast cancer metastasis model was used and the incidence of metastasis to distant organs was evaluated. We found that VEGF-C induced phosphorylation of VEGFR3 which is the most important signaling cascade to initiate the lymphangiogenesis was highly inhibited in the presence of LHbisD4. Also through other in vitro experiment such as proliferation assay, tubular formation assay, spheroid based sprouting assay, LHbisD4 was proven to be effective enough to suppress VEGF-C induced lymphangiogenesis. In orthotopic model, 10mg/kg/day dose of LHbisD4 was administered orally and we found the incidence of metastasis is highly decreased compared with control group. Especially, weight and volume of the lymph node is highly decreased in case of drug treated group, which means the lymphatic metastasis is significantly suppressed. Through this study, we demonstrate that our novel heparin conjugate could be a good candidate for targeting VEGF-C induced lymphangiogenesis as well as an effective anti-metastatic agent. Citation Format: Jeong Uk Choi, Youngro Byun. Orally active low molecular weight heparin conjugates as a tumoral lymphangiogenesis inhibitor. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B36.