Dermal Endothelial Cells Research Articles

Tazarotene-induced gene 3 may affect inflammatory angiogenesis in psoriasis by downregulating placental growth factor expression.

Tazarotene-induced gene 3 may affect inflammatory angiogenesis in psoriasis by downregulating placental growth factor expression.

Syndecan-3 is selectively pro-inflammatory in the joint and contributes to antigen-induced arthritis in mice.

IntroductionSyndecans are heparan sulphate proteoglycans expressed by endothelial cells. Syndecan-3 is expressed by synovial endothelial cells of rheumatoid arthritis (RA) patients where it binds chemokines, suggesting a role in leukocyte trafficking. The objective of the current study was to examine the function of syndecan-3 in joint inflammation by genetic deletion in mice and compare with other tissues.MethodsChemokine C-X-C ligand 1 (CXCL1) was injected in the joints of syndecan-3−/−and wild-type mice and antigen-induced arthritis performed. For comparison chemokine was administered in the skin and cremaster muscle. Intravital microscopy was performed in the cremaster muscle.ResultsAdministration of CXCL1 in knee joints of syndecan-3−/−mice resulted in reduced neutrophil accumulation compared to wild type. This was associated with diminished presence of CXCL1 at the luminal surface of synovial endothelial cells where this chemokine clustered and bound to heparan sulphate. Furthermore, in the arthritis model syndecan-3 deletion led to reduced joint swelling, leukocyte accumulation, cartilage degradation and overall disease severity. Conversely, CXCL1 administration in the skin of syndecan-3 null mice provoked increased neutrophil recruitment and was associated with elevated luminal expression of E-selectin by dermal endothelial cells. Similarly in the cremaster, intravital microscopy showed increased numbers of leukocytes adhering and rolling in venules in syndecan-3−/−mice in response to CXCL1 or tumour necrosis factor alpha.ConclusionsThis study shows a novel role for syndecan-3 in inflammation. In the joint it is selectively pro-inflammatory, functioning in endothelial chemokine presentation and leukocyte recruitment and cartilage damage in an RA model. Conversely, in skin and cremaster it is anti-inflammatory.

Dynamic Action of Myofibroblasts in Skin and Bone Marrow Transplantation

Skin is highly accessible and evaluable organ, which accelerate the understanding of novel medical innovation in association with organ transplantation, engineering, and wound healing, as well as the stage-specific adaptability of transplanted Bone Marrow (BM) cells. In skin transplantation biology, multi-stage/-angle damages occur in both grafted donor and perilesional host skin, and need to be repaired properly for the engraftment and later maintenance of the local homeostasis and characteristic skin architecture, such as stratified squamoid epithelium and dermal components. These local events are more unlikely to be regulated by the host immunity, because the donor (allogenic) skin engraftment mostly accomplishes onto the immunocompromised or immunosuppressive animals. Accumulating evidences have emerged the importance of alpha-Smooth Muscle Actin (SMA)-positive myofibroblasts, via stage- and cell type specific contribution of TGF-beta, PDGF, ET-1, CCN-2 signaling pathways and/or mastocyte-derived mediators (e.g. histamine and tryptase), for the functional reorganization of the grafted skin. Moreover, particular cell lineages from BM cells have been shown to harbor the differentiation capacity into multiple skin cell phenotypes, including epidermal keratinocytes, and dermal endothelial cells and pericytes, under controlled by chemokines or cytokines, but the trans-differentiation into alpha-SMA+ myofibroblasts is possibly reversed by inactivation of MEK/ERK signal cascade. We review the recent update of the myofibroblast biology in association with the reconstitution of the engrafted skin, and also work on translating this attractive action into the application of BM transplantation medicine in genetic skin diseases.

Downregulation Of Tissue Factor Expression In Human Pericytes In An Endothelial Cell Co-Culture Model

IntroductionTissue factor (TF) is a protein expressed by perivascular cells including pericytes (PCs) where it is responsible for initiation of blood coagulation. Many conditions have been associated with increased cellular TF expression, including inflammation, malignant transformation and tumor angiogenesis. However, our previous studies have shown that TF expression in PCs paradoxically decreases during wound healing.{McDonald:2008fy} To date, no studies have demonstrated the mechanism responsible for the downregulation of TF expression in any cell type. Because angiogenesis is a critical occurrence during wound healing, pro-angiogenic cytokines are likely candidates responsible for the downregulation of TF expression in PCs. Therefore, several well-characterized pro-angiogenic cytokines were studied for their effects on TF expression and activity in PCs. Materials and MethodsPCs (PromoCell, Heidielberg, Germany) were treated directly with each of five individual angiogenic factors: angiopoietin 2 (ANG2, 1-1000ng/mL), basic fibroblast growth factor (bFGF, 0.1-10 ng/mL), platelet derived growth factor (PDGF, 1-1000ng/mL), transforming growth factor beta (TGFB 0.2-200 ng/mL), and vascular endothelial growth factor (VEGF, 1-1000ng/mL). Serum rich media was used for all experiments and all treatments lasted 24 hours. PCs were assessed for TF activity using a coagulation factor X (FX) activation assay. In addition to direct treatment, PCs were assayed for TF protein expression following treatment in a co-culture model. Briefly, PCs were plated in a standard tissue culture treated plate. Human Dermal Microvessel Endothelial Cells (ECs) were placed inside a 0.4um polyester membrane transwell insert, which was placed into the well containing the PCs. This allowed for communication via soluble factors through the membrane, but not direct contact between the two cell types. Co-cultured ECs were treated with angiogenic factors as described above. Additionally, ECs cells were cultured for 24 hours with bFGF and the conditioned media was collected, cleared of cells, and applied to cultured pericytes. Following treatment PCs were lysed and whole cell lysate was separated by polyacrylamide gel electrophoresis and Western blot (WB). WB membranes were probed with anti-TF antibody for total protein determination. Actin was used as a loading control for all blots. ResultsFollowing direct stimulation for 24 hours, none of the angiogenic growth factors assayed produced a reduction in TF activity or antigen, even at supraphysiological concentrations. Following treatment of ECs in a co-culture model neither ANG2, PDGF, TGFB, nor VEGF produced a change in TF expression in pericytes. However, treatment of ECs with bFGF for 24 hours produced a marked decrease in total TF protein expression in co-cultured PCs. Four independent experiments confirmed the decreased expression of TF under these conditions. In contrast, bFGF conditioned EC media applied to PCs for 24 hours resulted in no change in TF protein expression. ConclusionsThe observation that bFGF stimulation of co-cultured ECs, but not direct stimulation, causes a decrease in TF protein expression in PCs suggests that a soluble mediator is responsible for the effect. The finding that a decrease in TF expression was not observed in PCs treated with bFGF-conditioned ECs media suggests that a labile mediator may be responsible. Disclosures:Hoffman:Novo Nordisk: Consultancy, Research Funding; CSL-Behring: Consultancy, Research Funding; Boehringer Ingelheim: Research Funding.

Norepinephrine and adenosine-5′-triphosphate synergize in inducing IL-6 production by human dermal microvascular endothelial cells

Endothelial cells (ECs) play important roles in cutaneous inflammation, in part, by release of inflammatory chemokines/cytokines. Because dermal blood vessels are innervated by sympathetic nerves, the sympathetic neurotransmitter norepinephrine (NE) and the co-transmitter adenosine-5′-triphosphate (ATP) may regulate expression of EC inflammatory factors. We focused on IL-6 regulation because it has many inflammatory and immune functions, including participation in Th17 cell differentiation. Strikingly, NE and ATP synergistically induced release of IL-6 by a human dermal microvascular endothelial cell line (HMEC-1). Adrenergic antagonist and agonist studies indicated that the effect of NE on induced IL-6 release is primarily mediated by β2-adrenergic receptors (ARs). By real-time PCR IL-6 mRNA was also synergistically induced in HMEC-1 cells. This synergistic effect of NE and ATP was reproduced in primary human dermal endothelial cells (pHDMECs) and is also primarily mediated by β2-ARs. Under conditions of stress, activation of the symphathetic nervous system may lead to release of ATP and NE by sympathetic nerves surrounding dermal blood vessels with induction of IL-6 production by ECs. IL-6 may then participate in immune and inflammatory processes including generation of Th17 cells. Production of IL-6 in this manner might explain stress-induced exacerbation of psoriasis, and perhaps, other skin disorders involving Th17-type immunity.

TGFβ signaling is required for sprouting lymphangiogenesis during lymphatic network development in the skin

Dermal lymphatic endothelial cells (LECs) emerge from the dorsolateral region of the cardinal veins within the anterior trunk to form an intricate, branched network of lymphatic vessels during embryogenesis. Multiple growth factors and receptors are required for specification and maintenance of LECs, but the mechanisms coordinating LEC movements and morphogenesis to develop three-dimensional lymphatic network architecture are not well understood. Here, we demonstrate in mice that precise LEC sprouting is a key process leading to stereotypical lymphatic network coverage throughout the developing skin, and that transforming growth factor β (TGFβ) signaling is required for LEC sprouting and proper lymphatic network patterning in vivo. We utilized a series of conditional mutants to ablate the TGFβ receptors Tgfbr1 (Alk5) and Tgfbr2 in LECs. To analyze lymphatic defects, we developed a novel, whole-mount embryonic skin imaging technique to visualize sprouting lymphangiogenesis and patterning at the lymphatic network level. Loss of TGFβ signaling in LECs leads to a severe reduction in local lymphangiogenic sprouting, resulting in a significant decrease in global lymphatic network branching complexity within the skin. Our results also demonstrate that TGFβ signaling negatively regulates LEC proliferation during lymphatic network formation. These data suggest a dual role for TGFβ signaling during lymphatic network morphogenesis in the skin, such that it enhances LEC sprouting and branching complexity while attenuating LEC proliferation.

Topical secretoneurin gene therapy accelerates diabetic wound healing by interaction between heparan-sulfate proteoglycans and basic FGF

Diabetic foot ulcers represent a therapeutic problem of high clinical relevance. Reduced vascular supply, neuropathy and diminished expression of growth factors strongly contribute to wound healing impairment in diabetes. Secretoneurin, an angiogenic neuropeptide, has been shown to improve tissue perfusion in different animal models by increasing the amount of vessels in affected areas. Therefore, topical secretoneurin gene therapy was tested in a full thickness wound healing model in diabetic db/db mice. Secretoneurin significantly accelerated wound closure in these mice and immunohistochemistry revealed higher capillary and arteriole density in the wounded area compared to control mice. In-vitro, the mechanism of action of secretoneurin on human dermal microvascular endothelial cells was evaluated in normal and diabetic cells. Secretoneurin shows positive effects on in vitro angiogenesis, proliferation and apoptosis of these cells in a basic fibroblast growth factor dependent manner. A small molecular weight inhibitor revealed fibroblast growth factor receptor 3 as the main receptor for secretoneurin mediated effects. Additionally, we could identify heparan-sulfates as important co-factor of secretoneurin induced binding of basic fibroblast growth factor to human dermal endothelial cells. We suggest topical secretoneurin plasmid therapy as new tool for delayed wound healing in patients suffering from diabetes.

Changes of Bcl-2, Bax and Caspase-3 expression in the dermal microvascular endothelial cells and the epidermal layers of the eschar (tache noire) in patients with Mediterranean spotted fever

Mediterranean spotted fever (MSF) is widely prevalent in many endemic regions in Bulgaria. The disease is still not quite thoroughly studied as to some aspects of its pathogenesis and especially to issues that concern the crucial signals for apoptosis in the target microvascular endothelial cells. To study the expression of Bcl-2 family proteins and Caspase-3 in the dermal capillary endothelial cells from skin papules and in the eschar (tache noire) epidermal layers of patients with MSF so that we can establish apoptotic processes and the time of their occurrence and deployment. Immunohistochemical reactions for Bcl-2, Bax and Caspase-3 were obtained in slices of punch-biopsies taken from papules of the skin rash and from the eschars of eight patients with MSF. The average intensity of the reactions was compared with that in control punch-biopsy slices from four healthy subjects. MSF was etiologically confirmed in all patients by positive antibody response to a specific antigen, Rickettsia conorii, with indirect immunofluorescent assay performed by the Rickettsial Reference Laboratory. The immune reaction for Bcl-2 was found to be poorly expressed in the capillary endothelial cells of skin papules of patients without any differences from controls. The expression of Bax and Caspase-3 was strongly upregulated in comparison with the controls. The Bcl-2/Bax ratio was significantly decreased. Microvascular endothelial cells of the eschar showed similar changes. While the Bcl-2/Bax ratio decreased in the epidermal layers of the eschar "tache noire", there were no changes in the intensity of the immunoreactivity of Caspase-3 as compared with controls. The upregulation of Bax and Caspase-3 is an indication of ongoing apoptotic processes in the dermal microvascular endothelial cells of MSF patients. The epidermal layers of the eschar showed increased sensitivity to apoptosis, however, executive phase of apoptosis did not occur.

IL‐27 Inhibits Lymphatic Endothelial Cell Proliferation by STAT1‐Regulated Gene Expression

IL-27 belongs to the IL-12 family of cytokines and is recognized for its role in Th cell differentiation and as an inhibitor of tumor angiogenesis. The purpose of this study was to investigate the effect of IL-27 on proliferation of lymphatic endothelial cells to gain insight into the interplay between the immune system and development of the lymphatic system. IL-27-stimulated signal transduction in human dermal lymphatic endothelial cells was measured by western blotting and synthesis of CXCL10 and CXCL11 by use of RT-PCR and ELISA. Proliferation was measured using MTT and BrdU kits and the role of STAT1 and chemokines was determined by use of siRNA and recombinant proteins. Stimulation of lymphatic endothelial cell cultures with IL-27 induced JAK dependent phosphorylation of STAT1 and STAT3 and inhibited lymphatic endothelial cell proliferation and migration. Expression of CXCL10 and CXCL11, both STAT1 target genes, was profoundly up-regulated upon IL-27 stimulation, and recombinant CXCL10 and CXCL11 inhibited FGF-2-induced proliferation in vitro. siRNA targeting of STAT1 almost completely abrogated CXCL10 and CXCL11 expression as well as the proliferative effect of IL-27. IL-27 function as an anti-lymphangiogenic regulator in vitro by up-regulating chemokines and interfering with the mitogenic effect of growth factors through STAT1 activation.

EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2's tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.

Atrial Natriuretic Peptide–Mediated Inhibition of Microcirculatory Endothelial Ca 2+ and Permeability Response to Histamine Involves cGMP-Dependent Protein Kinase I and TRPC6 Channels

Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and -independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI). We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial-restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca(2+)]i in vitro and of vascular leakage in vivo were markedly attenuated by the Ca(2+)-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr69 and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion. ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP.

Enhanced Lymph Vessel Density, Remodeling, and Inflammation Are Reflected by Gene Expression Signatures in Dermal Lymphatic Endothelial Cells in Type 2 Diabetes

Type 2 diabetes is associated with microvascular damage that causes frequent infections in the skin and chronic ulcers as a result of impaired wound healing. To trace the pathological changes, we performed a comprehensive analysis of lymphatic vessels in the skin of type 2 diabetic versus nondiabetic patients. The dermis revealed enhanced lymphatic vessel density, and transcriptional profiling of ex vivo isolated lymphatic endothelial cells (LECs) identified 160 genes differentially expressed between type 2 diabetic and nondiabetic LECs. Bioinformatic analysis of deregulated genes uncovered sets functionally related to inflammation, lymphatic vessel remodeling, lymphangiogenesis, and lipid and small molecule transport. Furthermore, we traced CD68+ macrophage accumulation and concomitant upregulation of tumor necrosis factor-α (TNF-α) levels in type 2 diabetic skin. TNF-α treatment of LECs and its specific blockade in vitro reproduced differential regulation of a gene set that led to enhanced LEC mobility and macrophage attachment, which was mediated by the LEC-derived chemokine CXCL10. This study identifies lymph vessel gene signatures directly correlated with type 2 diabetes skin manifestations. In addition, we provide evidence for paracrine cross-talk fostering macrophage recruitment to LECs as one pathophysiological process that might contribute to aberrant lymphangiogenesis and persistent inflammation in the skin.

THU0059 Decreased Expression of Neuropilin-1 in Systemic Sclerosis: Potential Contribution to Impaired Angiogenesis

BackgroundSystemic sclerosis (SSc) is a chronic, multisystem connective tissue disorder characterised by widespread microangiopathy, fibrosis and autoimmunity. Vascular damage is a primary event in SSc and is characterised by vascular...

AB0242 Evidence for caveolin-1 (CAV1) as a new susceptibility gene regulating tissue fibrosis in systemic sclerosis

BackgroundCaveolin-1 (CAV1) is an inhibitor of tissue fibrosis and has been implicated in the pathogenesis of systemic sclerosis (SSc).ObjectivesThe aim of our study was to analyse the possible association of...

Differential Gene Expression in Cholesteatoma by DNA Chip Analysis

In contrast to normal epithelium, the desquamating stratified squamous epithelium of temporal bone cholesteatoma characteristically exhibits sustained hyperproliferative growth and a capacity for bone erosion. We conducted genome-wide microarray analyses to determine the molecular nature of cholesteatoma's biological processes and identify disease-associated, altered gene activity. We tested the hypothesis that genes contributing to the pathophysiology of cholesteatoma are differentially expressed compared to control tissue. Prospective experimental analysis. Using new, enhanced microarray platforms and well-annotated human transcriptome probes, we measured global gene expression levels in surgical specimens of cholesteatoma and in the corresponding normal postauricular skin in four patients. Genes of interest were verified by quantitative real time reverse transcriptase polymerase chain reaction analyses using cholesteatoma and postauricular sample pairs (n = 13). External auditory canal skin from six additional patients was also evaluated as a normal control. Immunohistochemistry detected protein expression in tissue sections and the cells involved. DNA chip analyses identified 282 differentially expressed genes in cholesteatoma compared to control samples. Of these, 104 genes were upregulated and 178 were downregulated. Ontological classifications indicate relationships to cellular processes including receptor binding, cell communication and motion, vitamin metabolism, and cytokine-mediated inflammation. Based on potential involvement in disease pathology, 10 genes were selected and independently verified by quantitative polymerase chain reaction. Immunohistochemical detection of transcobalamin-1 and CCL27 implicates cholesteatoma keratinocytes and dermal endothelial cells as contributors in disease processes. We present a comprehensive, human genome-wide survey of disease-associated gene expression that extends the public database and provides new evidence for molecular mechanisms involved in cholesteatoma pathology. Laryngoscope, 123:S1-S21, 2013.

Activation of the epidermal growth factor receptor promotes lymphangiogenesis in the skin

BackgroundThe lymphatic vascular system regulates tissue fluid homeostasis and plays important roles in immune surveillance, inflammation and cancer metastasis. However, the molecular mechanisms involved in the regulation of lymphangiogenesis remain incompletely characterized. ObjectiveWe aimed to identify new pathways involved in the promotion of skin lymphangiogenesis. MethodsWe used a mouse embryonic stem cell-derived embryoid body vascular differentiation assay to investigate the effects of a selection of pharmacological agents with the potential to inhibit blood and/or lymphatic vessel formation. We also used a subcutaneous Matrigel assay to study candidate lymphangiogenesis factors as well as skin-specific transgenic mice. ResultsWe found that compounds inhibiting the epidermal growth factor (EGF) receptor (EGFR) led to an impaired formation of lymphatic vessel-like structures. In vitro studies with human dermal lymphatic endothelial cells (LECs), that were found to express EGFR, revealed that EGF promotes lymphatic vessel formation. This effect was inhibited by EGFR-blocking antibodies and by low molecular weight inhibitors of the EGFR associated tyrosine kinase. Incorporation of EGF into a mouse matrigel plug assay showed that EGF promotes enlargement of lymphatic vessels in the skin in vivo. Moreover, transgenic mice with skin-specific overexpression of amphiregulin, another agonistic ligand of the EGFR, displayed an enhanced size and density of lymphatic vessels in the skin. ConclusionThese findings reveal that EGFR activation is involved in lymphatic remodeling and suggest that specific EGFR antagonists might be used to inhibit pathological lymphangiogenesis.

Abstract B15: Invasive breast cancer amplifies lymphangiogenic signals in tumor microenvironment through integrin beta4 expressing cancer-associated fibroblasts

Abstract Lymphangiogensis is widely acknowledged as a major route of breast cancer metastasis and a critical alteration of tumor microenvironment (TME) for tumor progression. But, most of it has remained still unknown. Previously, we found that invasive ductal carcinoma (IDC), not ductal carcinoma in situ (DCIS), upregulates integrin alpha6beta4 in the fibrosis adjacent to tumor burden, and that MDA-MB-231 induces integrin beta4 neoexpression in cancer-associated fibroblasts (CAFs) via cell-to-cell contract. Integrin (alpha6)beta4 overexpression is often detected in malignant tumors and negatively associated with patient survivor. In addition, integrin alpha6beta4 was reported to regulate eIF-4E activity leading to vascular endothelial growth factor (VEGF) translation in cancer cells. It is, therefore, plausible that invasive breast cancer cells increase lymphangiogenic signals in TME through driving integrin beta4 expression in CAFs. To verify our hypothesis, we induced integrin beta4 overexpression in CAFs using co-culture with MDA-MB-231 or transfection with pRK5_beta4 plasmid, and then observed tube formation of human dermal lymphatic endothelial cells (HDLEC) by integrin beta4-expressing CAFs in 3D co-culture model. VEGF-D, known as a major factor of lymphangiogenesis, was highly upregulated in integrin beta4-expressing CAFs, which promoted tube formation of HDLEC compared to normal CAFs and MDA-MB-231. Interestingly, the level of VEGF-D expression in MDA-MB-231 was much lower than that of integrin beta4-expressing CAFs before and after co-culture. Taken together, invasive breast cancer may amplify lymphangiogenic signals in TME by inducing integrin beta4-expressing CAFs, and integrin beta4 neoexpression in CAFs by breast cancer may be a pivotal process for lymphangiogensis. Citation Format: Baek Gil Kim, Yoon Pyo Choi, Suki Kang, Joo Hyun Lee, Nam Hoon Cho. Invasive breast cancer amplifies lymphangiogenic signals in tumor microenvironment through integrin beta4 expressing cancer-associated fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B15.

CD200 Upregulation in Vascular Endothelium Surrounding Cutaneous Squamous Cell Carcinoma

To characterize the presence of CD200 and CD200 receptor (CD200R) in the human cutaneous squamous cell carcinoma (SCC) microenvironment and to define a possible role for the CD200 axis in immune evasion by SCC. Gene expression in SCC vs normal skin was studied. Laser capture microdissection was used to determine differential expression of CD200 in normal skin vs actinic keratosis vs SCC in situ vs invasive SCC. Immunofluorescence microscopy was used to define expression of CD200R on macrophages, myeloid dendritic cells, natural killer cells, and T cells in SCC vs normal skin. The effects of SCC supernatant on induction of CD200 in human blood endothelial cells was also examined. Academic Medical Center with an established Section of Mohs and Dermatologic Surgery and an active Cutaneous Biology Research Program. Surgical discard tissue from deidentified patients and samples of normal skin from healthy volunteers were used in this study. Expression of CD200 on SCC-associated blood vessels; expression of CD200 receptor on SCC-associated macrophages and T cells; and induction of CD200 on endothelial cells by SCC supernatants. CD200 gene and message were upregulated in SCC stroma. Immunostaining revealed a higher number of CD200(+) cells in SCC stroma than in normal dermis (180.8 cells/mm(2) vs 24.6 cells/mm(2)) (P<.01). CD200 was further identified mainly on blood vessel endothelium in SCC. Tumor supernatant was able to induce CD200 expression on human dermal blood endothelial cells in culture. CD200R was identified on macrophages and dendritic cells in SCC microenvironment. CD200 expression on local blood vessels may promote tumor progression by suppressing CD200R myeloid cells during diapedesis. These data highlight a previously unrecognized mechanism of immune evasion by SCC and may provide guidance for the development of targeted therapy.

Cadherin adhesome at a glance

This article is part of a Minifocus on Adhesion. For further reading, please see related articles: ‘Cycling around cell–cell adhesion with Rho GTPase regulators’ by Jessica McCormack et al. ( J. Cell Sci. 126 , 379-391). ‘E-cadherin–integrin crosstalk in cancer invasion and metastasis’

Gene expression analysis reveals marked differences in the transcriptome of infantile hemangioma endothelial cells compared to normal dermal microvascular endothelial cells

BackgroundInfantile hemangiomas are benign vascular tumors primarily found on the skin in 10% of the pediatric population. The etiology of this disease is largely unknown and while large scale genomic studies have examined the transcriptomes of infantile hemangioma tumors as a whole, no study to date has compared the global gene expression profiles of pure infantile hemangioma endothelial cells (HEMECs) to that of normal human dermal microvascular endothelial cells (HDMVECs).MethodsTo shed light on the molecular differences between these normal and aberrant dermal endothelial cell types, we performed whole genome microarray analysis on purified cultures of HEMECs and HDMVECs. We then utilized qPCR and immunohistochemistry to confirm our microarray results.ResultsOur array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated by greater than two fold in HEMECs compared to HDMVECs. Bioinformatics analysis revealed three major classifications of gene functions that were altered in HEMECs including cell adhesion, cell cycle, and arachidonic acid production. Several of these genes have been reported to be critical regulators and/or mutated in cancer, vascular tumors, and vascular malformations. We confirmed the expression of a subset of these differentially expressed genes (ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1) using qPCR and utilized immunohistochemistry on a panel of paraffin embedded infantile hemangioma tumor tissues to demonstrate that the cancer/testis antigen CTAG2 is highly abundant in vessel-dense proliferating infantile hemangiomas and with significantly reduced levels during tumor involution as vascular density decreases.ConclusionOur data reveal that the transcriptome of HEMECs is reflective of a pro-proliferative cell type with altered adhesive characteristics. Moveover, HEMECs show altered expression of many genes that are important in the progression and prognosis of metastatic cancers.