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Enhanced Lymph Vessel Density, Remodeling, and Inflammation Are ORIGINAL ARTICLE Enhanced Lymph Vessel Density, Remodeling, and Inflammation Are Reflected by Gene Expression Signatures in Dermal Lymphatic Endothelial Cells in Type 2 Diabetes Monika Haemmerle,1 Thomas Keller,1 Gerda Egger,1 Helga Schachner,1 Carl Walter Steiner,2 Dejan Stokic,3 Christoph Neumayer,4 Markus K. Brown,1 Dontscho Kerjaschki,1 and Brigitte Hantusch1 Type 2 diabetes is associated with microvascular damage that organ damage. Large vessels react to the chronically in- causes frequent infections in the skin and chronic ulcers as creased glucose and glucose-driven metabolites with a result of impaired wound healing. To trace the pathological enhanced arteriosclerosis. Diabetic microangiopathy grad- changes, we performed a comprehensive analysis of lymphatic ually evokes retinopathy, leading to subsequent blindness, vessels in the skin of type 2 diabetic versus nondiabetic patients. and nephropathy, the most frequent reason of renal in- The dermis revealed enhanced lymphatic vessel density, and sufficiency. In the skin, microvasculopathy causes pro- transcriptional profiling of ex vivo isolated lymphatic endothelial fl fi longed in ammation, impaired healing of wounds, and cells (LECs) identi ed 160 genes differentially expressed be- ulcers (6). tween type 2 diabetic and nondiabetic LECs. Bioinformatic – analysis of deregulated genes uncovered sets functionally related Type 2 diabetes induced microvascular lesions are to inflammation, lymphatic vessel remodeling, lymphangiogene- characterized by aberrant matrix component deposition, sis, and lipid and small molecule transport. Furthermore, we traced resulting in narrowing of the vascular lumen that causes CD68+ macrophage accumulation and concomitant upregulation of ischemia. Concurrently, the affected blood vessel endo- tumor necrosis factor-a (TNF-a) levels in type 2 diabetic skin. thelium shows imbalances of vasoconstrictors and -dilators, TNF-a treatment of LECs and its specific blockade in vitro repro- secretion of pro- and anti-inflammatory cytokines, and in- duced differential regulation of a gene set that led to enhanced creased prothrombotic activity (7), which leads to leaki- LEC mobility and macrophage attachment, which was mediated by the LEC-derived chemokine CXCL10. This study identifies ness and sustained effusion of leukocytes and plasma lymph vessel gene signatures directly correlated with type 2 di- components into the tissue. In contrast to blood vessels, abetes skin manifestations. In addition, we provide evidence for nothing is known so far about the involvement of the paracrine cross-talk fostering macrophage recruitment to LECs lymphatic vasculature in human type 2 diabetes, although as one pathophysiological process that might contribute to aber- dermal lymphatic vessels are known to play important rant lymphangiogenesis and persistent inflammation in the skin. roles in tissue fluid homeostasis, lipid absorption, and Diabetes 62:2509–2529, 2013 immune surveillance (8). Of note, lymphatic vessels func- tion as collectors and export conduits of inflammatory cells, representing gatekeepers for macrophage and lym- phocyte abundance in different tissues (9). Pathological he incidence of type 2 diabetes and obesity is fl rapidly increasing worldwide (1). Currently, gen- processes of in ammation, wound healing, and adipo- eralized insulin insensitivity is considered the genesis, all relevant for type 2 diabetes, have been linked central pathogenic event (2) that is frequently to functional defects of the lymphatic system in animal T experiments (9). However, for human patients, it is cur- linked to a systemic metabolic syndrome, a state of chronic low-level inflammation involving macrophage ac- rently unknown whether lymphatics remain unchanged, tivation in adipose tissue (3). Recent insights also indicate are passive bystanders, or participate actively in the skin genetic factors in the development of the disease (4). lesions of type 2 diabetes. However, chronic hyperglycemia induces extensive macro- In this article, we report on enhanced lymphatic and microvascular alterations (5) that lead to systemic microvessel density in the skin of type 2 diabetic patients. By comparing the gene expression profiles of freshly iso- lated dermal lymphatic endothelial cells (LECs) from From the 1Clinical Institute of Pathology, Medical University of Vienna, 2 patients with type 2 diabetes with those of normoglycemic Vienna, Austria; the Department of Internal Medicine, Division of Rheuma- fi tology, Medical University of Vienna, Vienna, Austria; the 3Section for Sci- controls, we identi ed molecular and cellular processes ence of Complex Systems, Medical University of Vienna, Vienna, Austria; regulated in lymphatic vessels, in particular, proin- and the 4Division of Vascular Surgery, Department of Surgery, Medical Uni- flammatory, lymphangiogenic, and enhanced lipid shut- versity of Vienna, Vienna, Austria. Corresponding author: Brigitte Hantusch, [email protected] tling properties, accompanied by downregulated immune .at. defense, apoptosis mediators, and small compound trans- Received 22 June 2012 and accepted 9 February 2013. porters. Concomitantly, we traced a strong dermal CD68+ DOI: 10.2337/db12-0844 fi This article contains Supplementary Data online at http://diabetes macrophage in ltration, which elicited elevated tumor .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0844/-/DC1. necrosis factor-a (TNF-a) levels. A subset of diabetic LEC Ó 2013 by the American Diabetes Association. Readers may use this article as (dLEC) deregulated genes was TNF-a responsive and long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by correlated with lymphatic vessel remodeling and in- -nc-nd/3.0/ for details. flammation, including the chemokine CXCL10, which diabetes.diabetesjournals.org DIABETES, VOL. 62, JULY 2013 2509 DERMAL LYMPHATIC VESSELS IN TYPE 2 DIABETES specifically led to macrophage attraction and adhesion to Experiments were performed in triplicate, and statistical differences between LECs in vitro. Hence, we have obtained the first indica- groups were analyzed as detailed later. tions to our knowledge that the dermal lymphatic system is LEC motility and population doubling assay. For migration assay, a scratch wound was created in confluent LEC monolayers grown in 24-well plates. actively involved in the progression of skin manifestations Nonadherent cells were washed away, fresh medium with or without TNF-a as in type 2 diabetes. described previously (n = 3) was added, and wound closure was monitored every hour with an inverted live cell microscope (Axiovert 200M; Zeiss). For population doubling analysis, identical LEC numbers were grown in six wells RESEARCH DESIGN AND METHODS with or without addition of TNF-a (three in each group). After defined time Skin samples from type 2 diabetic and nondiabetic patients. The study points, cells were washed, trypsinized, and counted in a hemocytometer. was approved by the local ethics committee (proposal no. 449/2001; 81/2008), Population doublings were calculated as ln(cell concentration counted/cell and all patients (described in Supplementary Table 1) gave informed consent. concentration seeded). Skin samples (n = 4 in each group) were taken from the proximal region of Macrophage adhesion and chemotaxis assay. Human monocytic leukemia amputated legs or abdominoplastic tissue, and care was taken to excise areas cell line THP-1 (American Type Culture Collection no. TIB-202) was grown in at maximal distance from inflammatory or ulcerous changes (;15 cm). RPMI 1640/10% FCS (Gibco no. 10108)/1% Pen/Strep and labeled with Cell- Immunohistochemical analyses. Immunohistochemical stainings of paraffin- Tracker Green CMFDA (5-chloromethylfluorescein diacetate) Molecular embedded or cryofixed skin sections were performed as described previously Probes 1:5,000 (Invitrogen no. C2925). Five by 105 cells were stimulated with (10). Supplementary Table 2 summarizes the antibodies and respective dilu- 10 ng/mL phorbol myristic acid (PMA) (Sigma no. P8139) for 24 h, diluted in tions applied. For quantifications, under exclusion of empty areas, non- 500 mL EBM-2/0.5% FCS, and immediately added to LEC monolayers (n = 3). In overlapping microscopic fields (regions of interest [ROIs]) of 100 mm2 (30 blocking experiments, LECs were preincubated for 1 h with inhibitory anti- fields per patient) were captured with an Olympus VANOX AHBT3 micro- CXCL10 and anti-vascular cell adhesion molecule 1 (VCAM-1) antibodies be- scope. Positively stained vessels, cellular nuclei, and macrophages were fore addition of THP-1 cells. After 3 h, nonadherent cells were washed away, counted in these ROIs, and the cross-sectional dimension (referred to as di- and adherent THP-1 cells were photographed with an inverted live cell mi- ameter) of the vessels was measured. Average numbers were calculated per croscope (Axiovert 200M). Agarose spot assay was performed as described patient group and statistically analyzed as detailed later. previously (12). Briefly, 1% agarose solution (Gibco no. 18300-012) was mixed Ex vivo isolation of dermal LECs. Micropreparation of LECs was performed 1:1 with concentrated LEC culture supernatants (see previous). Two 10-mL as described previously (10). Briefly, human skin was dermatomized and spots of this mixture were pipetted into 24-well plates and allowed to gel for epidermis and dermis dislocated by incubation in dispase solution (Roche no.
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