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Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of Atherosclerosis in Diabetes Aino Soro-Paavonen 1 , Anna M.D. Watson 1 , Jiaze Li 1 , Karri Paavonen 1 , Audrey Koitka 1 , Anna C. Calkin 1 , David Barit 1 , Melinda T. Coughlan 1 , Brian G. Drew 2 , Graeme I. Lancaster 3 , Merlin Thomas 1 , Josephine M. Forbes 1 , Peter P. Nawroth 4 , Angelika Bierhaus 4 , Mark E. Cooper 1 and Karin A. Jandeleit-Dahm 1 1 Albert Einstein Juvenile Diabetes Research Foundation Centre for Diabetes Complications, Diabetes Metabolism Division, Baker Heart Research Institute, Melbourne, Australia 2 Clinical Physiology Laboratory, Baker Heart Research Institute, Melbourne, Australia 3 Cellular and Molecular Metabolism Laboratory, Baker Heart Research Institute, Melbourne, Australia 4 Department of Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany Corresponding author: Karin Jandeleit-Dahm, karin.jandeleit-dahm{at}baker.edu.au Abstract OBJECTIVE— Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE −/− model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS— ApoE −/− and RAGE −/− /apoE −/− double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS— Although diabetic apoE −/− mice showed increased plaque accumulation (14.9 ± 1.7%), diabetic RAGE −/− /apoE −/− mice had significantly reduced atherosclerotic plaque area (4.9 ± 0.4%) to levels not significantly different from control apoE −/− mice (4.3 ± 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-κB subunit p65 , VCAM-1 , and MCP-1 ; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox , p47phox , and rac-1 . Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE −/− mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE −/− /apoE −/− mice. CONCLUSIONS— This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications. Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 28 May 2008. Readers may use this article as 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-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted May 20, 2008. Received December 21, 2007. DIABETES

Vascular endothelial growth factor receptor-3 (VEGFR-3) is essential for embryonic cardiovascular development, but thereafter becomes confined to the lymphatic endothelium in adult tissues. We have here studied VEGFR-3 expression in experimental wounds of pigs and chronic inflammatory wounds of humans. In healing incisional and punch biopsy wounds made in the dorsal skin of pigs, angiogenic blood vessels, identified by use of the blood vascular endothelial markers vWF and PAL-E and the basal lamina protein laminin, developed into the granulation tissue stroma from day 4 onward, being most abundant on days 5 and 6 and regressing thereafter. VEGFR-3-positive vessels were observed in the granulation tissue from day 5 onward. These vessels were distinct from the PAL-E/laminin/vWF-positive vessels and fewer in number, and they appeared to sprout from pre-existing VEGFR-3-positive lymphatic vessels at the wound edge. Unlike the blood vessels, very few VEGFR-3-positive lymphatic vessels persisted on day 9 and none on day 14. In chronic wounds such as ulcers and decubitus wounds of the lower extremity of. humans, VEGFR-3 was also weakly expressed in the vascular endothelium. Our results suggest that transient lymphangiogenesis occurs in parallel with angiogenesis in healing wounds and that VEGFR-3 becomes up-regulated in blood vessel endothelium. in chronic inflammatory wounds.

The vascular endothelial growth factor (VEGF) family has recently been expanded by the isolation of two additional growth factors, VEGF-B and VEGF-C. Here we compare the regulation of steady-state levels of VEGF, VEGF-B and VEGF-C mRNAs in cultured cells by a variety of stimuli implicated in angiogenesis and endothelial cell physiology. Hypoxia, Ras oncoprotein and mutant p53 tumor suppressor, which are potent inducers of VEGF mRNA did not increase VEGF-B or VEGF-C mRNA levels. Serum and its component growth factors, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) as well as transforming growth factor-beta (TGF-beta) and the tumor promoter phorbol myristate 12,13-acetate (PMA) stimulated VEGF-C, but not VEGF-B mRNA expression. Interestingly, these growth factors and hypoxia simultaneously downregulated the mRNA of another endothelial cell specific ligand, angiopoietin-1. Serum induction of VEGF-C mRNA occurred independently of protein synthesis; with an increase of the mRNA half-life from 3.5 h to 5.5-6 h, whereas VEGF-B mRNA was very stable (T 1/2>8 h). Our results reveal that the three VEGF genes are regulated in a strikingly different manner, suggesting that they serve distinct, although perhaps overlapping functions in vivo.

Sox18 as a lymphatic switch Mutations in the Sox18 transcription factor gene cause lymphatic dysfunction in the rare human syndrome hypotrichosis-lymphoedema-telangiectasia, suggesting that it may play a role in lymphatic development of function. That role has now been identified: Sox18 acts as a molecular switch to induce differentiation of lymphatic endothelial cells via the direct activation of transcription of the Prox1 lymphatic marker in lymphatic precursor cells of the embryonic venous system. This suggests possible new strategies for therapeutic stimulation or suppression of lymphoangiogenesis. A role for Sox18 transcription factor has been suggested by lymphatic dysfunction in the human syndrome hypotrichosis-lymphedema-telangiectasia (HLT), which is caused by mutations in Sox18 . This paper shows that Sox18 directly activates Prox1 transcription. Sox18 -null embryos show a complete absence of Prox1 -positive lymphatic endothelial cells emanating from the cardinal vein. The lymphatic system plays a key role in tissue fluid regulation and tumour metastasis, and lymphatic defects underlie many pathological states including lymphoedema, lymphangiectasia, lymphangioma and lymphatic dysplasia 1 , 2 , 3 . However, the origins of the lymphatic system in the embryo, and the mechanisms that direct growth of the network of lymphatic vessels, remain unclear. Lymphatic vessels are thought to arise from endothelial precursor cells budding from the cardinal vein under the influence of the lymphatic hallmark gene Prox1 (prospero homeobox 1; ref. 4 ). Defects in the transcription factor gene SOX18 (SRY (sex determining region Y) box 18) cause lymphatic dysfunction in the human syndrome hypotrichosis-lymphoedema-telangiectasia 5 , suggesting that Sox18 may also play a role in lymphatic development or function. Here we use molecular, cellular and genetic assays in mice to show that Sox18 acts as a molecular switch to induce differentiation of lymphatic endothelial cells. Sox18 is expressed in a subset of cardinal vein cells that later co-express Prox1 and migrate to form lymphatic vessels. Sox18 directly activates Prox1 transcription by binding to its proximal promoter. Overexpression of Sox18 in blood vascular endothelial cells induces them to express Prox1 and other lymphatic endothelial markers, while Sox18 -null embryos show a complete blockade of lymphatic endothelial cell differentiation from the cardinal vein. Our findings demonstrate a critical role for Sox18 in developmental lymphangiogenesis, and suggest new avenues to investigate for therapeutic management of human lymphangiopathies.

Tumor metastasis to lymph nodes is a key indicator of patient survival, and is enhanced by the neo-lymphatics induced by tumor-secreted VEGF-C or VEGF-D, acting via VEGFR-3 signalling. These targets constitute important avenues for anti-metastatic treatment. Despite this new understanding, clinical observations linking metastasis with tumor depth or location suggest that lymphangiogenic growth factors are not the sole determinants of metastasis. Here we explored the influence of tumor proximity to lymphatics capable of responding to growth factors on nodal metastasis in a murine VEGF-D over-expression tumor model. We found that primary tumor location profoundly influenced VEGF-D-mediated lymph node metastasis: 89 % of tumors associated with the flank skin metastasised, in contrast with only 19 % of tumors located more deeply on the body wall ( p  < 0.01). Lymphatics in metastatic tumors arose from small lymphatics, and displayed distinct molecular and morphological profiles compared with those found in normal lymphatics. Smaller lymphatic subtypes were more abundant in skin (2.5-fold, p  < 0.01) than in body wall, providing a richer source of lymphatics for VEGF-D + skin tumors, a phenomenon also confirmed in human samples. This study shows that the proximity of a VEGF-D + primary tumor to small lymphatics is an important determinant of metastasis. These observations may explain why tumor location relative to the lymphatic network is prognostically important for some human cancers.

In mice, vascular endothelial growth factor-C (VEGF-C) plays an important role in development of the lymphatic system and in pathogenesis of pulmonary inflammation. Its role in development of the lymphatic system in human lung and in lung injury in newborns remains unclear. We studied the role of VEGF-C in developing human lung, and in acute and chronic lung injury in preterm infants. Included in the immunohistochemistry study were 10 fetuses, 15 control neonates without primary lung disease, 15 preterm infants with respiratory distress syndrome, and 8 infants with bronchopulmonary dysplasia. Tracheal aspirate fluid samples of intubated very-low-birth-weight infants during Postnatal Weeks 1-5 were analyzed with ELISA. Bronchiolar staining for VEGF-C was observed in all 48 samples. Alveolar epithelial staining was seen in most fetuses (8/10). In addition, staining was observed in alveolar macrophages in bronchopulmonary dysplasia (4/8), and late respiratory distress syndrome (2/7). VEGF receptor-3 (VEGFR-3) staining was observed in lymphatic endothelium adjacent to vascular endothelium. VEGF-C was expressed consistently in tracheal aspirate fluid, being highest during the first 2 postnatal days. Antenatal administration of glucocorticoids was associated with higher VEGF-C in tracheal aspirate fluid. The pattern of pulmonary VEGF-C and VEGFR-3 protein expression and consistent VEGF-C protein appearance in tracheal aspirate fluid in human preterm infants indicate a role for VEGF-C in the physiologic development of the lymphatic system of the lung.

Sox18 as a lymphatic switch