Hyaluronan synthase 3 promotes plaque inflammation and atheroprogression.

OBJECTIVE: Hyaluronan (HA) is a prominent component of the provisional extracellular matrix (ECM) present in the neointima of atherosclerotic plaques. Here the role of HA synthase 3 (HAS3) in atheroprogression was studied. APPROACH AND RESULTS: It is demonstrated here that HAS isoenzymes 1, -2 and -3 are expressed in human atherosclerotic plaques of the carotid artery. In Apolipoprotein E (Apoe)-deficient mice Has3 expression is increased early during lesion formation when macrophages enter atherosclerotic plaques. Importantly, HAS3 expression in vascular smooth muscle cells (VSMC) was found to be regulated by interleukin 1 ß (IL-1ß) in an NFkB dependent manner and blocking antibodies to IL-1ß abrogate Has3 expression in VSMC by activated macrophages. Has3/Apoe double deficient mice developed less atherosclerosis characterized by decreased Th1-cell responses, decreased IL-12 release, and decreased macrophage-driven inflammation. CONCLUSIONS: Inhibition of HAS3-dependent synthesis of HA dampens systemic Th1 cell polarization and reduces plaque inflammation. These data suggest that HAS3 might be a promising therapeutic target in atherosclerosis. Moreover, because HAS3 is regulated by IL-1ß, our results suggest that therapeutic anti-IL-1ß antibodies, recently tested in human clinical trials (CANTOS), may exert their beneficial effects on inflammation in post-myocardial infarction patients in part via effects on HAS3. TOC categorybasic study TOC subcategoryarteriosclerosis.

Deletion of Hyaluronan Synthase 3 Inhibits Neointimal Hyperplasia in Mice.

OBJECTIVE: Hyaluronan (HA) is a polymeric glucosaminoglycan that forms a provisional extracellular matrix in diseased vessels. HA is synthesized by 3 different HA synthases (HAS1, HAS2, and HAS3). Aim of this study was to unravel the role of the HAS3 isoenzyme during experimental neointimal hyperplasia. APPROACH AND RESULTS: Neointimal hyperplasia was induced in Has3-deficient mice by ligation of the carotid artery. HA in the media of Has3-deficient mice was decreased 28 days after ligation, and neointimal hyperplasia was strongly inhibited. However, medial and luminal areas were unaffected. Cell density, proliferation, and apoptosis were not altered, suggesting a proportional decrease of both, the number of cells and extracellular matrix. In addition, endothelial function as determined by acetylcholine-induced relaxation of aortic rings, immunoblotting of endothelial nitric oxide synthase, and arterial blood pressure were not affected. Furthermore, the oxidative stress response was not affected as determined in total protein extracts from aortae. Transcriptome analysis comparing control versus ligated carotid arteries hinted toward a mitigated differential regulation of various signaling pathways in Has3-deficient mice in response to ligation that were related to vascular smooth muscle cell (VSMC) migration, including focal adhesions, integrins, mitogen-activated protein kinase, and phosphatidylinositol signaling system. Lentiviral overexpression of HAS3 in VSMC supported the migratory phenotype of VSMC in response to platelet-derived growth factor BB in vitro. Accordingly, knockdown of HAS3 reduced the migratory response to platelet-derived growth factor BB and in addition decreased the expression of PDGF-B mRNA. CONCLUSIONS: HAS3-mediated HA synthesis after vessel injury supports seminal signaling pathways in activation of VSMC, increases platelet-derived growth factor BB-mediated migration, and in turn enhances neointimal hyperplasia in vivo.

Deficiency in lymphotoxin ß receptor protects from atherosclerosis in apoE-deficient mice.

RATIONALE: Lymphotoxin ß receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE: The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1ß2, increased Ccl5 mRNA expression. CONCLUSIONS: These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.

Chronic ultraviolet B irradiation causes loss of hyaluronic acid from mouse dermis because of down-regulation of hyaluronic acid synthases.

Remodeling of the dermal extracellular matrix occurs during photoaging. Here, the effect of repetitive UVB irradiation on dermal hyaluronic acid (HA) was examined. C57/BL6 mice were chronically (182 days) irradiated with UVB, and consecutive skin biopsies were collected during the irradiation period and afterward (300 and 400 days of age). UVB caused marked loss of HA from the papillary dermis and down-regulation of HA synthase 1 (HAS1), HAS2, and HAS3 mRNA expression. In contrast, hyaluronidases (HYAL) 1, HYAL2, and HA receptor CD44 were unchanged. Furthermore, transforming growth factor beta-1 (TGF-beta1) and TGF-beta1-receptor II expression were decreased in UVB-irradiated biopsies, and TGF-beta1 strongly induced HAS1 and HAS2 expression in cultured dermal fibroblasts. Therefore, TGF-beta1 might be one factor involved in UVB-induced down-regulation of HAS enzymes. In addition, total cell number and the percentage of proliferating fibroblasts in the papillary dermis of UVB-irradiated mice were decreased. Down-regulation of HAS2 by lentiviral overexpression of short hairpin RNA in vitro caused inhibition of HA synthesis, DNA synthesis, and migration of dermal fibroblasts. In conclusion, chronic UVB irradiation induces loss of HA from the dermis, thereby contributing to the quiescent phenotype of dermal fibroblasts.

Specific occlusion of murine and human tumor vasculature by VCAM-1-targeted recombinant fusion proteins.

BACKGROUND: The tumor vasculature is increasingly recognized as a target for cancer therapy. We developed and evaluated recombinant fusion proteins targeting the coagulation-inducing protein soluble tissue factor (sTF) to the luminal tumor endothelial antigen vascular cell adhesion molecule 1 (VCAM-1, CD106). METHODS: We generated fusion proteins consisting of sTF fused to antibody fragments directed against mouse or human VCAM-1 and characterized them in vitro by flow cytometry, surface plasmon resonance, and two-stage coagulation assays. Their therapeutic effects were tested in three human xenograft tumor models: L540rec Hodgkin lymphoma, Colo677 small-cell lung carcinoma, and Colo677/HDMEC small-cell lung carcinoma with human vasculature. Toxicity was analyzed by histologic examination of organs and determination of laboratory blood parameters. RESULTS: The fusion proteins bound VCAM-1 with nanomolar affinities and had the same coagulation activity as an sTF standard. Xenograft tumor-bearing mice treated with fusion protein (FP) alone or in combination with lipopolysaccharide (FP/L) or doxorubicin (FP/D) exhibited tumor-selective necrosis (L540rec tumors: 74% tumor necrosis [95% confidence interval {CI} = 55% to 93%] with FP/L versus 13% tumor necrosis [95% CI = 4% to 22%] with vehicle; Colo677 tumors: 26% [95% CI = 16% to 36%] with FP versus 8% [95% CI = 2% to 14%] with vehicle); tumor growth delay (Colo677/HDMEC: mean tumor weights after 3 days = 42 mg in FP-treated mice versus 71 mg in vehicle-treated mice, difference = 29 mg, 95% CI = 8 to 100, Mann-Whitney P = .008); and some tumor regressions (one of seven FP-treated Colo677 tumor-bearing mice and two of seven FP/D-treated mice). The fusion protein was well tolerated. CONCLUSIONS: Recombinant tissue factor-based fusion proteins directed against an intraluminal tumor endothelial cell marker induce tumor-selective intravascular coagulation, tumor tissue necrosis, and tumor growth delay.