Nanna Junker

Regulation of YKL-40 expression during genotoxic or microenvironmental stress in human glioblastoma cells

YKL‐40 is a 40 kDa secreted glycoprotein belonging to the family of ‘mammalian chitinase‐like proteins’, but without chitinase activity. YKL‐40 has a proliferative effect on fibroblasts, chondrocytes and synoviocytes, and chemotactic effect on endothelium and vascular smooth muscle cells. Elevated YKL‐40 levels are found in serum of patients with diseases characterized by inflammation, fibrosis and tissue remodeling. Several studies have reported that high serum YKL‐40 levels in patients with cancer are associated with poor prognosis. YKL‐40 expression is strongly elevated in serum and biopsy material from glioblastomas patients. We investigated the expression of YKL‐40 in three human malignant glioma cell lines exposed to different types of stress. Whereas a polymerase chain reaction transcript was detectable in all three cell lines, only U87 produced measurable amounts of YKL‐40 protein. In U87, hypoxia and ionizing radiation induced a significant increase in YKL‐40 after 24–48 h. The hypoxic induction of YKL‐40 was independent of HIF1. Etoposide, ceramide, serum depletion and confluence all led to elevated YKL‐40. Inhibition of p53 augmented the YKL‐40 expression indicating that YKL‐40 is attenuated by p53. In contrast, both basic fibroblast growth factor and tumor necrosing factor‐α repressed YKL‐40. These are the first data on regulation of YKL‐40 in cancer cells. Diverse types of stress resulted in YKL‐40 elevation, which strongly supports an involvement of YKL‐40 in the malignant phenotype as a cellular survival factor in an adverse microenvironment. (Cancer Sci 2005; 96: 183–190)

Hypoxia-induced secretion of macrophage migration-inhibitory factor from MCF-7 breast cancer cells is regulated in a hypoxia-inducible factor-independent manner.

The cytokine MIF is over-expressed in tumors and is associated with tumor proliferation, angiogenesis and metastasis. Hypoxia, a hallmark feature of tumors, increases MIF expression from tumor cells. We examined the role of hypoxia-inducible transcription factors on MIF secretion from MCF-7 breast carcinoma cells. Secretion of MIF was induced by hypoxia after 24h but up-regulation of MIF mRNA was minimal. Inhibition of HIF-1alpha, HIF-2alpha, NF-kappaB and C/EBPbeta using siRNA had no effect on hypoxia-induced MIF secretion. However, inhibition of transcription and translation significantly decreased MIF production, suggesting that hypoxia-induced secretion of MIF in MCF-7 cells is via an alternative pathway.

Ykl-40 Expression in Benign and Malignant Lesions of the Breast: A Methodologic Study

Elevated serum levels of the protein YKL-40 are associated with a poor prognosis in patients with solid and hematologic malignancies including breast cancer. The aim of this study was to develop a valid reproducible immunohistochemical method to visualize YKL-40 expression in normal breast tissue as well as in benign and malignant breast lesions. The presence of YKL-40 in breast tissue was verified by in situ hybridization and protein extraction procedures. An immunohistochemical method was developed and 4 different antibodies directed against YKL-40 were tested. Ten patients with normal breast tissue and benign breast lesions and 53 patients with localized breast carcinomas were analyzed immunohistochemically. The presence of YKL-40 in normal epithelial cells as well as in malignant tumor cells of the breast was established; however, a difference in staining intensity and staining pattern was observed. In normal breast tissue, a weak YKL-40 immunoreactivity was found in the cytoplasm of the epithelial cells with an additional strong dotlike staining between the nucleus and the gland lumen. In malignant lesions, 81% of the in situ carcinomas and 64% of the invasive carcinomas showed strong diffuse cytoplasmic YKL-40 immunoreactivity. No nuclear and membrane staining was found. A subpopulation of cells of macrophage morphology in normal breast tissue and in malignant lesions showed strong YKL-40 immunoreactivity.

Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis

Objective—Atherosclerotic lesions contain hypoxic areas, but the pathophysiological importance of hypoxia is unknown. Hypoxia-inducible factor-1&agr; (HIF-1&agr;) is a key transcription factor in cellular responses to hypoxia. We investigated the hypothesis that HIF-1&agr; has effects on macrophage biology that promotes atherogenesis in mice. Approach and Results—Studies with molecular probes, immunostaining, and laser microdissection of aortas revealed abundant hypoxic, HIF-1&agr;–expressing macrophages in murine atherosclerotic lesions. To investigate the significance of macrophage HIF-1&agr;, Ldlr−/− mice were transplanted with bone marrow from mice with HIF-1&agr; deficiency in the myeloid cells or control bone marrow. The HIF-1&agr; deficiency in myeloid cells reduced atherosclerosis in aorta of the Ldlr−/− recipient mice by ≈72% (P=0.006).In vitro, HIF-1&agr;–deficient macrophages displayed decreased differentiation to proinflammatory M1 macrophages and reduced expression of inflammatory genes. HIF-1&agr; deficiency also affected glucose uptake, apoptosis, and migratory abilities of the macrophages. Conclusions—HIF-1&agr; expression in macrophages affects their intrinsic inflammatory profile and promotes development of atherosclerosis.

Osteopontin deficiency dampens the pro-atherogenic effect of uraemia

AIMS Uraemia is a strong risk factor for cardiovascular disease. Osteopontin (OPN) is highly expressed in aortas of uraemic apolipoprotein E knockout (E KO) mice. OPN affects key atherogenic processes, i.e. inflammation and phenotypic modulation of smooth muscle cells (SMCs). We explored the role of OPN on vascular pathology in uraemic mice. METHODS AND RESULTS Uraemia was induced by 5/6 nephrectomy in E KO and in OPN and E double KO mice (E/OPN KO). In E KO mice, uraemia increased the relative surface plaque area in the aortic arch (from 28 ± 2% [n = 15], to 37 ± 3% [n = 20] of the aortic arch area, P < 0.05). A positive correlation was observed between plasma OPN and aortic atherosclerosis in uraemic E KO mice (r(2) = 0.48, P = 0.001). In contrast, aortic atherosclerosis was not increased by uraemia in E/OPN KO mice. OPN deficiency in haematopoietic cells (including macrophages) did not affect development of uraemic atherosclerosis, even though OPN-deficient foam cells had decreased inflammatory capacity. Gene expression analyses indicated that uraemia de-differentiates SMCs in the arterial wall. This effect was dampened in whole-body OPN-deficient mice. CONCLUSION The data suggest that OPN promotes development of uraemic atherosclerosis possibly by changing the phenotype of vascular smooth muscle cells.

Interaction between VEGF and Calcium-Independent Phospholipase A2 in Proliferation and Migration of Retinal Pigment Epithelium

Purpose: Inhibition of VEGF in the eye is an important treatment modality for reducing proliferation and migration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Additionally, previous studies suggest calcium-independent phospholipase A2 group VIA (iPLA2-VIA) to be a potential regulator of cell proliferation and migration, and evidence show abundant expression of iPLA2-VIA in RPE cells. The aim of the present study was to evaluate the potential role of iPLA2-VIA in VEGF-induced proliferation and migration of RPE cells. Materials and methods: The human RPE cell line, ARPE-19, was used in all assays. To explore the role of iPLA2-VIA in VEGF-induced RPE proliferation and migration, iPLA2-VIA inhibition by the iPLA2-VIA specific inhibitor, bromoenol lactone, was done. RPE cell proliferation and migration were evaluated by measurements of incorporated radioactive thymidine in DNA and by a Boyden chamber technique, respectively. A luciferase assay monitored the VEGF-induced iPLA2-VIA transcriptional activity. Western blot analysis and an activity assay were used to detect the protein levels and activity of iPLA2-VIA respectively after treatment with VEGF. Results: RPE cells treated with VEGF showed significant increased proliferation and migration. Furthermore, inhibition of iPLA2-VIA significantly reduced the spontaneous proliferation and migration as well as the VEGF-induced proliferation and migration. Finally, inhibition of iPLA2-VIA reduced the VEGF-induced iPLA2-VIA-activity, -protein level, and -promoter activity. Conclusions: A significant interaction between VEGF and iPLA2-VIA in the regulation of RPE cells appears to be relevant in elucidating the exact mechanisms of action in the proliferative and migratory phenotype of RPE cells in AMD.