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Increased responsiveness of hypoxic endothelial cells to FGF2 is mediated by HIF-1-dependent regulation of enzymes involved in synthesis of heparan sulfate FGF2-binding sites

¹ Division of Cardiology, Department of Medicine, the Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA
² Section of Cardiology and Angiogenesis Research Center, Department of Medicine, Dartmouth Hitchcock Medical Center and Dartmouth Medical School, Lebanon, NH 03756, USA

View larger version (69K): [in a new window]   Fig. 1. Hypoxia-induced changes in endothelial cell HS matrix. Changes in the cell-surface-associated GAG content (A), heparan sulfate (B), HS/CS ratio (C) and FGF2-HS binding sites (D) are shown as a ratio between hypoxic and normoxic states for cardiac microvascular endothelial cells (striped bars) and human umbilical vein endothelial cells (gray bars). * denotes a significant change in the index under hypoxic versus normoxic conditions (P<0.05). Data are shown as mean±s.d.

View larger version (45K): [in a new window]   Fig. 2. Northern blot analysis of HS core protein and FGF receptor expression. Total RNA blots of CMEC and HUVEC cultured under hypoxic (+) or normoxic (-) conditions probed for expression of syndecans 1,2 and 4, glypican-1, perlecan and FGF receptor 1 (FGF R1). Ethidium bromide staining of the 18S RNA band is shown as the loading control. Note the decreased expression of syndecan-1, 4 and glypican-1 under hypoxic conditions and the lack of significant change in expression with other tested genes.

View larger version (34K): [in a new window]   Fig. 3. Effect of hypoxia and HIF-1 gene expression on mRNA levels of HS biosynthetic enzyme expression. (A) RT-PCR analysis of mRNA levels of enzymes involved in GAG chain biosynthesis was carried out on HUVEC (left panels) and CEMC (right panel) cultured under normoxic (C) or hypoxic conditions for 6 hours (H6) or 24 hours (H24). In addition, expression of the same enzymes was measured in cells transduced with an empty adenoviral vector (C) or an adenoviral vector carrying the HIF1-VP16 construct (HIF). The internal PCR loading control with 18S ribosomal RNA is show on the bottom of each panel. (B,C) Quantitative analysis of GAG chain biosynthesis enzymes expression in HUVEC (B) and CMEC (C) cells. The levels were determined after cell culture under normoxic conditions (gray bars), after 6 hours (striped bars) or 24 hours (light striped bars) of hypoxia or following infection with a control (empty vector) adenovirus (black bars) or a HIF1-VP16 adenovirus (stippled bars). Densitometric analysis of RT-PCR-determined expression levels of enzymes shown in (A) adjusted for loading are shown as mean±s.d. of three independent experiments. Note the increased expression of GlcNAcT-I, NDST1 and HS2ST in both HUVEC and CMEC cultured under hypoxic conditions or in cells transduced with HIF1-VP16 adenovirus. GlcNAcT-I, N-Acetyl glucosamine transferase; NDST, N-deacetyl/N-sulfotransferase; HS2ST, heparan sulfate 2-O sulfotransferase; CS/DS2ST, chondroitin sulfate/dermatan sulfate 2-O sulfotransferase; HS6ST, heparan sulfate 6-O sulfotransferase. * P<0.05 by Student's t-test.

View larger version (17K): [in a new window]   Fig. 4. Effect of hypoxia on FGF2-induced cell growth. Growth of CMEC and HUVEC in 0.25% serum in the presence (+) or absence (-) of FGF2 (10 ng/ml) was determined after 48 hours of hypoxia (+) or normoxia (-). For each condition, the cells counts are normalized to the baseline number taken as 100%. Note increased growth response to FGF2 for both cell types cultured under hypoxic conditions. *P<0.05 hypoxia versus normoxia.

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