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Fig. 3. Effect of hypoxia and HIF-1{alpha} 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{alpha}-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{alpha}-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{alpha}-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.