Fig. 1.
Fig. 1. Mechanisms responsible for hypoxia-induced activation of TGF-β2 gene expression in HUVECs. / (A) TSP-1–dependent increases in bioactive TGF-β2 levels in hypoxic HUVEC supernatants. Confluent HUVECs, cultured without serum, were exposed to 20% or 1% O2 in the presence or absence of 10 μg/mL anti–TSP-1 monoclonal antibody 133; 28 μM LSKL peptide, corresponding to TSP-1–binding amino acid residues 54-57 from TGF-β1 LAP; 28 μM scrambled peptide SLLK; 10 μg/mL anti–TGF-β2 antibody; or 10 μg/mL mouse IgG. After 18 hours, supernatants were collected, and bioactive TGF-β levels were quantitated by MLEC bioassay. Results from 3 independent experiments are expressed as fold change (±SD) in bioactive TGF-β levels in hypoxic HUVECs. Values were derived by dividing the levels obtained from hypoxic supernatants by each condition's normoxic counterpart (not shown). Asterisks indicate significant differences (P ≤ .05, Studentt, 2-tailed) from the untreated condition (leftmost bar). (B) TSP-1–dependent increases in TGF-β2 mRNA levels in hypoxic HUVECs. Confluent HUVECs, cultured without serum, were exposed to 20% or 1% O2 in the presence or absence of 10 μg/mL anti–TSP-1 monoclonal antibody 133, 28 μM LSKL (inhibitory) peptide, 28 μM SLLK (scrambled) peptide, 10 μg/mL anti–TGF-β2 antibody, 10 μg/mL anti–TGF-β type II receptor (anti–TGF-β RII) antibody, 10 μg/mL mouse IgG, or 10 μg/mL anti–TGF-β1 antibody for 18 hours and then subjected to total RNA extraction. For RNase protection assays, 10 μg total RNA was hybridized to an antisense RNA probe cocktail containing the templates for TGF-β2 and L-32 (ribosomal protein subunit) genes. Representative results from 1 of 3 independent experiments are shown in the top panel, in which protected fragments of TGF-β2 and L-32 mRNAs are indicated by arrows. The bottom panel presents quantification of results from the 3 experiments. TGF-β2 signal was normalized to that from L32. Each bar represents the increase in mRNA levels from hypoxic HUVECs compared with their own normoxic controls at 18 hours. Asterisks indicate significant differences (P ≤ .05, Student t, 2-tailed) from the untreated condition (leftmost bar). N indicates normoxic conditions; H, hypoxic conditions.

Mechanisms responsible for hypoxia-induced activation of TGF-β2 gene expression in HUVECs.

(A) TSP-1–dependent increases in bioactive TGF-β2 levels in hypoxic HUVEC supernatants. Confluent HUVECs, cultured without serum, were exposed to 20% or 1% O2 in the presence or absence of 10 μg/mL anti–TSP-1 monoclonal antibody 133; 28 μM LSKL peptide, corresponding to TSP-1–binding amino acid residues 54-57 from TGF-β1 LAP; 28 μM scrambled peptide SLLK; 10 μg/mL anti–TGF-β2 antibody; or 10 μg/mL mouse IgG. After 18 hours, supernatants were collected, and bioactive TGF-β levels were quantitated by MLEC bioassay. Results from 3 independent experiments are expressed as fold change (±SD) in bioactive TGF-β levels in hypoxic HUVECs. Values were derived by dividing the levels obtained from hypoxic supernatants by each condition's normoxic counterpart (not shown). Asterisks indicate significant differences (P ≤ .05, Studentt, 2-tailed) from the untreated condition (leftmost bar). (B) TSP-1–dependent increases in TGF-β2 mRNA levels in hypoxic HUVECs. Confluent HUVECs, cultured without serum, were exposed to 20% or 1% O2 in the presence or absence of 10 μg/mL anti–TSP-1 monoclonal antibody 133, 28 μM LSKL (inhibitory) peptide, 28 μM SLLK (scrambled) peptide, 10 μg/mL anti–TGF-β2 antibody, 10 μg/mL anti–TGF-β type II receptor (anti–TGF-β RII) antibody, 10 μg/mL mouse IgG, or 10 μg/mL anti–TGF-β1 antibody for 18 hours and then subjected to total RNA extraction. For RNase protection assays, 10 μg total RNA was hybridized to an antisense RNA probe cocktail containing the templates for TGF-β2 and L-32 (ribosomal protein subunit) genes. Representative results from 1 of 3 independent experiments are shown in the top panel, in which protected fragments of TGF-β2 and L-32 mRNAs are indicated by arrows. The bottom panel presents quantification of results from the 3 experiments. TGF-β2 signal was normalized to that from L32. Each bar represents the increase in mRNA levels from hypoxic HUVECs compared with their own normoxic controls at 18 hours. Asterisks indicate significant differences (P ≤ .05, Student t, 2-tailed) from the untreated condition (leftmost bar). N indicates normoxic conditions; H, hypoxic conditions.

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