Impairment of hypoxia-induced angiogenesis by LDL involves a HIF-centered signaling network linking inflammatory TNFα and angiogenic VEGF

Figures

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  Figure 1. The GEP analyses map the potential correlations among the HIFs, TNFα, VEGFs, and NF-κB signaling pathways in ECs. (A, B) By analyzing the datasets involving gene expression profiling (GEP) in ECs using the R2: Genomics Analysis and Visualization Platform, the heatmaps were generated based on P values for analyses of correlations (red and blue indicating positive and negative correlations, respectively) between each pair of genes as indicated on longitudinal and transverse axes, in (A; dataset, Exp HUVEC vs ESC - James - 12 - MAS5.0 - u133p2) human stem cells (hESC) vs human umbilical vein endothelial cells (HUVEC) and (B; dataset, Normal Endothelial Cells HUAEC/HUVEC - Luttun - 38 - MAS5.0 - u133p2) HUVEC vs human umbilical artery endothelial cells (HUAEC). Numbers in the heatmaps indicate the areas (outlined by dash line) clustered for each pathway. (C, D) Gene ontology (GO) analyses were performed to categorize (C) HIF-1α- and (D) HIF-2α-related genes according to their functions, in all types of ECs, including (dataset, Normal Endothelial Cells HUAEC/HUVEC - Luttun - 38 - MAS5.0 - u133p2).

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  Figure 2. Hypoxia induces angiogenesis of ECs, in association with VEGF production and HIF activation. (A, B) HUVECs were cultured in ECGM medium containing 2% serum for the indicate intervals under either hypoxic (1% O2) or normoxic (21% O2) condition, after which cells were subjected to the following assays, including (A) colony formation assay (72 hrs) and (B) Matrigel-based tube formation assay (24 hrs). Representative microscopic images for at least three independent experiments were shown. Arrowheads indicate unclosed loops of vascular structure. (C) When HUVECs were cultured as described above under hypoxic (1% O2) or normoxic (21% O2) condition, medium was harvested at the indicated intervals and subjected to an ELISA assay to determine absolute amount of VEGF (pg/ml). Values represent the means ± SD for at least three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 for comparison with control (72 hrs under 21% O2); ns = not significant. (D) HUVECs were cultured under hypoxic (1% O2) condition (left panels) or exposed to the chemical hypoxia mimetic lactic acid (3 mM) for the indicated intervals (6 - 24 hrs), after which Western blot analysis was performed…

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  Figure 3. Knockdown of either HIF-1α or HIF-2α impairs hypoxia-induced angiogenesis and VEGF production in ECs. (A) HUVECs were transfected with pLKO.1 constructs encoding shRNA specifically targeting HIF-1α and HIF-2α, as well as scramble sequence as negative control (shNC). As the basal levels of HIF-1α or HIF-2α in HUVECs were relatively low, these transfected cells were exposed to 1% O2 (21% O2 as normoxia control) for 24 hrs, after which Western blot analysis was performed to confirm shRNA knockdown of HIF-1α and HIF-2α, respectively. (B, C) HUVECs expressing HIF-1α or HIF-2α shRNA were then exposed to 1% O2 for the indicated intervals, followed by colony formation assay (B, 72 hrs) and Matrigel-based tube formation assay (C, 24 hrs). Representative microscopic images for at least three independent experiments were shown. Arrowheads indicate unclosed loops of vascular structure. (D) In parallel, the VEGF level was measured by ELISA assay after cultured for 72 hrs under 1% O2. Values represent the means ± SD for at least three independent experiments performed in triplicate. **P < 0.01 for comparison with shNC control.

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  Figure 4. Native LDL, but not oxLDL, inhibits activation of HIF and NF−κB signals induced by hypoxia. HUVECs were treated as follows: (A) pre-treated with LDL (100 μg/ml) for 48 hrs, followed by incubation under hypoxic (1% O2) condition for additional 48 hrs; (B) pre-treated with LDL (100 μg/ml) in the presence or absence of the free radical scavenger PBN (2 mM) for 48 hrs, and then exposed to the PHD inhibitor DMOG (1 μM) for additional 72 hrs; (C) pre-treated with the indicated concentrations of oxidized LDL (oxLDL, μg/ml) for 48 hrs, followed by DMOG (1 μM) for additional 72 hrs. After treatment, Western blot analysis was performed to monitor expression of HIF-1α, HIF-2α, and HIF-1β, as well as phosphorylation of NF-κB p65 (S536). (D) Alternatively, HUVECs were treated as described in panel 4B, after which cytoplasmic and nuclear fractions were separated and subjected to Western blot analysis for monitoring nuclear translocation of HIF-1α, HIF-2α, and HIF-1β. Blots were reprobed for β-actin and laminin A as loading controls for cytoplasmic and nuclear fractions, respectively. (E) HUVECs were pre-incubated with the NF-κB inhibitor PDTC (100 μM) for 4 hrs, followed by DMOG (1 μM) for…

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  Figure 5. Hypoxia induces autocrine of TNFα in ECs, which in turn activates HIFs and NF-κB to produce VEGF. (A) Using the same approach as described in Figure 1A and 1B, the heatmap was generated by analyzing a dataset involving gene expression profiling (GEP) in HUVECs stimulated by TNFα (dataset, Exp HUVEC TNF-alpha - Kodama - 25 - MAS5.0 - u133p2). Numbers in the heatmaps indicate the areas (outlined by dash line) clustered for each pathway. (B) HUVECs were cultured under hypoxic (1% O2) or normoxic (21% O2) condition as described in Figure 2C, an ELISA assay was performed to determine absolute amount of TNFα (pg/ml) in medium harvested at the indicated intervals. Values represent the means ± SD for at least three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 for comparison with control (72 hrs under 21% O2). (C, D) HUVECs were pre-treated with LDL (100 μg/ml) for 48 hrs, followed by TNFα (50 ng/ml) for additional intervals as below, after which Western blot analysis was performed to monitor S536 phosphorylation of NF-κB p65 (5 min) and expression of HIFs (C, 4 hrs) and TNF-R1 (D, 4 hrs). Blots for TNF-R1 were densitometrically quantified by using ImageJ…

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  Figure 6. LDL inactivates TNFα-induced NF-κB activation via down-regulation of its key signaling components. (A) HUVECs were treated with 3 mM lactic acid as described in Figure 2D, after which Western blot analysis was performed to assess activation of the canonical (e.g., TRAF2 expression as well as IKKα/β and p65 phosphorylation) and non-canonical (e.g., expression of TRAF3 and NIK) NF-κB pathways. (B, C) HUVECs were pre-treated with LDL (100 μg/ml) for 48 hrs, followed by TNFα (50 ng/ml) for additional intervals as below, after which Western blot analysis was carried out to monitor phosphorylation of IKKα/β (Ser176/180, 5 min) as well as expression of multiple key components of both canonical and non-canonical NF-κB pathways, including IKKα, IKKβ, IκΒα (5 min), p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), RelB, and c-Rel (4 hrs). All blots were densitometrically quantified, and values indicate fold increase after normalization to β-actin.

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  Figure 7. LDL-mediated impairment of hypoxia-induced angiogenesis involves a broad spectrum of HIF-dependent signaling pathways. (A) HUVECs were exposed to indicated concentrations of LDL (25 - 100 μg/ml) for 48 hrs, and then cultured under hypoxic (1% O2) condition for additional 48 hrs. (B) HUVECs with shRNA knockdown of HIF-1α or HIF-2α were exposed to 1% O2 (21% O2 as normoxia control) for 24 hrs. After treatment, Western blot analysis was performed to monitor phosphorylation of AKT (S473), ERK1/2 (T202/Y204), CXCR4, VEGFR1, and VEGFR2. Blots were reprobed for β-actin as loading control. (C) A schematic diagram for the mechanism by which hypoxia induces angiogenesis via an autocrine loop of TNFα and resulting activation of the self-regulatory TNFα/NF-κB/HIF/VEGF signaling network in ECs, as well as the potential mechanism of action for anti-angiogenic property of LDL by which a) LDL might impair the autocrine loop of TNFα via down-regulation of its receptor TNF-R1, rather than TNFα itself; and b) LDL disrupts the TNFα-NF-κB-HIF-VEGF signaling cascade via down-regulation of multiple key components of both canonical and non-canonical NF-κB pathways.