LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118

Abstract

Since disturbance of angiogenesis predisposes to ischemic injuries, attempts to promote angiogenesis have been made to improve clinical outcomes of patients with many ischemic disorders. While hypoxia inducible factors (HIFs) stimulate vascular remodeling and angiogenesis, hyperlipidemia impairs angiogenesis in response to various pro-angiogenic factors. However, it remains uncertain how HIFs regulate angiogenesis under hyperlipidemia. Here, we report that exposure to low-density lipoprotein (LDL) suppressed in vitro angiogenesis of human brain microvascular endothelial cells. Whereas LDL exposure diminished expression of HIF-1α and HIF-2α induced by hypoxia, it inhibited DMOG- and TNFα-induced HIF-1α and HIF-2α expression in normoxia. Notably, in both hypoxia and normoxia, LDL markedly reduced expression of HIF-1β, a constitutively stable HIF subunit, an event associated with NF-κB inactivation. Moreover, knockdown of HIF-1β down-regulated HIF-1α and HIF-2α expression, in association with increased HIF-1α hydroxylation and 20S proteasome activity after LDL exposure. Significantly, the proteasome inhibitor BSc2118 prevented angiogenesis attenuation by LDL through restoring expression of HIFs. Together, these findings argue that HIF-1β might act as a novel cross-link between the HIF and NF-κB pathways in suppression of angiogenesis by LDL, while proteasome inhibitors might promote angiogenesis by reactivating this signaling cascade under hyperlipidemia.

Keywords: HIF; NF-κB; angiogenesis; low-density lipoprotein; proteasome inhibitor.

Figure 1. LDL attenuates cell proliferation and tube formation of hCMEC/D3 cells under hypoxic condition

The immortalised human brain microvascular endothelial cell line hCMEC/D3 cells cultured in Microvascular Endothelial Cell Medium-2, containing either 0.5% FBS for proliferation assay or 5% FBS for tube formation assay, were treated (72 hr) with the indicated concentrations of native LDL under hypoxic (1% oxygen) condition, after which cells were subjected to the following assays: A. cell proliferation assay using the BrdU Flow Kit; B–C. Matrigel-based tube formation assay. Three independent experiments (n = 3) were performed. *p < 0.05, **p < 0.01 versus controls. Scale bars, 200 μm.

Figure 2. LDL down-regulates HIF-1α, HIF-2α, and HIF-1β in hCMEC/D3 cells in both hypoxia and normoxia

A. hCMEC/D3 cells were exposed to the indicated concentrations of native LDL (25–100 μg/ml) under hypoxic (1% O₂) condition for 72 hr, after which Western blot analysis was performed to monitor protein levels of HIF-1α and HIF-2α. B–D. hCMEC/D3 cells were exposed to the indicated concentrations of LDL for 24 (B), 48 (C), and 72 hr (D) under normoxic (21% O₂) condition, after which Western blot analysis was performed to monitor protein levels of HIF-1β. E. hCMEC/D3 cells were treated with 1 μM DMOG for 4 hr, followed by 50–100 μg/ml LDL for additional 72 hr, after which expression of HIF-1α, HIF-2α, and HIF-1β in the cytoplasmic and nuclear fractions was assessed by Western blot analysis. Blots re-probed for β-actin and laminin A were used as loading controls for cytoplasmic and nuclear fractions, respectively. All blots were quantified densitometrically using ImageJ software. The relative protein abundance was calculated by comparing to either β-actin or Laminin A and expressed as fold increase over controls (without LDL treatment). Values for controls were arbitrarily set to 1.0. At least three independent experiments (n ≥ 3) were performed. *p < 0.05, **p < 0.01, ***p < 0.001 (for whole cell lysates or cytoplasmic fraction); ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 (for nuclear fraction) versus their controls; ns, not significant.

Figure 3. LDL inhibits NF-κB-dependent expression of HIF-1β induced by TNFα in hCMEC/D3 cells, resulting in HIF-1α and HIF-2α down-regulation in normoxia

A. hCMEC/D3 cells were exposed to either LDL (100 μg/ml) alone or in combination with TNFα (20 ng/ml), after which Western blot analysis was performed to monitor expression of NF-κB p65, HIF-1α, HIF-2α, and HIF-1β. B. hCMEC/D3 cells were incubated with the indicated concentrations of LDL for 72 hours, after which protein levels of NF-κB p65 in whole cell lysates was determined by Western blot analysis. C. hCMEC/D3 cells were transiently transduced with pLKO. 1 NF-κB p65 shRNA (shp65), followed by Western blot analysis for detecting protein levels of NF-κB p65 and HIF-1β. D. hCMEC/D3 cells were transiently transduced with pLKO.1 HIF-1β shRNA (shHIF-1β), and then exposed to TNFα, after which Western blot analysis was performed to monitor protein levels of HIF-1β, NF-κB p65, HIF-1α, HIF-2α, and AHR. At least three independent experiments (n ≥ 3) performed. *p < 0.05, **p < 0.01, ***p < 0.001 versus controls without LDL treatment; ⁺p < 0.05, ⁺⁺p < 0.01 versus controls with TNFα treatment alone; ns, not significant.

Figure 4. LDL induces HIF-1α hydroxylation at Pro402 and Pro564 sties, while increases 20S proteasome activity in hCMEC/D3 cells

A–B. hCMEC/D3 cells were exposed to LDL (100 μg/ml) in hypoxia, after which Western blot analysis was performed to monitor hydroxylation of HIF-1α using antibodies specifically recognizing hydroxylated HIF-1α at Pro402 (A) and Pro564 (B) respectively. C–D. CT-L activity of 20S proteasome was analysed in hCMEC/D3 after exposed (24 hr) to the indicated concentrations of LDL in either hypoxia (C) or normoxia (D). Cells were treated with MG132 as control. E–F. hCMEC/D3 cells were exposed to 100 μg/ml LDL for 48 hr in the absence or presence of pre-treatment with 100 nM BSc2118 (4 hr prior to LDL) in normoxia (E) or hypoxia (F), after which Western blot analysis was performed to assess expression of NF-κB p65 and HIF-1β. At least three independent experiments (n ≥ 3) were performed. *p < 0.05, **p < 0.01 versus controls without LDL treatment; ⁺p < 0.05 versus controls with LDL treatment alone.

Figure 5. The proteasome inhibitor BSc2118 induces tube formation of hCMEC/D3 cells, in association with HIF-1α and HIF-2α accumulation

A. hCMEC/D3 cells were exposed to the indicated concentrations of BSc2118 for 24 hr, followed by cell viability assay. B–C. hCMEC/D3 cells were exposed to BSc2118 (100–200 μg/ml) with or without DMOG for 4 hr, after which Western blot analysis was performed to monitor protein levels of for HIF-1α, HIF-2α, and HIF-1β. D. hCMEC/D3 cells were treated with 100–200 nM BSc2118, followed by Matrigel-based tube formation assay. Scale bars, 200 μm. Three independent experiments (n = 3) were performed. *p < 0.05, **p < 0.01 versus control without BSc2118; ^#p < 0.05, ^##p < 0.01 versus controls with DMOG alone; ns, not significant.

Figure 6. The proteasome inhibitor BSc2118 restores LDL-attenuated proliferation and tube formation of hCMEC/D3 cells in both normoxia and hypoxia

A–B. hCMEC/D3 cells were exposed to 50–100 μg/ml of LDL in the absence or presence of 100 nM BSc2118 in normoxia, after which cell proliferation analysis (A) and Matrigel-based tube formation assay were carried out. C–D. Alternatively, hCMEC/D3 cells were treated as described in panel A-B in hypoxia, and then subjected to analyses of cell proliferation and tube formation. Three independent experiments (n = 3) were performed. *p < 0.05, **p < 0.05 versus controls without LDL treatment; ⁺p < 0.05 versus controls with LDL treatment alone. Scale bars, 200 μm.