Heme oxygenase-2 deletion causes endothelial cell activation marked by oxidative stress, inflammation, and angiogenesis

Abstract

In previous studies, we have shown that heme oxygenase (HO)-2 null [HO-2(-/-)] mice exhibit a faulty response to injury; chronic inflammation and massive neovascularization replaced resolution of inflammation and tissue repair. Endothelial cells play an active and essential role in the control of inflammation and the process of angiogenesis. We examined whether HO-2 deletion affects endothelial cell function. Under basal conditions, HO-2(-/-) aortic endothelial cells (mAEC) showed a 3-fold higher expression of vascular endothelial growth factor receptor 1 and a marked angiogenic response compared with wild-type (WT) cells. Compared with WT cells, HO-2(-/-) mAEC showed a 2-fold reduction in HO activity and marked increases in levels of gp91(phox)/NADPH oxidase isoform, superoxide, nuclear factor kappaB activation, and expression of inflammatory cytokines, including interleukin (IL)-1alpha and IL-6. HO-2 deletion transforms endothelial cells from a "normal" to an "activated" phenotype characterized by increases in inflammatory, oxidative, and angiogenic factors. This switch may be the result of reduced HO activity and the associated reduction in the cytoprotective HO products, carbon monoxide and biliverdin/bilirubin, because addition of biliverdin to HO-2(-/-) cells attenuated angiogenesis and reduced superoxide production. This transformation underscores the importance of HO-2 in the regulation of endothelial cell homeostasis.

Fig. 1.

A, representative gels depicting genotyping (top), HO-2 mRNA (middle), and protein (bottom) in HO-2(−/−) (KO) cells. B, representative blot and densitometry analysis of HO-1 in WT and HO-2(−/−) (KO) mAECs (n = 4; *, p < 0.05). C, HO-dependent CO levels in WT and KO cells (n = 3; *, p < 0.05). D, representative blot and densitometry analysis of HO-1 protein in nuclear (N) and cytosolic (C) extracts from mAECs (n = 4; **, p < 0.01); glyceraldehyde-3-phosphate dehydrogenase and histone h3 were used to assess purity of cytosolic and nuclear fractions, respectively.

Fig. 2.

A, representative images of DHE staining of WT and HO-2(−/−) (KO) mAECs in the absence and presence of Tiron (10 mM) or biliverdin (10 μM). B, quantitative analysis of DHE fluorescence intensity (arbitrary units). Results are expressed as the Tiron-sensitive DHE fluorescence corrected for the number of cells (n = 5–9; ***, p < 0.001 compared with WT; ‡, p < 0.01 to control).

Fig. 3.

Expression of NOX-2 (gp91phox) (A) and NOX-4 (B) mRNA in HO-2(−/−) (KO) and WT mAECs (n = 4–11; *, p < 0.05). Western blots and densitometry analysis of gp91phox (C), EC-SOD (D), and eNOS and peNOS (E) at serine 1177 (n = 3–4; *, p < 0.05; **, p < 0.01).

Fig. 4.

NF-κB activation in WT and HO-2(−/−) (KO) mAECs. Representative photographs showing the cellular localization of NF-κB (red). Nuclei were stained with 4′,6-diamino-2-phenylindole (blue). Quantitative image analysis presents the percentage of cells with nuclear NF-κB expression (n = 4; *, p < 0.05).

Fig. 5.

NF-κB expression in cytosolic and nuclear extracts of mAECs from HO-2(−/−) (KO) and WT mice. Representative Western blot of NF-κB and its phosphorylated form and densitometry analysis showing the nuclear to cytosol ratio of phosphorylated NF-κB and the phosphorylated NF-κB to NF-κB ratio (n = 3; *, p < 0.05).

Fig. 6.

Inflammatory markers in serum and mAECs from WT and HO-2(−/−) (KO) mice. Serum levels of tumor necrosis factor-α (A), IL-1α (B), and IL-6 (C) (n = 4; *, p < 0.05 and **, p < 0.01). Real-time PCR analysis of IL-1α (D) and IL-6 (E) mRNA (n = 11; *, p < 0.05). F, Western blot and densitometry analysis of VCAM-1 (n = 3).

Fig. 7.

Expression of VEGF-A, -C, and -D and VEGFR-1, -2, and -3 in mAECs from HO-2(−/−) (KO) and WT mice. Real-time PCR analysis is given as relative expression of VEGFR-1, -2, and -3 and VEGF-A, -C, and -D mRNA (n = 4–6; *, p < 0.05; **, p < 0.01).

Fig. 8.

In vitro angiogenesis in mAECs from HO-2(−/−) (KO) and WT mice. Cells plated onto Matrigel were cultured in the presence and absence of biliverdin (10 μM), SN50 (10 μM), or VEGFR-1 inhibitory antibody (20 μg/ml) and assayed for capillary length and lumen formation. A, representative photographs of WT and KO mAECs. B, effect of VEGF and biliverdin on total length of network and number of lumen in KO and WT cells (n = 8; *, p < 0.05 and ***, p < 0.001 compared with untreated; ‡, p < 0.05 compared with WT untreated). C, effect of SN50 and VEGFR-1 inhibitory antibody on total length of network and number of lumen in KO and WT cells (n = 6; *, p < 0.05 compared with untreated).