Macrophages transmit potent proangiogenic effects of oxLDL in vitro and in vivo involving HIF-1α activation: a novel aspect of angiogenesis in atherosclerosis

Abstract

Neovascularization has been linked to the progression and vulnerability of atherosclerotic lesions. Angiogenesis is increased in lipid-rich plaque. Hypoxia-inducible factor alpha (HIF-1α) is a key transcriptional regulator responding to hypoxia and activating genes, which promote angiogenesis, among them vascular endothelial growth factor (VEGF). Oxidized low-density lipoprotein (oxLDL) is generated in lipid-rich plaque by oxidative stress. It triggers an inflammatory response and was traditionally thought to inhibit endothelial cells. New data, however, suggest that oxLDL can activate HIF-1α in monocytes in a hypoxia-independent fashion. We hypothesized that HIF-1α activation in monocyte-macrophages could transmit proangiogenic effects of oxLDL linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. First, we examined the effect of oxLDL on HIF-1α and VEGF expression in monocyte-macrophages and on their proangiogenic effect on endothelial cells in vitro in a monocyte-macrophage/endothelial co-culture model. OxLDL strongly induced HIF-1α and VEGF in monocyte-macrophages and significantly increased tube formation in co-cultured endothelial cells. HIF-1α inhibition reversed this effect. Second, we demonstrated a direct proangiogenic effect of oxLDL in an in vivo angiogenesis assay. Again, HIF-1α inhibition abrogated the proangiogenic effect of oxLDL. Third, in a rabbit atherosclerosis model, we studied the effect of dietary lipid lowering on arterial HIF-1α and VEGF expression. The administration of low-lipid diet significantly reduced the expression of both HIF-1α and VEGF, resulting in decreased plaque neovascularization. Our data point to oxLDL as a proangiogenic agent linking hyperlipidemia, inflammation, and angiogenesis in atherosclerosis. This effect is dependent on macrophages and, at least in part, on the induction of the HIF-1α pathway.

Figures

Fig. 1

Panel 1: Effect of oxLDL on HIF-1α and VEGF in monocytes. a–h Representative pictures of HIF-1α (a–d) and VEGF (e–h) expression in monocytes cultured under normoxia without oxLDL (a, e), under normoxia with oxLDL (b, f), under hypoxia without oxLDL (c, g), and under hypoxia with oxLDL (d, h). A strong induction of HIF-1α and VEGF by oxLDL was observed independent of the presence of hypoxia (×400). i–k Double labeling for HIF-1α (i) and VEGF (j) showed clear co-localization of both antigens in single oxLDL-treated monocytes (overlay in k) (× 1,000). l Quantitative evaluation confirmed the significant increase in HIF-1α and VEGF expression with oxLDL treatment to a degree similar to hypoxia-induced expression. Combining oxLDL and hypoxia further enhanced the expression of both HIF-1α and VEGF (ANOVA, P<0.05). m Transcriptional activation of HIF-1α as measured by the Trans-AM assay was elevated significantly in monocytes treated with oxLDL. Of note, co-treatment with the antioxidant (tiron) abrogated HIF-1α transcriptional activation induced by oxLDL (ANOVA, P<0.05). Panel 2: Effect of oxLDL on monocyte-mediated angiogenesis. a–f HUVECs were seeded onto growth factor-depleted Matrigel in a transwell co-culture setup (shown in g) in the presence of monocytes (b, d, f) or without monocytes (a, c, e). The monocytes were either left untreated (a, b), treated with oxLDL (c, d), treated with the HIF-1α inhibitor chetomin (e), or with oxLDL and chetomin (f). HUVECs grown alone on growth factor-depleted Matrigel did not show significant tube formation (a) and were used as negative control. HUVECs under the same conditions grown in the presence of untreated monocytes did not show tube formation either (b). Likewise, HUVECs grown alone but treated with oxLDL did also not show signs of tube formation (c). However, HUVECs grown in the presence of oxLDL-treated monocytes did show extensive tube formation (arrows) reflecting increased angiogenic activity (d). Of note, co-treatment of monocytes with the HIF-1α inhibitor chetomin significantly suppressed the proangiogenic effect of oxLDL treatment (f) and chetomin did not have an effect on HUVECs alone (e). g Schematic drawing of the monocyte endothelial co-culture system for determining capillary tube-like structure formation hBar graphs comparing the length of endothelial tube formation as measured in micrometers. In summary, groups 1–3 and 5 served as various controls, and groups 4 and 6 were the two treatment groups as described from a to f (ANOVA P<0.01; error bars ± SEM)

Fig. 2

Effect of oxLDL on in vivo angiogenesis in Matrigel plug assay, a–j Growth factor-depleted Matrigel was used in an in vivo angiogenesis assay (Matrigel plug assay). Representative pictures for each condition were taken at low (×100) (a–e) and at intermediate (×200) magnification (f–j). As negative control, growth factor-depleted Matrigel was used alone and no capillary networks were observed (a, f). As positive control, growth factor-depleted Matrigel containing 300 ng/ml VEGF was used, and extensive capillary networks were seen (b, g). When combining VEGF treatment with the HIF-1α inhibitor chetomin, the extensive capillary network formation induced by VEGF was unchanged (c, h). Using growth factor-depleted Matrigel containing oxLDL, capillary networks as extensive as those seen with VEGF treatment were noted (d, i). Importantly, when combining oxLDL treatment with the HIF-1α inhibitor chetomin, the increased capillary network formation induced by oxLDL was nearly completely abrogated (e, j). k–n High power magnification (× 1,000) of serial sections of tissue containing oxLDL-treated Matrigel plug stained with anti-CD31 antibody (k, m) reveals that erythrocyte-filled capillary networks seen with H&E staining (l, n) are lined by endothelial cells, oBar graph comparing the number of capillary networks identified by visual field for each condition. In summary, oxLDL-induced capillary network formation even exceeded that induced by VEGF and, most notably, in contrast to VEGF, was abrogated by treatment with the HIF-1α inhibitor chetomin (ANOVA, P<0.01; error bars ± SEM). pBar graph comparing Hgb concentration of Matrigel plugs identified by Drabkin’s reagent for each condition. In summary, oxLDL resulted in the highest Hgb concentration and its effect was abrogated by treatment with the HIF-1α inhibitor chetomin (ANOVA, P<0.01; error bars ± SEM)

Fig. 3

Effect of dietary lipid lowering on in vivo angiogenesis in experimental atherosclerosis, a–l Representative pictures of the effect of dietary lipid lowering on intimal neovascularization in a rabbit atherosclerosis model (a–d arteries of animals with high-lipid diet; i–l arteries of animals on low-lipid diet). With increasing magnification, the extent of neovascularization (arrows) especially in shoulder areas (asterisk) of large and foam cell-rich atherosclerotic lesions of animals with hyperlipidemia becomes visible on serial sections stained with Masson Trichrome and vWF antibody (a–d). In contrast, after normalization of serum lipids, the microvascular structures are nearly completely absent from a largely fibrotic intima and vWF immunostaining is only seen at the luminal endothelial layer (i–1). mBar graphs comparing neovessel content (percent intimal area covered with neovessels) and neovessel density (intimal neovessels per square millimeter) with high- vs. low-lipid diet. Hyperlipidemia resulted in strikingly enhanced neovascularization compared to animals with normolipidemic conditions (independent sample t test, P<0.01; error bars ± SEM). f–g High power magnification showing double immunofluorescence labeling for CD31 (f) and for vWF (g) with significant overlay (h) of both signals confirming the endothelial lining of microvascular structures of hyperlipidemic animals.

Fig. 4

Effect of dietary lipid lowering on in vivo macrophage expression of HIF-1α and VEGF in experimental atherosclerosis. Representative pictures of the effect of dietary lipid lowering on the expression of HIF-1α and VEGF in macrophages in a rabbit atherosclerosis model (a–h arteries of animals with continued high-lipid diet; i–o arteries of animals on low-lipid diet), a–h Nuclear HIF-1α (a, e) and cytoplasmic VEGF (c, g) expressions (arrows) are both located in intimal areas (ni) with a strong macrophage presence as indicated by RAM-11 immunostaining (b, f) (arrows) in atheroma of rabbits on high-lipid diet, i–o In atheroma of rabbits on low-lipid diet, HIF-1α (i, m) and VEGF (k, o) were nearly absent and only a few macrophages labeled by RAM-11 could be found (j, n). lBar graph comparing the percentage of intimal cells expressing HIF-1α, VEGF, and RAM-11 in atheroma of rabbits on high- vs. low-lipid diet (independent sample t test, P<0.01; error bars indicate ± SEM). p–t High power magnification showing triple immunofluorescence labeling for HIF-1α blue (q) (note the nuclear staining pattern also seen by conventional peroxidase labeling as shown in a), for VEGF in red (r) and for macrophages in green (s) with significant overlay (t) of all three signals in foam cell-rich intima of hypolipidemic animals, u, v Arterial protein analysis using Western blot shows a strong reduction in the amount of HIF-1α(u) and VEGF (v) protein as a result of dietary lipid normalization with low-lipid diet (lanes C and D) compared to continued high-lipid diet (lanes A and B). Beta-actin is shown as a loading control