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Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodeling¹

SHAW-FANG YET^*,2, MATTHEW D. LAYNE^*, XIAOLI LIU^*, YEN-HSU CHEN^*,, BONNA ITH^*, NICHOLAS E. S. SIBINGA and MARK A. PERRELLA^*

^* Pulmonary and Critical Care Division, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA;
Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; and
Cardiovascular Division, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA

²Correspondence: Pulmonary and Critical Care Division, Brigham and Women’s Hospital, 75 Francis St., Thorn 1333, Boston, MA 02115, USA. E-mail: syet{at}rics.bwh.harvard.edu.

SPECIFIC AIMS

The present study was designed to investigate the importance of heme oxygenase-1 (HO-1) in the pathophysiology of hypercholesterolemia-induced atherosclerosis and to determine the role of HO-1 in vein graft disease using a murine model of vein graft stenosis.

PRINCIPAL FINDINGS

1. Increased atherosclerotic lesion formation in HO-1^–/–apoE^–/– mice
To examine the role of HO-1 in the development of atherosclerosis, we generated mice deficient in both HO-1 and apoE (HO-1^–/–apoE^–/–). HO-1^+/+apoE^–/– and HO-1^–/–apoE^–/– mice were fed Western diet starting at 4 wk of age, and the right brachiocephalic arteries were subsequently analyzed. After 8 wk on a Western diet, both HO-1^+/+apoE^–/– and HO-1^–/–apoE^–/– mice had elevated total plasma cholesterol levels (832±62 and 914±48 mg/dL, respectively, n=6 each group; P=0.32). Despite similar high cholesterol levels, HO-1^–/–apoE^–/– mice developed more severe atherosclerotic lesions than HO-1^+/+apoE^–/– mice. Verhoeff’s elastin staining (Fig. 1 A, B) revealed that lesions from HO-1^–/–apoE^–/– mice were increased in size and more occlusive (43.7±7.7% luminal occlusion, n=5) than HO-1^+/+apoE^–/– mice (12.2±3.8% luminal occlusion, n=4; P<0.05). The lesions from HO-1^+/+ apoE^–/– mice were composed primarily of macrophages (Fig. 1C , brown). In contrast, lesions from HO-1^–/–apoE^–/– mice were not only more occlusive but also more advanced, with increased accumulation of macrophages (Fig. 1D , brown), which appeared to be lipid-filled foam cells (Fig. 1B , arrows). In HO-1^+/+ apoE^–/– mice, SM -actin-positive cells were present in the medial smooth muscle layers of the arteries, but not in the lesions (Fig. 1E ). After 8 wk on a Western diet, smooth muscle cells—the hallmark of advanced lesions—were present in the HO-1^–/–apoE^–/– mouse lesions (Fig. 1F , arrow). The HO-1^+/+apoE^–/– mice had less advanced lesions that did not contain a fibrous cap (Fig. 1G , arrowhead), in contrast to smooth muscle cell-rich fibrous caps in the lesions from HO-1^–/–apoE^–/– mice (Fig. 1H , arrow).

View larger version (95K): [in this window] [in a new window]   Figure 1. Increased atherosclerotic lesion formation in HO-1–/–apoE–/– mice. Representative histological analysis of HO-1+/+apoE–/– and HO-1–/–apoE–/– vessel sections after 8 wk on a Western diet. A, B) Verhoeff’s staining for elastin (black) was performed on sections from the proximal brachiocephalic arteries from HO-1+/+apoE–/– (A) and HO-1–/–apoE–/– (B) mice. C, D) MOMA-2 staining for macrophages (brown) in proximal brachiocephalic arteries (x100) from HO-1+/+apoE–/– (C) and HO-1–/–apoE–/– (D) mice. E–H) SM -actin–stained (red) vessels from HO-1+/+apoE–/– (E, G) and HO-1–/–apoE–/– (F, H) mice. B) Arrows indicate foam cells. F) Arrow indicates smooth muscle cells in the lesion. G) Arrowhead marks a small noncomplex lesion. H) Arrows mark the fibrous cap. Lu, lumen; Med, medial smooth muscle layers. Original magnification x100 (A–F) and x200 (G, H).

2. Increased neointima formation in HO-1^–/– mice 10 days after vein graft surgery
To investigate the role of HO-1 in vein graft stenosis, wild-type and HO-1^–/– mice were subjected to a mouse model of vein graft stenosis. Ten days after surgery, in contrast to the minimal neointimal formation in wild-type mice (13.3±2.5% luminal occlusion, n=5), there was a robust neointima formation in HO-1^–/– mice (34.2±8.7% luminal occlusion, n=4; P <0.05 vs. wild-type). In wild-type composite vessels, SM -actin-positive cells were detected mainly in the medial smooth muscle layers of the arterial portion and the small population of neointimal cells. In HO-1^–/– composite vessels, however, both arterial medial smooth muscle layers and the large vein graft neointima stained positive for SM -actin, indicating that the neointima consisted primarily of VSMC.

3. VSMC death and calcification in HO-1^–/– vein grafts
Fourteen days after surgery, in composite vessels from wild-type mice there was robust formation of neointima (Fig. 2 A; 45.6±3.9% luminal occlusion, n=4). In comparison, 62.6±6.6% of the lumen was occluded in HO-1^–/– composite vessels (Fig. 2B , n=3). At a higher magnification, robust neointimal cells were present between the lumen and internal elastic lamina in wild-type vein grafts (Fig. 2C ) vs. acellular material in HO-1^–/– vein grafts (Fig. 2D ). Vein graft neointima from wild-type composite vessels consisted of -actin-positive VSMC (Fig. 2E ). Very few SM -actin-positive VSMC were detected in the neointima of HO-1^–/– vein grafts (Fig. 2F ), indicating the death of VSMC. We began to observe circumferentially oriented elastic laminae in wild-type vein grafts (Fig. 2G , arrowheads), indicating arterialization of the graft. In the HO-1^–/– neointima, we observed closely layered elastic laminae toward the lumen (Fig. 2H , arrowheads), a characteristic of advanced lesions. von Kossa calcium staining showed that only a few cells from wild-type neointima stained positive (Fig. 2I , brown), indicating calcium deposition in limited neointimal cells. In contrast, von Kossa staining revealed that the acellular material in the neointima from HO-1^–/– mice stained strongly for the deposition of calcium (Fig. 2J , brown). These data are consistent with dystrophic calcification, extracellular calcium deposition in areas of cell death.

View larger version (97K): [in this window] [in a new window]   Figure 2. Histological analysis of the jugular vein/carotid artery composite vessels in wild-type and HO-1–/– mice 14 days after surgery. Sections are oriented vein patch on the top and arterial side on the bottom. A–D) Elastin stain (black) of cross sections of jugular vein/carotid artery composite vessels from A) HO-1+/+ and B) HO-1–/– mice. Original magnification, x63. C, D) Higher magnification (x200) from panels A and B, respectively. E, F) Immunostaining for smooth muscle -actin (red) of adjacent sections from panels A and B, respectively. Original magnification, x63. G, H) Higher magnification (x400) from panels E and F, respectively. G, H) Arrowheads indicate circumferentially oriented elastic laminae. I, J) von Kossa calcium staining (brown) of representative vessel sections from HO-1+/+ and HO-1–/– mice, respectively. I) Arrows point to calcium staining (brown). Original magnification, x100. The two lines indicate the approximate junction of the vein patch and the arterial wall. A–H) Arrows indicate internal elastic lamina (IEL). Lu, lumen.

4. VSMC from HO-1^–/– mice are more susceptible to oxidative stress
To elucidate the potential molecular mechanism leading to VSMC death, we isolated aortic smooth muscle cells from wild-type and HO-1^–/– mice and treated them with hydrogen peroxide (H₂O₂). Wild-type and HO-1^–/– VSMC tolerated H₂O₂ concentrations up to 800 µM. With an additional small increase (50 µM) in concentration to 850 µM, 80% of wild-type VSMC were still viable. The viability of HO-1^–/– VSMC decreased to <10%. These data suggested that HO-1^–/– VSMC were more susceptible to oxidant H₂O₂-induced cell death than were VSMC isolated from wild-type mice.

CONCLUSIONS AND SIGNIFICANCE

In response to hypercholesterolemia, mice deficient in HO-1 and apoE developed larger and more advanced lesions than mice deficient in apoE alone. Our results clearly show that the loss of HO-1 alone is sufficient to accelerate atherosclerosis and indicate that the activities of other HO isoforms do not fully compensate for the loss of HO-1.

To examine the effects of the loss of HO-1 activity on vascular remodeling in a different form of obstructive vascular disease, we used a mouse jugular vein/carotid artery autograft model to mimic the vein grafts used in bypass surgery. Ten days after surgery, HO-1^–/– mice had much larger neointima than wild-type mice. Instead of developing even larger lesions 14 days after surgery, there was massive cell death in the neointima of HO-1^–/– mice. Calcification in areas of VSMC death was observed in HO-1^–/– vein grafts. Our data suggest that the vein graft had a maladaptive response to increased pressure in the absence of HO-1. Increased hemodynamic pressure induces oxidative stress and HO-1 expression in VSMC, suggesting a protective role of HO-1 in the cell’s defense against oxidative damage in the vasculature. Consistent with this hypothesis, VSMC from HO-1^–/– mice are more prone to oxidant-induced cell death, indicating that increased susceptibility to oxidative stress is one of the mechanisms leading to cell death in the absence of HO-1.

Our data demonstrate that HO-1 plays an essential, protective role in vascular remodeling in the settings of hypercholesterolemia and vein graft stenosis and that an absence of HO-1 exacerbates cardiovascular disease (Fig. 3 ). Future studies will investigate the molecular mechanisms by which HO-1 exerts its role in the development of atherosclerotic lesions and vein graft stenosis.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram of the role of HO-1 in the atherosclerotic lesion formation and vein graft stenosis. Our data suggest that HO-1 plays an important role in vascular remodeling. After 8 wk on a Western diet, HO-1+/+apoE–/– mice had small, uncomplicated lesions consisting of lipid-filled macrophages (small circles). HO-1–/–apoE–/– mice developed more severe atherosclerotic lesions containing lipid-filled macrophages, -actin-positive VSMC (black oval), and a VSMC-rich fibrous cap (black). In a vein graft stenosis model, HO-1 deficiency resulted in neointimal VSMC death (indicated by x’s) and calcification of the vein graft.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0187fje; doi: 10.1096/fj.03-0187fje

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