Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis

Abstract

Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocyte-type 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.

Figure 1

Expression of LO in atherosclerotic lesions. (a) LO transcripts in AAO, CAD, and CHD lesions. First column, transcript species; cycle numbers of samples in third through sixth columns are indicated in brackets; second row, sizes of PCR products in bp; third through sixth columns, ethidium bromide-stained PCR products of AAO, CAD, CHD lesions, and amplified external standards (eSt); seventh column, numbers of standard cDNA molecules added to PCR buffer before amplification (n[eSt]). (b) LO and FLAP immunoblots of CAD type V lesions. For controls, 2 ng of each 5- and 15-LO proteins was applied; for FLAP, an unspecified amount of leukocyte membrane protein was applied. Positions of MW standards are indicated by arrows at right.

Figure 2

Comparison of 15-LO transcripts in human tissue RNA extracts. Multiple-tissue RNAs or tracheal and bronchial ring RNA were subjected to 15-LO and GAPDH real-time RT-PCR analyses. Data for cardiovascular tissues represent the mean of real-time RT-PCR assays performed on tissues of 38 patients (42 cardiovascular specimens) afflicted with all lesion types of atherosclerosis.

Figure 3

Identification of 5-LO⁺ cells in CAD lesions. (Bars = 20 μm.) (A–D) 5-LO is expressed in macrophages and/or DCs. (A) 5-LO (Cy3 red). (B) CD68 (Cy2 green). (C) DNA stained with DAPI blue. (D) Merge of A–C. (E) Preadsorption of antiserum abolishes nuclear 5-LO staining of a foam-cell-rich area: CD68 (Cy2 green), DAPI blue. Note absence of Cy2 red 5-LO staining. (F and G) Cells containing 5-LO express high levels of HLA-DR. (F) 5-LO (Cy3 red). (G) Same as F, plus HLA-DR (Cy2 green) and DAPI blue. (H) A subpopulation of 5-LO-expressing cells has a DC phenotype; colocalization of 5-LO (Cy3 red), CD1a (Cy2 green), and DAPI blue. (I and K) Foam cells express 5-LO. (I) 5-LO (streptavidin-biotin dark brown) and lipid (Sudan IV red). (K) Same as I, plus DAPI blue. 5-LO⁺ cells are indicated by arrows and 5-LO⁻ cells are indicated by arrowheads. (L–O) 5-LO is located in the nucleus and in the cytoplasm of macrophages; 3-dimensional reconstruction of 12-μm stacks of confocal images. (L) 5-LO (Cy3 red) and DAPI blue. (M) DAPI blue only of L. (N) 5-LO (Cy3 red) and DAPI blue. The cytoplasmic 5-LO⁺ compartment is indicated by an arrow. (O) DAPI blue only of N. (P) 5-LO-expressing cells are located in the vicinity of CD3⁺ lymphocytes. A 5-LO⁺ (Cy3 red) cell is indicated by the arrow and CD3⁺ (Cy2 green) cells by the arrowheads (DAPI blue). (R) 5-LO is expressed in lesion mast cells. 5-LO (Cy3 red), mast cell tryptase (Cy2 green), and DAPI blue. (S) Positive control showing 15-LO staining in human lung and 5-LO staining in lung macrophages. A normal medium-sized human bronchus from a patient afflicted with lung carcinoma was analyzed for 15-LO (Cy3 red), 5-LO (Cy2 green; indicated by the arrow), and DAPI blue. (T) Advanced CAD lesions lack 15-LO-expressing cells. A foam-cell-rich area of a CAD lesion was analyzed for CD68 (Cy2 green) and DAPI blue. Note the absence of Cy2 red 15-LO staining.

Figure 4

5-LO⁺ cells increase during transition from early to advanced CHD. The numbers of CD68⁺ macrophages/foam cells/DCs (open bars) and 5-LO⁺ cells (filled bars) were determined in arterial wall laminae of type 0–V lesions and grouped for combined 0–I (n = 13), II–III (n = 9), and IV–V (n = 7) lesions. Cell numbers were determined in specimens of the left-descending circumflex coronary artery from 29 patients. A total of 7,300 CD68⁺ macrophages/foam cells/DCs, 7,902 5-LO⁺ cells, 713 mast cell tryptase⁺ cells, and 120 CD66b⁺ cells were counted. Data represent means of 29 specimens ± SEM. n.s., not significant. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.