PARP inhibition in atherosclerosis and its effects on dendritic cells, T cells and auto-antibody levels

Abstract

Objective: Atherosclerosis is a chronic inflammatory process. Poly(ADP-ribose) polymerase-1 (PARP), a nuclear enzyme linked to DNA repair, has been shown to be involved in atherogenesis; however, the effects on dendritic cells, T cells and serum auto-antibody levels are not fully understood.

Methods: Male Apoe-/- mice on a western diet were treated with the PARP inhibitor INO-1001 (n = 15), while the control group (n = 15) received 5% glucose solution for 10 weeks.

Results: Inhibition of PARP markedly reduced atherosclerotic lesion development (p = 0.001). Immunohistochemistry and mRNA analysis revealed a reduced inflammatory compound inside the lesion. Focusing on dendritic cells, INO-1001 reduced number of cells (p = 0.04), grade of activation, represented by Il12 (p = 0.04) and Cd83 (p = 0.03), and grade of attraction, represented by Mip3α (p = 0.02) in the plaque. Furthermore, INO-1001 decreased number of T lymphocyte (p = 0.003) in the lesion and grade of activation after stimulation with oxLDL in vitro. Moreover, serum IgM antibody levels to oxLDL were significantly lower in INO-1001 treated mice (p = 0.03).

Conclusions: Functional blockade of PARP by INO-1001 reduces atherosclerotic lesion development. The anti-atherogenic effect is beside already known mechanisms also moderated due to modulation of DC and T cell invasion and activation, DC attraction as well as IgM antibody levels to oxLDL.

Figure 1

Antibody serum levels to oxLDL. On the day of tissue harvesting (18 weeks) IgM (A) and IgG (B) antibody serum levels to oxLDL were determined by EL1SA (n = 30). Results are shown as box plots displaying medians and 25^th and 75^th percentiles as boxes and 10th and 90th percentiles as whiskers, n.s. - not significant.

Figure 2

Effects of PARP-l inhibition on atherosclerotic plaques in Apoe^-/- mice. Morphomctric quantification of fractional stenosis [%] (A) and maximum stenosis [%] (A) and maximum stenosis [%] (B) of early atherosclerotic lesions were compared between INO-1001 treated (n = 15) and control mice (n = 15). Results are shown as box plots displaying medians and 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers. Representative Oil Red O immunohistostainings from aortic root of control (C) and INO-1001 treated (D) mice are shown.

Figure 3

Cellular composition in atherosclerotic lesions of Apoe^-/- mice. Representative photomicrographs of immunohistochemistry stainings of collagen (yellow, MOVATs (A+B)), T cells (CD3 (C-F)) as well as activation of the tissue (VCAM-1 (G-J)) and of activated endothelial cells (VCAM-l (1+J)) in atherosclerotic lesions (n = 30), A-D and G-H show 4× and E-F and I-J 20× magnification.

Figure 4

Quantification of immunohistochemistry analysis. Immunohistochemistry analysis of lesion composition (n = 30) of INO-1001 treated mice compared to controls were performed. Number of positive cells were related to lesion area (CD3 (A)), related per plaque (CD3 (B), VCAM-1 (D)), to total number of cells in the adventitia (CD11c (C)) and to DAPI+ cells in the lesion (TUNEL (E)) in INO-1001 treated and control mice. Results are shown as box plots displaying medians and 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers, n.s. = not significant.

Figure 5

Quantitative tissue PCR results for different inflammatory markers in atherosclerotic lesions. cDNA was generated from thoracic aorta tissue extracts (n = 30) and molecule transcripts of various molecules were compared between INO-1001 treated und control animals. Copy numbers were adjusted for copies of the housekeeping gene β-actin. Results are shown as plots displaying medians and 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers.

Figure 6

DCs in the adventitia of Apoe^-/- mice. Representative photomicrographs of immunohistochemistry staining of adventitial DCs (CD11c) in controls (A) and INO-1001 treated mice (B); 10× magnification.

Figure 7

In vitro effects of PARP-1 inhibition on peripheral blood lymphocytes. Isolated spleen cells were incubated with 20 μg/ml oxLDL. The effects of addition of specific PARP-1 inhibitor INO-1001 on the expression of activation-associated cytokines were measured by qPCR after 8 h. cDNA was generated of incubated cells and molecule transcripts of various molecules were compared between INO-1001 treated und control animals. Copy numbers were adjusted for copies of the housekeeping gene β-actin. Representative data from one of three independent experiments with similar results.