Stage-Dependent Impact of RIPK1 Inhibition on Atherogenesis: Dual Effects on Inflammation and Foam Cell Dynamics - PubMed (original) (raw)
doi: 10.3389/fcvm.2021.715337. eCollection 2021.
Huihui Li 1, Yonghu Huang 1, Hong Chen 1, Haojie Rao 1, Guoli Yang 1, Qing Wan 1, Zekun Peng 1, John Bertin 2, Brad Geddes 2, Michael Reilly 2, Jean-Luc Tran 2, Miao Wang 1
Affiliations
- PMID: 34760938
- PMCID: PMC8572953
- DOI: 10.3389/fcvm.2021.715337
Stage-Dependent Impact of RIPK1 Inhibition on Atherogenesis: Dual Effects on Inflammation and Foam Cell Dynamics
Yuze Zhang et al. Front Cardiovasc Med. 2021.
Abstract
Objective: Atherosclerosis is an arterial occlusive disease with hypercholesterolemia and hypertension as common risk factors. Advanced-stage stenotic plaque, which features inflammation and necrotic core formation, is the major reason for clinical intervention. Receptor interacting serine/threonine-protein kinase 1 (RIPK1) mediates inflammation and cell death and is expressed in atherosclerotic lesions. The role of RIPK1 in advanced-stage atherosclerosis is unknown. Approach and Results: To investigate the effect of RIPK1 inhibition in advanced atherosclerotic plaque formation, we used ApoE SA/SA mice, which exhibit hypercholesterolemia, and develop angiotensin-II mediated hypertension upon administration of doxycycline in drinking water. These mice readily develop severe atherosclerosis, including that in coronary arteries. Eight-week-old ApoE SA/SA mice were randomized to orally receive a highly selective RIPK1 inhibitor (RIPK1i, GSK547) mixed with a western diet, or control diet. RIPK1i administration reduced atherosclerotic plaque lesion area at 2 weeks of treatment, consistent with suppressed inflammation (MCP-1, IL-1β, TNF-α) and reduced monocyte infiltration. However, administration of RIPK1i unexpectedly exacerbated atherosclerosis at 4 weeks of treatment, concomitant with increased macrophages and lipid deposition in the plaques. Incubation of isolated macrophages with oxidized LDL resulted in foam cell formation in vitro. RIPK1i treatment promoted such foam cell formation while suppressing the death of these cells. Accordingly, RIPK1i upregulated the expression of lipid metabolism-related genes (_Cd36, Ppara, Lxr_α, Lxrb, Srebp1c) in macrophage foam cells with ABCA1/ABCG1 unaltered. Furthermore, RIPK1i treatment inhibited ApoA1 synthesis in the liver and reduced plasma HDL levels. Conclusion: RIPK1 modulates the development of atherosclerosis in a stage-dependent manner, implicating both pro-atherosclerotic (monocyte infiltration and inflammation) and anti-atherosclerotic effects (suppressing foam cell accumulation and promoting ApoA1 synthesis). It is critical to identify an optimal therapeutic duration for potential clinical use of RIPK1 inhibitor in atherosclerosis or other related disease indications.
Keywords: RIPK1; atherosclerosis; cell death; foam cells; inflammation; macrophages; reverse cholesterol transport.
Copyright © 2021 Zhang, Li, Huang, Chen, Rao, Yang, Wan, Peng, Bertin, Geddes, Reilly, Tran and Wang.
Conflict of interest statement
JB, BG, MR, and JT are current or previous employee at GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
Figure 1
Pharmacokinetics of GSK547 and its potent inhibition of RIPK1 in mice. (A) Chemical structure of GSK547 and its reported in vitro IC50 (19). (B) Pharmacodynamics dose-response in an acute TNF/zVAD shock model on body temperature at 0.01, 0.1, 1, and 10 mg/kg GSK547 administered orally. mpk = mg/kg. n = 3–7 mice per group. Mice were administered GSK547 orally at 0.01, 0.1, 1.0, and 10 mg/kg, then blood drug levels at serial time points were shown (C,D). The average drug level from 0.5 h post-dose was used to calculate the predicted RIPK1 inhibition, and actual body temperature change was calculated as percent change corrected to the TNF/zVAD group. (C) Pharmacokinetics of GSK547 at 0.01, 0.1, 1, and 10 mg/kg orally. n = 5 mice per group. (D) Correlation of PK drug level to the RIPK1inhibtion. The drug concentration is correlated with the acute in vivo PK data on (D) at 0.1 mpk (11 ng/ml), 1 mpk (98 ng/ml), 10 mpk (886 ng/ml). The drug dose of 0.1 mpk is equivalent to the IC50 of the in vitro data. The drug concentration increased dose-dependently, which correlated well to the predicted and observed inhibition as indicated in the table (B,D). At 1.0 and 10 mpk doses, the GSK547 inhibited RIPK1 activation at 99%. (E) Administration of GSK547 in food base PK study achieved a steady-state concentration. GSK547 PK (E, left)/PD study (E, right) was conducted and blood was sampled at peak and trough in mice fed the diet for 1 week at 9.6 and 96 mg/kg/day. Mice were challenged on day 8 with TNF/zVAD (16.7 mg/kg). n=3-5 mice per group. (F) Plasma levels of GSK547 in ApoE SA/SA mice that were administered with a western diet (21% fat and 0.2% cholesterol) mixed with GSK547 at a dose of 10 mg/kg/day, and with Doxycycline (Dox)-containing water to induce hypertension. Sample was collected at ~10 a.m. of the day after 2- and 4-week dosing. n = 4 mice per group. All the data represented as mean ± SEM; ***P < 0.001. Statistical analysis: Ordinary one-way ANOVA test.
Figure 2
Differential impacts of RIPK1 inhibition on early and late atherogenesis in mice. Male ApoE SA/SA mice were fed a western diet contained RIPK1 inhibitor GSK547 (RIPK1i, 10 mg/kg) or not (Ctl) for 2 or 4 weeks with Dox-containing water to induce hypertension. Representative histological analysis of cross-sections from the aortic sinus stained with Oil Red O (A) and quantification of aortic sinus Oil Red O-positive area of male mice (B). Scale bar = 500 μm. Representative histological analysis of heart cross-sections stained with Oil Red O (C) and quantification of coronary artery (D) Oil Red O-positive area of male mice. Panel C, scale bar = 500 μm, top; scale bar = 100 μm, bottom. Dashed lines showed vessel media. Blue symbols = Ctl group, pink symbols = RIPK1i group. n = 6–10 mice per group. All the data represented as mean ± SEM; *P < 0.05, **P < 0.01. Statistical analysis: unpaired student's t test.
Figure 3
RIPK1i treatment decreased plasma HDL and suppressed liver ApoA1 expression level. (A) Plasma HDL-C level in male ApoE SA/SA mice fed with WD and Dox for 2 and 4 weeks. n = 5–8 mice per group. (B) Representative Western blot images of ApoA1 from liver of male ApoE SA/SA mice fed with WD and Dox for 2 weeks. (C) Quantification of liver ApoA1 protein level (n = 9.7). (D) qRT-PCR analysis of Apoa1, Apom, Ppara in liver of male ApoE SA/SA mice fed with WD and Dox for 2 weeks (n = 9.8). All of the data represented as mean ± SEM; *P < 0.05, **P < 0.01. Statistical analysis: unpaired student's _t_-test.
Figure 4
RIPK1i accelerated macrophage accumulation and foam cell formation. (A–D) Histological analysis of cross-sections from aortic sinus of ApoE SA/SA mice receiving RIPK1i for 4 weeks. n = 5 mice per group. (A) Representative IHC images of F4/80 in aortic sinus plaque. Scale bar = 100 μm. (B) Quantification of macrophages (F4/80 positive) are represented. (C) Representative IHC images of α-SMA in aortic sinus plaque. Scale bar = 100 μm. (D) Quantification of smooth muscle cells (α-SMA positive) are represented. (E–H) Peritoneal macrophages isolated from male ApoE SA/SA mice or male C57BL/6J mice were pretreated with RIPK1i (50 ng/ml) or DMSO for 3 h, then stimulated with oxidized LDL (50 μg/ml) for 24 h. (E) Representative images of Oil Red O stained foam cells for male ApoE SA/SA mice. (F) Percentage of foam cells in ox-LDL induced peritoneal macrophages for male ApoE SA/SA mice (left, n = 5) and male wild-type mice (right, n = 3). (G) Relative Cd36 mRNA level in ox-LDL induced peritoneal macrophages for male ApoE SA/SA mice (n = 6). (H) Lipid metabolism-related genes expression levels in ox-LDL induced peritoneal macrophages for male ApoE SA/SA mice (n = 5–6 per group). All of the data represented as mean ± SEM; *P < 0.05, **P < 0.01. Statistical analysis: unpaired student's _t_-test (B,G), non-parametric Mann-Whitney U test (F,H).
Figure 5
RIPK1i suppressed cell apoptosis and necroptosis in plaque. (A–D) Histological analysis of cross-sections from aortic sinus of ApoE SA/SA mice. (A) Representative immunofluorescence images of Cleaved Caspase-3 staining (red) in aortic sinus. Scale bar = 500 μm. (B) Quantification of apoptosis (Cleaved Caspase-3 positive) area (n = 5). (C) Representative IHC images of RIP3 in aortic sinus plaque. Scale bar = 100 μm. (D) Quantification of necroptosis (RIP3 positive) area (n = 6). (E) and (F) Peritoneal macrophages isolated from male ApoE SA/SA mice were pretreated with RIPK1i (50 ng/ml) or DMSO for 3 h, then stimulated with oxidized LDL (50 μg/ml) for 24 h and stained for TUNEL. (E) Representative immunofluorescence images of TUNEL staining (green). TUNEL positive cells are indicated by the white arrows. Scale bar = 50 μm. (F) Percentage of TUNEL positive cell in peritoneal macrophages (n = 6). All of the data represented as mean ± SEM; *P < 0.05, **P < 0.01. Statistical analysis: unpaired student's _t_-test (B), non-parametric Mann-Whitney U test (D,F).
Figure 6
RIPK1i treatment alleviated systemic inflammation and macrophage infiltration in early atherogenesis. (A) Heat map analysis of inflammatory cytokines levels in ApoE SA/SA mice in early stage (left, 2 weeks) and late stage (right, 4 weeks). (B–D) Plasma levels of TNF-α (B), IL-1β (C) and MCP-1 (D) in early stage (2 weeks) and late stage (4 weeks) of ApoE SA/SA mice. n = 4–6 mice per group. (E) Representative IHC images of F4/80 in aortic sinus plaque from ApoE SA/SA mice in early stage (left). Quantification of macrophages (F4/80 positive) are represented (right). Scale bar = 100 μm. n = 7.9 mice per group. All of the data represented as mean ± SEM; *P < 0.05; **P < 0.01. Statistical analysis: non-parametric Mann-Whitney U test (B,E), unpaired student's _t_-test (C,D).
Figure 7
Schematic illustration of the stage-dependent impact of RIPK1i in atherosclerosis.
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