Rac2 Modulates Atherosclerotic Calcification by Regulating Macrophage Interleukin-1β Production

Abstract

Objective: The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood.

Approach and results: Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1β (IL-1β) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1β expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1β levels. Moreover, we found that elevated IL-1β was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden.

Conclusions: Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1β expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.

Keywords: atherosclerosis; biomarkers; coronary artery disease; inflammation; macrophages.

Figure 1. Calcified plaque is associated with increased IL-1β and decreased Rac2 expression

(A) Ex vivo near-infrared calcium imaging from ApoE^−/− mice fed a HFD over 21 weeks along with quantification (B) of relative calcification signal (**, P<0.05; n=5 animals per time point). (C) Quantification (relative to time 0) of mRNA expression for Rac1, Rac2, F4/80, and IL-1β in aortic arches from ApoE^−/− mice fed a HFD over 21 weeks (**, P<0.05, n=5 animals per time point). (D) CT micrographs of 1 cm human proximal LAD segments in longitudinal axis illustrating calcified plaque as identified by the Agatston method. White bar, 0.5 cm. White small arrows, microcentrifuge tube wall. Yellow arrow, atherosclerotic wall segment without calcification. Red arrow, atherosclerotic wall segment with calcification. (E) Elastic Van Gieson staining of human left coronary artery sections (proximal to segments in D) from explanted hearts along with morphometric analysis (F), quantifying intima:media (I:M) thickness ratio (n=5 individual subjects). Black bar, 500 µm. Black arrow, calcification. Real-time PCR quantification of Rac1 (G), Rac2 (H), CSF1R (I) and IL-1β (J) mRNA expression from noncalcified vs. calcified human LAD coronary segments from D (***, P<0.001; **, P<0.05; n=5 individual subjects). All data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Figure 2. Rac2 deletion results in increased plaque calcification

(A) Ex vivo MicroCT images (Upper Panels; Bar, 1000 µm) of aortic arches from mice fed HFD for 14 weeks along with micrographs (Lower Panels; Bar, 250 µm) of lesser arch tissue sections stained for Alizarin red. Black arrows indicate the dark luminal irregularities of lipid accumulation. Yellow arrows denote white areas of calcification. (B) Quantification of percent wall area positive for Alizarin Red Staining from aortic arches in A (**, P<0.05; n=8 animals). (C) Ex vivo near-infrared calcium imaging from mice on normal chow or HFD for 14 weeks along with quantification (D) of relative calcification signal (**, P<0.05; n=8 animals). (E) Real-time PCR quantification of mRNA isolated from aortic arch tissue after mice were fed a HFD for 14 weeks for the transcription factors, RUNX2, SOX9, OSX, and MSX2, as well as ALP (**, P<0.05; n=8 animals). (F) Immunofluorescence micrographs of aortic plaques from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice demonstrating expression of ALP (green), CD68 (red), and SMA (magenta). Nuclei counterstained with DAPI (blue). Bar, 50 µm. Lumen, L. Intimal lesion, I. Media, M. (G) Quantification of relative ALP expression in aortic plaques from F (**, P<0.05; n=8 animals). All data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Figure 3. Vascular calcification in Rac2^−/− mice depends on the hematopoietic compartment and is associated with increased macrophage IL-1β expression

(A) Ex vivo near-infrared calcium imaging of aortas along quantification of relative calcification (B) signal and serum IL-1β levels (C) from reciprocal bone marrow transplants, using Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice (**, P<0.05 n=6 animals). (D) Real-time PCR quantification of mRNA isolated from aortic arch plaque tissue after mice were fed a HFD for 14 weeks for the following cytokines: IL-1β, TNF-α, INFγ, IL-18, and IL-6 (**, P<0.05; n=8 animals). (E) Serum IL-1β, IL-1α, and TNF-α protein concentrations by ELISA from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− and mice after HFD for 14 weeks (**, P<0.05; n=8 animals with each data point being an average of two technical replicates). (F) Immunofluorescence micrographs of aortic plaques from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice demonstrating expression of IL-1β (green), CD68 (red), and SMA (magenta). Nuclei counterstained with DAPI (blue). Bar, 170 µm. Lumen, L. Intimal lesion, I. Media, M. (G) Quantification of percent cells positive for IL-1β in aortic plaques from F (**, P<0.05; n=8 animals). All data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Figure 4. Rac2^−/− deletion is associated with increased NF-κB- and ROS-dependent IL-1β expression

(A) Resting or LPS-primed BMDMs from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice were treated with cholesterol crystals at indicated concentrations for 24 hours, and ELISA for IL-1β was performed on culture supernatants (**, P<0.05; n=3 animals with each animal data point being an average of 3 technical repeats). (B) Representative Rac2, actin, and Rac1 immunoblots of BMDM lysates from Rac2^−/−ApoE^−/− mice after BMDMS were transfected with GFP alone (control) or GFP with wild-type Rac2 (Rac2) or with constitutively active Rac2 (Q61L). (C) BMDMs, transfected as in (B), were primed with LPS and treated with 1000 µg/mL cholesterol crystals for 24 hours, and ELISA for IL-1β was performed on culture supernatants (**, P<0.05; n=3 animals with each animal data point being an average of 3 technical repeats). (D) Real-time PCR quantification of IL-1β mRNA isolated from resting or LPS-primed BMDMs that were treated with 1000 µg/mL cholesterol crystals for 24 hours (**, P<0.05; n=3 animals with each animal data point being an average of 3 technical repeats). (E) Relative luciferase activity in Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− BMDMs transfected with a NF-κB responsive luciferase construct and then stimulated by LPS-primed cholesterol crystal exposure (**, P<0.05; 3 animals with each animal data point being an average of 3 technical repeats). (F) Relative luminescence as a measure of ROS production in Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− BMDMs stimulated by LPS-primed cholesterol crystal exposure (**, P<0.05; n=3 animals with each animal data point being an average of 3 technical repeats). (G) Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− BMDMs were pretreated with DMSO (vehicle control), SB203580 (MAPK inhibitor, 10 µM), celastrol (NF-κB inhibitor, 5 µM) or Diphenyleneiodonium (DPI; reactive oxygen species inhibitor, 10 µM) and then LPS-primed and treated with cholesterol crystals for 3 hours, and ELISA for IL-1β was performed on culture supernatants (** P<0.05; n=6 mice per group). (H) Real-time PCR quantification of mRNA isolated from BMDMs in G for IL-1β (**, P<0.05 Rac2^−/−ApoE^−/− relative to Rac2^+/+ApoE^−/−; n=6 mice per group). All data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Figure 5. Macrophage Rac2 regulates Rac1-dependent IL-1β expression

(A) Immunofluorescence micrographs of aortic plaques from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice demonstrating expression of Rac2 (green), CD68 (red), and SMA (magenta). Nuclei counterstained with DAPI (blue). Bar, 50 µm. Lumen, L. Intimal lesion, I. Media, M. (B) Quantification of percent cells positive for Rac2 in aortic plaques from A (***, P<0.001; n=8 animals). (C) Immunofluorescence micrographs of aortic plaques from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice demonstrating expression of Rac1 (green), CD68 (red), and SMA (magenta). Nuclei counterstained with DAPI (blue). Bar, 50 µm. Lumen, L. Intimal lesion, I. Media, M. (D) Quantification of percent cells positive for Rac1 in aortic plaques from C (n=8 animals). (E) Rac1 immunoblot of BMDM lysates from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice loaded with increasing GTPγS concentrations for 5 minutes at 37°C, after which GTP-Rac1 was affinity precipitated by PBD pulldown along with quantification of GTP-bound Rac1 (F) (**, P<0.05; n=3 animals with each animal data point being an average of 3 technical repeats). (G) LPS-primed BMDMs from Rac2^+/+ApoE^−/− and Rac2^−/−ApoE^−/− mice were treated with 1000 µg/ml cholesterol crystals in the absence or presence of Rac1 inhibitor, EHT 1864, at indicated concentrations (**, P<0.05; n=3 animals with each animal data point being an average of 3 technical repeats). (H) LPS-primed BMDMs from ApoE^−/−CSF1R^mcmRac1^fl/fl and Rac2^−/− ApoE^−/−CSF1R^mcmRac1^fl/fl mice pre-treated with or without 4-hydroxytamoxifen (4-OHT) were exposed to 1000 µg/mL cholesterol crystals for 24 hours, and ELISA for IL-1β was performed on culture supernatants (**, P<0.05; n=3 animals with each animal data point being an average of 3 technical repeats). Lower panel, Rac1 and actin immunoblots of BMDM lysates. All data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Figure 6. IL-1β stimulates vascular smooth muscle cells calcification and atherosclerotic calcification is dependent on IL-1β signaling

(A) Photomicrographs along with quantification (B) of Alizarin red staining for mineralized calcium from cultured primary mouse aortic smooth muscle cells (ApoE^−/−) that were treated with increasing concentrations of IL-1β for 21 days (**, P<0.05; n=6 animals with each animal data point being an average of 3 technical repeats). Scale bar 1000 µm. (C) Real-time PCR quantification of RUNX2, SOX9, OSX, MSX2, and ALP in mRNA samples isolated from primary mouse aortic smooth muscle cells (ApoE^−/−) that were treated with 0 or 100 pg/mL of recombinant IL-1β for 21 days (**, P<0.05; n=6 animals with each animal data point being an average of 3 technical repeats). (D) Near-infrared calcium imaging along with quantification (E) of calcification signal in ex vivo aortas from Rac2^−/−ApoE^−/− mice after HFD for 9 weeks to establish baseline and then after an additional 5 weeks of HFD coupled to treatment with vehicle control or IL-1ra (**, P<0.05 n=8 animals). (F) Serum IL-1β protein concentrations by ELISA (**, P<0.05; n=8 animals). (G) Hematoxylin and eosin staining of adjacent aortic sinus sections at the level of the aortic valve after HFD for 9 weeks to establish baseline and then after an additional 5 weeks of HFD coupled to treatment with vehicle control or IL-1ra. Bar, 200 µm. (H) Quantification of average aortic plaque area (**, P<0.05; n=9 aortic valve sinuses from 3 animals). All data are representative of at least 3 independent experiments. Quantitative data are displayed as mean ± SEM.

Figure 7. Increased serum IL-1β level is associated with higher coronary calcium burden and is predictive of cardiovascular outcomes in patients

(A) The Pearson correlation between IL-1β protein concentrations and CACS (***, P=0.001; n=79). (B) Noncontrast CT scan images illustrating the LAD territory calcification in coronary disease patients who were divided by CACS into low and high calcium burden groups. Bar, 3 cm. (C) Serum IL-1β protein concentrations for each patient (gray dots) along with median and interquartile ranges within each level of calcium burden (***, P=0.0040; n=39–40). (D) Kaplan-Meier event curve for hard cardiac events of sudden cardiac death, myocardial infarction, and acute coronary syndrome in patients divided by calcium burden (**, P=0.0259, n=39–40, long-rank test). (E) Kaplan-Meier event curve for hard cardiac events of sudden cardiac death, myocardial infarction, and acute coronary syndrome in patients divided by both calcium burden and serum IL-1β concentration (**, P=0.0119, n=10–28, long-rank test).

Figure 8. Summary Schematic

Pattern recognition receptor signaling (i.e. Toll-like receptor signaling) coupled to cholesterol crystal induced lysosomal destabilization by phagocytic cells like macrophages promotes Rac-modulated expression of IL-1β. Rac2 is a major determinant of the degree of macrophage IL-1β expression by regulating Rac1 activity upstream of NF-κB, and ROS. Macrophage Rac2 and Rac1 exist in a state of balance with Rac2 serving as a brake that tempers Rac1-dependent IL-1β expression. When the Rac2 brake is released, as in the chronic inflammation associated with atherosclerosis or as modeled in the Rac2^−/−ApoE^−/− mouse, IL-1β expression is enhanced, resulting in the upregulation of mesenchymal cell osteogenic transcription factors and the consequent mineralization of calcium.