The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development

Abstract

Objective: To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs).

Background: AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies.

Methods: The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II.

Results: Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-β regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages.

Conclusions: These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Figure 1.. SETDB2 is Increased Murine AAA Macrophages and Increases the Repressive H3K9 Trimethylation on Timp Gene Promoters.

A. Male C57BL/6J mice were injected intraperitoneally with an AAV containing mouse PCSK9D377Y and fed saturated fat diet for 6 wk. Mice were infused with saline or AngII (1,000 ng/min/kg) for 4 weeks. Quantitative PCR analysis of Timp1, Timp2, and Timp3 isolated from in vivo macrophages (MΦ) (CD11b⁺[CD3⁻CD19⁻Nk1.1⁻Ly6G⁻]) of mice exposed to saline or AngII for 28 days (n =3/group run in triplicate). *p<0.05, **p<0.01 for Welch’s t-test. B. Representative Verhoeff–van Gieson elastin staining of abdominal aortic sections at 10X and 40X showing disrupted aortic structure in AngII mice compared with saline control mice; scale bar is 50 μm or 10 μm in Verhoeff–van Gieson stain; arrows represent elastin fragmentation. C, D. Quantitative PCR analysis of Setdb2 isolated from aortas or MΦs (CD11b⁺[CD3⁻CD19⁻Nk1.1⁻Ly6G⁻]) in mice infused with either saline or Ang II for 28 days (n = 3–4/group run in triplicate). *p<0.05 for Mann-Whitney U test. E. ChIP analysis for H3K9me3 at Timp1, Timp2, and Timp3 promoter was performed (n=3/group run in triplicate). For all ChIP experiments, isotype-matched IgG was run in parallel. Dotted line represents isotype-matched control. **p<0.01 for Mann-Whitney U test.

Figure 2.. Human Aortic Single Cell Transcription Profiling Reveals Elevated SETDB2 and Extracellular Matrix Organization Pathways in Infiltrating Monocyte/Macrophages.

A. Aortic tissue from patients with AAA (n=19) and atherosclerotic controls (n=6) were collected. No statistical differences were found between groups with respect to sex, age, or comorbid conditions. SETDB2 gene expression was measured by qPCR with log scale. *p<0.05 by Welch’s t test replicated twice. B. Immunohistochemistry was performed for SETDB2 in human control and AAA samples. Representative slides are shown at 40X and scale bar is 60 μm. C. Cluster analysis using the uniform manifold approximation and projection (UMAP) technique of single cell sequencing from human AAA (n=4) and nonaneurysmal (n=2) samples revealed 21 distinct cell clusters (representative). D. Feature plots displaying the single cell gene expression of SETDB2 across cell clusters. E. Gene Ontology biological process or cellular component enrichment analysis of differentially expressed genes SETDB2⁺ vs. SETDB2⁻ cells. The combined score metric corresponds to the P value (two-tailed Fisher’s exact test) multiplied by the Z-score of the deviation from the expected rank, and q values determined by Benjamini–Hochberg correction.

Figure 3.. AAA Formation is Inhibited in Macrophage-specific SETDB2-deficient Mice.

A. Experimental design of macrophage-specific SETDB2-deficiency in murine AAA model. Wild-type (WT M𝛷) mice and mice with macrophage-specific SETDB2 deficiency (Setdb2^−/− M𝛷) were fed high fat diet for 6 weeks and infused with saline or AngII infusion (1,000 ng/min/kg) for 4 weeks. B. Representative ultrasound images of the abdominal aorta at day 28 in WT M𝛷 or Setdb2^−/− M𝛷 following either saline or AngII infusion. Dotted line represents aortic contour and arrows represent aortic wall diameter. C, D. Maximal abdominal aortic diameter and aneurysm incidence as determined by ultrasound measured by two observers in WT M𝛷 or Setdb2^−/− M𝛷 infused with either saline or AngII (n= 6 in saline infused cohorts and 20 in AngII infused cohorts). *p<0.05; **p<0.001 by ANOVA with Newman-Keuls Multiple Comparison test. Data are presented as the mean±SEM. E. Representative Verhoeff–van Gieson elastin staining of abdominal aortic sections showing preserved aortic structure in Setdb2^−/− M𝛷 + AngII compared with WT M𝛷 + AngII mice; scale bar is 200 μm in Verhoeff–van Gieson stain; arrows represent elastin fragmentation. Average number of elastin fragmentation per high power field. F. ChIP analysis for H3K9me3 at Timp1, Timp2, and Timp3 promoter was performed on macrophages (CD11b⁺[CD3⁻CD19⁻Nk1.1⁻Ly6G⁻]) isolated from AngII + WT M𝛷 and Setdb2^−/− M𝛷 at day 28 (n=5 mice/group pooled and run in triplicate). For all ChIP experiments, isotype-matched IgG was run in parallel. Dotted line represents isotype-matched control. **p<0.01 for Mann-Whitney U test. G. Timp1, Timp2, and Timp3 expression were measured by quantitative PCR in in vivo M𝛷s isolated from WT M𝛷 or Setdb2^−/− M𝛷 following AngII infused mice on day 28 (n=3/group run in triplicate). *p<0.05, **p<0.01 by Mann-Whitney U Test. H. MMP activity was measured in protein abdominal aorta extracts by fluorometry and measured in fluorometric units. Data represent the mean±SEM from n=6 animals per group. Statistical analysis was performed by 1-way ANOVA (Newman-Keuls post hoc test). *p<0.05. I. MMP activity in abdominal aortic sections by in situ zymography. Representative images are shown with green signal corresponding to active MMPs; blue signal corresponding to DAPI staining for cell nuclei.

Figure 4.. IFNβ/JAK/STAT3 pathway induced Setdb2/H3K9me3 on Timp Promoters in Macrophages.

A. Setdb2 expression in BMDM treated ex vivo with IFNβ for 8 hrs (n = 6 mice per group). B. ChIP analysis of BMDMs from controls treated ex vivo with IFNβ (100U) for 6 hrs and analyzed for H3K9me3 at the NF-κB binding site on the Timp1, Timp2, and Timp3 promoter (n = 6 mice per group). C. Timp1, Timp2, and Timp3 expression in BMDM treated ex vivo with IFNβ for 8 hrs (n = 6 mice per group). *p<0.05 D. Quantitative PCR analysis of Setdb2 was conducted in macrophages (MΦ) (CD11b⁺[CD3⁻CD19⁻Nk1.1⁻Ly6G⁻]) isolated from Ifnar^−/− and littermate controls (Ifnar^+/+) (n = 3 mice/group run in triplciated). **p<0.01 for Mann-Whitney U test. E. Setdb2 expression in BMDMs treated with IFNβ +/− tofacitinib (JAK inhibitor; 50 nM) (n = 3 mice per group). **p<0.01 for Mann-Whitney U test. F. Quantitative PCR analysis of Setdb2 was conducted in macrophages (MΦ) (CD11b⁺[CD3⁻CD19⁻Nk1.1⁻Ly6G⁻]) isolated from Stat3^f/fLyz2^Cre- and littermate controls (Stat3^f/fLyz2^Cre+) (n = 3 mice/group run in triplciated). **p<0.01 for Mann-Whitney U test.

Figure 5.

Schematic of SETDB2-mediated regulation of macrophage Timp expression and MMP during AAA development.