Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Artery Disease.

Monocytes are associated with human cardiovascular disease progression. Monocytes are segregated into three major subsets: classical (cMo), intermediate (iMo), and nonclassical (nMo). Recent studies have identified heterogeneity within each of these main monocyte classes, yet the extent to which these subsets contribute to heart disease progression is not known. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects within the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using the Gensini Score (GS). We employed high-dimensional single-cell transcriptome and protein methods to define how human monocytes differ in subjects with low to severe coronary artery disease. We analyzed 487 immune-related genes and 49 surface proteins at the single-cell level using Antibody-Seq (Ab-Seq). We identified six subsets of myeloid cells (cMo, iMo, nMo, plasmacytoid DC, classical DC, and DC3) at the single-cell level based on surface proteins, and we associated these subsets with coronary artery disease (CAD) incidence based on Gensini score (GS) in each subject. Only frequencies of iMo were associated with high CAD (GS > 32), adj.p = 0.024. Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r = 0.314, p = 0.014) and further showed a robust sex-dependent positive correlation in female subjects (r = 0.663, p = 0.004). cMo frequencies did not correlate with CAD severity. Key gene pathways differed in iMo among low and high CAD subjects and between males and females. Further single-cell analysis of iMo revealed three iMo subsets in human PBMC, distinguished by the expression of HLA-DR, CXCR3, and CD206. We found that the frequency of immunoregulatory iMo_HLA-DR+CXCR3+CD206+ was associated with CAD severity (adj.p = 0.006). The immunoregulatory iMo subset positively correlated with GS in both females (r = 0.660, p = 0.004) and males (r = 0.315, p = 0.037). Cell interaction analyses identified strong interactions of iMo with CD4+ effector/memory T cells and Tregs from the same subjects. This study shows the importance of iMo in CAD progression and suggests that iMo may have important functional roles in modulating CAD risk, particularly among females.

Sex-specific effects of injury and beta-adrenergic activation on metabolic and inflammatory mediators in a murine model of post-traumatic osteoarthritis.

OBJECTIVE: Metabolic processes are intricately linked to the resolution of innate inflammation and tissue repair, two critical steps for treating post-traumatic osteoarthritis (PTOA). Based on lipolytic and immunoregulatory actions of norepinephrine, we hypothesized that intra-articular ß-adrenergic receptor (ßAR) stimulation would suppress PTOA-associated inflammation in the infrapatellar fat pad (IFP) and synovium. DESIGN: We used the ßAR agonist isoproterenol to perturb intra-articular metabolism 3.5 weeks after applying a non-invasive single-load compression injury to knees of 12-week-old male and female mice. We examined the acute effects of intra-articular isoproterenol treatment relative to saline on IFP histology, multiplex gene expression of synovium-IFP tissue, synovial fluid metabolomics, and mechanical allodynia. RESULTS: Injured knees developed PTOA pathology characterized by heterotopic ossification, articular cartilage loss, and IFP atrophy and fibrosis. Isoproterenol suppressed the upregulation of pro-fibrotic genes and downregulated the expression of adipose genes and pro-inflammatory genes (Adam17, Cd14, Icam1, Csf1r, and Casp1) in injured joints of female (but not male) mice. Analysis of published single-cell RNA-seq data identified elevated catecholamine-associated gene expression in resident-like synovial-IFP macrophages after injury. Injury substantially altered synovial fluid metabolites by increasing amino acids, peptides, sphingolipids, phospholipids, bile acids, and dicarboxylic acids, but these changes were not appreciably altered by isoproterenol. Intra-articular injection of either isoproterenol or saline increased mechanical allodynia in female mice, whereas neither substance affected male mice. CONCLUSIONS: Acute ßAR activation altered synovial-IFP transcription in a sex and injury-dependent manner, suggesting that women with PTOA may be more sensitive than men to treatments targeting sympathetic neural signaling pathways.

Mycobacterium bovis Bacille-Calmette-Guérin Infection Aggravates Atherosclerosis.

Tuberculosis has been associated with increased risk of atherosclerotic cardiovascular disease. To examine whether mycobacterial infection exacerbates atherosclerosis development in experimental conditions, we infected low-density lipoprotein receptor knockout (Ldlr-/-) mice with Mycobacterium bovis Bacille-Calmette-Guérin (BCG), an attenuated strain of the Mycobacterium tuberculosis complex. Twelve-week old male Ldlr-/- mice were infected with BCG (0.3-3.0x106 colony-forming units) via the intranasal route. Mice were subsequently fed a western-type diet containing 21% fat and 0.2% cholesterol for up to 16 weeks. Age-matched uninfected Ldlr-/- mice fed with an identical diet served as controls. Atherosclerotic lesions in aorta were examined using Oil Red O staining. Changes induced by BCG infection on the immunophenotyping profile of circulating T lymphocytes and monocytes were assessed using flow cytometry. BCG infection increased atherosclerotic lesions in en face aorta after 8 weeks (plaque ratio; 0.021±0.01 vs. 0.013±0.01; p = 0.011) and 16 weeks (plaque ratio, 0.15±0.13 vs. 0.06±0.02; p = 0.003). No significant differences in plasma cholesterol or triglyceride levels were observed between infected and uninfected mice. Compared to uninfected mice, BCG infection increased systemic CD4/CD8 T cell ratio and the proportion of Ly6Clow non-classical monocytes at weeks 8 and 16. Aortic plaque ratios correlated with CD4/CD8 T cell ratios (Spearman's rho = 0.498; p = 0.001) and the proportion of Ly6Clow non-classical monocytes (Spearman's rho = 0.629; p < 0.001) at week 16. In conclusion, BCG infection expanded the proportion of CD4+ T cell and Ly6Clow monocytes, and aggravated atherosclerosis formation in the aortas of hyperlipidemic Ldlr-/- mice. Our results indicate that mycobacterial infection is capable of enhancing atherosclerosis development.

ApoER2 (Apolipoprotein E Receptor-2) Deficiency Accelerates Smooth Muscle Cell Senescence via Cytokinesis Impairment and Promotes Fibrotic Neointima After Vascular Injury.

OBJECTIVE: Genome-wide studies showed that mutation in apoER2 (apolipoprotein E receptor-2) is additive with ε4 polymorphism in the APOE gene on cardiovascular disease risk in humans. ApoE or apoER2 deficiency also accelerates atherosclerosis lesion necrosis in hypercholesterolemic mice and promotes neointima formation after vascular injury. This study tests the hypothesis that apoE and apoER2 modulate vascular occlusive diseases through distinct mechanisms. Approach and Results: Carotid endothelial denudation induced robust neointima formation in both apoE-/- and apoER2-deficient Lrp8-/- mice. The intima in apoE-/- mice was rich in smooth muscle cells, but the intima in Lrp8-/- mice was cell-poor and rich in extracellular matrix. Vascular smooth muscle cells isolated from apoE-/- mice were hyperplastic whereas Lrp8-/- smooth muscle cells showed reduced proliferation but responded robustly to TGF (transforming growth factor)-ß-induced fibronectin synthesis indicative of a senescence-associated secretory phenotype, which was confirmed by increased ß-galactosidase activity, p16INK4a immunofluorescence, and number of multinucleated cells. Western blot analysis of cell cycle-associated proteins showed that apoER2 deficiency promotes cell cycle arrest at the metaphase/anaphase. Coimmunoprecipitation experiments revealed that apoER2 interacts with the catalytic subunit of protein phosphatase 2A. In the absence of apoER2, PP2A-C (protein phosphatase 2A catalytic subunit) failed to interact with CDC20 (cell-division cycle protein 20) thus resulting in inactive anaphase-promoting complex and impaired cell cycle exit. CONCLUSIONS: This study showed that apoER2 participates in APC (anaphase-promoting complex)/CDC20 complex formation during mitosis, and its absence impedes cytokinesis abscission thereby accelerating premature cell senescence and vascular disease. This mechanism is distinct from apoE deficiency, which causes smooth muscle cell hyperplasia to accelerate vascular disease.

Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

AIMS: The MEK5/Erk5 pathway mediates beneficial effects of laminar flow, a major physiological factor preventing vascular dysfunction. Forced Erk5 activation induces a protective phenotype in endothelial cell (EC) that is associated with a dramatically decreased migration capacity of those cells. Transcriptional profiling identified the Krüppel-like transcription factors KLF2 and KLF4 as central mediators of Erk5-dependent gene expression. However, their downstream role regarding migration is unclear and relevant secondary effectors remain elusive. Here, we further investigated the mechanism underlying Erk5-dependent migration arrest in ECs. METHODS AND RESULTS: Our experiments reveal KLF2-dependent loss of the pro-migratory Rac/Cdc42 mediator, p21-activated kinase 1 (PAK1), as an important mechanism of Erk5-induced migration inhibition. We show that endothelial Erk5 activation by expression of a constitutively active MEK5 mutant, by statin treatment, or by application of laminar shear stress strongly decreased PAK1 mRNA and protein expression. Knockdown of KLF2 but not of KLF4 prevented Erk5-mediated PAK1 mRNA inhibition, revealing KLF2 as a novel PAK1 repressor in ECs. Importantly, both PAK1 re-expression and KLF2 knockdown restored the migration capacity of Erk5-activated ECs underscoring their functional relevance downstream of Erk5. CONCLUSION: Our data provide first evidence for existence of a previously unknown Erk5/KLF2/PAK1 axis, which may limit undesired cell migration in unperturbed endothelium and lower its sensitivity for migratory cues that promote vascular diseases including atherosclerosis.