Faculty Bibliography

Platelets are best known as mediators of hemostasis and thrombosis; however, their inflammatory effector properties are increasingly recognized. Atherosclerosis, a chronic vascular inflammatory disease, represents the interplay between lipid deposition in the artery wall and unresolved inflammation. Here, we reveal that platelets induce monocyte migration and recruitment into atherosclerotic plaques, resulting in plaque platelet-macrophage aggregates. In Ldlr^-/- mice fed a Western diet, platelet depletion decreased plaque size and necrotic area and attenuated macrophage accumulation. Platelets drive atherogenesis by skewing plaque macrophages to an inflammatory phenotype, increasing myeloid suppressor of cytokine signaling 3 (SOCS3) expression and reducing the Socs1:Socs3 ratio. Platelet-induced Socs3 expression regulates plaque macrophage reprogramming by promoting inflammatory cytokine production (Il6, Il1b, and Tnfa) and impairing phagocytic capacity, dysfunctions that contribute to unresolved inflammation and sustained plaque growth. Translating our data to humans with cardiovascular disease, we found that women with, versus without, myocardial infarction have up-regulation of SOCS3, lower SOCS1:SOCS3, and increased monocyte-platelet aggregate. A second cohort of patients with lower extremity atherosclerosis demonstrated that SOCS3 and the SOCS1:SOCS3 ratio correlated with platelet activity and inflammation. Collectively, these data provide a causative link between platelet-mediated myeloid inflammation and dysfunction, SOCS3, and cardiovascular disease. Our findings define an atherogenic role of platelets and highlight how, in the absence of thrombosis, platelets contribute to inflammation.

BACKGROUND:Despite robust cholesterol lowering, cardiovascular disease risk remains increased in patients with diabetes mellitus. Consistent with this, diabetes mellitus impairs atherosclerosis regression after cholesterol lowering in humans and mice. In mice, this is attributed in part to hyperglycemia-induced monocytosis, which increases monocyte entry into plaques despite cholesterol lowering. In addition, diabetes mellitus skews plaque macrophages toward an atherogenic inflammatory M1 phenotype instead of toward the atherosclerosis-resolving M2 state typical with cholesterol lowering. Functional high-density lipoprotein (HDL), typically low in patients with diabetes mellitus, reduces monocyte precursor proliferation in murine bone marrow and has anti-inflammatory effects on human and murine macrophages. Our study aimed to test whether raising functional HDL levels in diabetic mice prevents monocytosis, reduces the quantity and inflammation of plaque macrophages, and enhances atherosclerosis regression after cholesterol lowering. METHODS:mice were transplanted into either wild-type, diabetic wild-type, or diabetic mice transgenic for human apolipoprotein AI, which have elevated functional HDL. Recipient mice all had low levels of low-density lipoprotein cholesterol to promote plaque regression. After 2 weeks, plaques in recipient mouse aortic grafts were examined. RESULTS:Diabetic wild-type mice had impaired atherosclerosis regression, which was normalized by raising HDL levels. This benefit was linked to suppressed hyperglycemia-driven myelopoiesis, monocytosis, and neutrophilia. Increased HDL improved cholesterol efflux from bone marrow progenitors, suppressing their proliferation and monocyte and neutrophil production capacity. In addition to reducing circulating monocytes available for recruitment into plaques, in the diabetic milieu, HDL suppressed the general recruitability of monocytes to inflammatory sites and promoted plaque macrophage polarization to the M2, atherosclerosis-resolving state. There was also a decrease in plaque neutrophil extracellular traps, which are atherogenic and increased by diabetes mellitus. CONCLUSIONS:Raising apolipoprotein AI and functional levels of HDL promotes multiple favorable changes in the production of monocytes and neutrophils and in the inflammatory environment of atherosclerotic plaques of diabetic mice after cholesterol lowering and may represent a novel approach to reduce cardiovascular disease risk in people with diabetes mellitus.

Background: Women with early-stage breast cancer are at excess risk of cardiovascular disease (CVD) due to deleterious therapy-induced direct as well as indirect perturbations across the entire cardiovascular system. CVD events such as acute myocardial infarction (AMI) induce a systemic (host) inflammatory response that accelerates underlying atherosclerotic disease. Whether an AMIinduced systemic response affects cancer progression is not known.
Method(s): In a prospective case cohort study, we evaluated the relationship between a new onset, post-diagnosis CVD event (e.g., AMI, stroke, heart failure) and recurrence in 3802 patients with early-stage breast cancer. To assess causality, we tested the effects of surgically-induced AMI on breast cancer progression and metastasis in mouse models of breast cancer.
Result(s): A new onset CVD event was associated with increased risk of recurrence compared to patients not experiencing an event (HR: 1.69; 95% confidence interval, 1.15 to 2.50). In preclinical models, surgically-induced AMI significantly accelerated tumor growth compared to sham surgery controls (p<0.001), as well as metastatic burden (p<0.05). Tumors of AMI mice had an altered tumor microenvironment and tumor immune cell landscape, driven by the increased availability, recruitment, intratumoral accumulation and immunosuppressive phenotype of CD11b⁺ Ly6C^high myeloid cells.
Conclusion(s): A CVD event accelerates breast cancer progression in humans and mice. These data provide new mechanistic insight into cross-disease communication as a mediator of breast cancer pathogenesis

OBJECTIVE:. In adipose, less macrophage lipid accumulation was found with global but not myeloid-specific LpL deficiency. Neither deletion affected the expression of inflammatory genes. Global LpL deficiency also reduced the numbers of elicited peritoneal macrophages. Finally, we assessed gene expression in macrophages from atherosclerotic lesions during regression; LpL deficiency did not affect the polarity of plaque macrophages. CONCLUSIONS:The phenotypic changes observed in macrophages upon deletion of Lpl in vitro is not mimicked in tissue macrophages.

Macrophages accumulate prominently in the visceral adipose tissue (VAT) of obese humans and high fat diet (HFD) fed mice, and this is linked to insulin resistance and type II diabetes. While the mechanisms regulating macrophage recruitment in obesity have been delineated, the signals directing macrophage persistence in VAT are poorly understood. We previously showed that the neuroimmune guidance cue netrin-1 is expressed in the VAT of obese mice and humans, where it promotes macrophage accumulation. To better understand the source of netrin-1 and its effects on adipose tissue macrophage (ATM) fate and function in obesity, we generated mice with myeloid-specific deletion of netrin-1 (Ntn1^fl/flLysMCre^+/-; Ntn1^Δmac). Interestingly, Ntn1^Δmac mice showed a modest decrease in HFD-induced adiposity and adipocyte size, in the absence of changes in food intake or leptin, that was accompanied by an increase in markers of adipocyte beiging (Prdm16, UCP-1). Using single cell RNA-seq, combined with conventional histological and flow cytometry techniques, we show that myeloid-specific deletion of netrin-1 caused a 50% attrition of ATMs in HFD-fed mice, particularly of the resident macrophage subset, and altered the phenotype of residual ATMs to enhance lipid handling. Pseudotime analysis of single cell transcriptomes showed that in the absence of netrin-1, macrophages in the obese VAT underwent a phenotypic switch with the majority of ATMs activating a program of genes specialized in lipid handling, including fatty acid uptake and intracellular transport, lipid droplet formation and lipolysis, and regulation of lipid localization. Furthermore, Ntn1^Δmac macrophages had reduced expression of genes involved in arachidonic acid metabolism, and targeted LCMS/MS metabololipidomics analysis revealed decreases in proinflammatory eicosanoids (5-HETE, 6-trans LTB₄, TXB₂, PGD₂) in the obese VAT. Collectively, our data show that targeted deletion of netrin-1 in macrophages reprograms the ATM phenotype in obesity, leading to reduced adipose inflammation, and improved lipid handling and metabolic function.

Nanomedicine research produces hundreds of studies every year, yet very few formulations have been approved for clinical use. This is due in part to a reliance on murine studies, which have limited value in accurately predicting translational efficacy in larger animal models and humans. Here, we report the scale-up of a nanoimmunotherapy from mouse to large rabbit and porcine atherosclerosis models, with an emphasis on the solutions we implemented to overcome production and evaluation challenges. Specifically, we integrated translational imaging readouts within our workflow to both analyze the nanoimmunotherapeutic's in vivo behavior and assess treatment response in larger animals. We observed our nanoimmunotherapeutic's anti-inflammatory efficacy in mice, as well as rabbits and pigs. Nanoimmunotherapy-mediated reduction of inflammation in the large animal models halted plaque progression, supporting the approach's translatability and potential to acutely treat atherosclerosis.

Obesity can lead to type 2 diabetes and is an epidemic. A major contributor to its adverse effects is inflammation of the visceral adipose tissue (VAT). Life-long caloric restriction (CR), in contrast, results in extended lifespan, enhanced glucose tolerance/insulin sensitivity, and other favorable phenotypes. The effects of CR following obesity are incompletely established, but studies show multiple benefits. Many leukocyte types, macrophages predominantly, reside in VAT in homeostatic and pathological states. CR following obesity transiently increases VAT macrophage content prior to resolution of inflammation and obesity, suggesting that macrophage content and phenotype play critical roles. Here, we examined the heterogeneity of VAT leukocytes and the effects of obesity and CR. In general, our single-cell RNA-sequencing data demonstrate that macrophages are the most abundant and diverse subpopulation of leukocytes in VAT. Obesity induced significant transcriptional changes in all 15 leukocyte subpopulations, with many genes showing coordinated changes in expression across the leukocyte subpopulations. Additionally, obese VAT displayed expansion of one major macrophage subpopulation, which, in silico, was enriched in lipid binding and metabolic processes. This subpopulation returned from dominance in obesity to lean proportions after only 2 weeks of CR, although the pattern of gene expression overall remained similar. Surprisingly, CR VAT is dominated by a different macrophage subpopulation, which is absent in lean conditions. This subpopulation is enriched in genes related to phagocytosis and we postulate that its function includes clearance of dead cells, as well as excess lipids, contributing to limiting VAT inflammation and restoration of the homeostatic state.

BACKGROUND:While progress in the prevention of cardiovascular disease (CVD) has been noted over the past several decades, there are still those who develop CVD earlier in life than others. OBJECTIVE:We investigated traditional and lifestyle CVD risk factors in young to middle-aged patients compared to older ones with obstructive coronary artery disease (CAD). METHODS:A retrospective analysis of patients with a new diagnosis of obstructive CAD undergoing coronary intervention was performed. Young to middle-aged patients were defined as those in the youngest quartile (n = 281, mean age 50 ± 6 years, 81% male) compared to the other three older quartiles combined (n = 799, mean age 69 ± 7.5 years, 71% male). Obstructive CAD was determined by angiography. RESULTS:Young to middle-aged patients compared to older ones were more likely to be male (p < 0.01), smokers (21 vs. 9%, p < 0.001), and have a higher body mass index (31 ± 6 vs. 29 ± 6 kg/m2, p < 0.001). Younger patients were less likely to eat fruits, vegetables, and fish and had fewer controlled CVD risk factors (2.7 ± 1.2 vs. 3.0 ± 1.0, p < 0.001). Compared to older patients, higher levels of psychological stress (aOR 1.6, 95% CI 1.1-2.4), financial stress (aOR 1.8, 95% CI 1.3-2.5), and low functional capacity (aOR 3.3, 95% CI 2.4-4.5) were noted in the young to middle-aged population as well. CONCLUSION/CONCLUSIONS:Lifestyle in addition to traditional CVD risk factors should be taken into account when evaluating risk for development of CVD in a younger population.