Faculty Bibliography

Atherosclerosis is characterized by the pathological accumulation of cholesterol-laden macrophages in the arterial wall. Atherosclerosis is also the main underlying cause of cardiovascular diseases (CVDs), and its development is largely driven by elevated plasma cholesterol. Strong epidemiological data find an inverse association between plasma β-carotene with atherosclerosis, and we recently showed that β-carotene oxygenase 1 (BCO1) activity, responsible for β-carotene cleavage to vitamin A, is associated with reduced plasma cholesterol in humans and mice. In this study, we explore whether intact β-carotene or vitamin A affect atherosclerosis progression in the atheroprone low-density lipoprotein receptor (LDLR) - deficient mice. In comparison to control-fed Ldlr-/- mice, β-carotene-supplemented mice showed reduced atherosclerotic lesion size at the level of the aortic root and reduced plasma cholesterol levels. These changes were absent in Ldlr-/-/Bco1-/- mice, despite accumulating β-carotene in plasma and atherosclerotic lesions. We discarded the implication of myeloid BCO1 in the development of atherosclerosis by performing bone marrow transplant experiments. Lipid production assays found that retinoic acid, the active form of vitamin A, reduced the secretion of newly synthetized triglyceride and cholesteryl ester in cell culture and mice. Overall, our findings provide insights into the role of BCO1 activity and vitamin A in atherosclerosis progression through the regulation of hepatic lipid metabolism.

Rationale: Treatment efficacy for diabetes is largely determined by assessment of HbA1c levels, which poorly reflects direct glucose variation. People with pre-diabetes and diabetes spend >50% of their time outside the optimal glucose range. These glucose variations, termed transient intermittent hyperglycemia (TIH) appear to be an independent risk-factor for cardiovascular disease (CVD) but the pathological basis for this association is unclear. Objective: To determine whether TIH per se promotes myelopoiesis to produce more monocytes and consequently adversely affects atherosclerosis. Methods and Results: To create a mouse model of TIH we administered 4 bolus doses of glucose at 2hr intervals intraperitoneally once to wild-type (WT) or once weekly to atherosclerotic prone mice. TIH accelerated atherogenesis without an increase in plasma cholesterol, seen in traditional models of diabetes. TIH promoted myelopoiesis in the bone marrow, resulting in increased circulating monocytes, particularly the inflammatory Ly6-C^hi subset, and neutrophils. Hematopoietic-restricted deletion of S100a9, S100a8 or its cognate receptor Rage, prevented monocytosis. Mechanistically, glucose uptake via GLUT-1 and enhanced glycolysis in neutrophils promoted the production of S100A8/A9. Myeloid-restricted deletion of Slc2a1 (GLUT-1) or pharmacological inhibition of S100A8/A9 reduced TIH-induced myelopoiesis and atherosclerosis. Conclusions: Together, these data provide a mechanism as to how TIH, prevalent in people with impaired glucose metabolism, contributes to CVD. These findings provide a rationale for continual glucose control in these patients and may also suggest that strategies aimed at targeting the S100A8/A9-RAGE axis could represent a viable approach to protect the vulnerable blood vessels in diabetes.

Disruption of systemic homeostasis by either chronic or acute stressors, such as obesity¹ or surgery², alters cancer pathogenesis. Patients with cancer, particularly those with breast cancer, can be at increased risk of cardiovascular disease due to treatment toxicity and changes in lifestyle behaviors^3-5. While elevated risk and incidence of cardiovascular events in breast cancer is well established, whether such events impact cancer pathogenesis is not known. Here we show that myocardial infarction (MI) accelerates breast cancer outgrowth and cancer-specific mortality in mice and humans. In mouse models of breast cancer, MI epigenetically reprogrammed Ly6C^hi monocytes in the bone marrow reservoir to an immunosuppressive phenotype that was maintained at the transcriptional level in monocytes in both the circulation and tumor. In parallel, MI increased circulating Ly6C^hi monocyte levels and recruitment to tumors and depletion of these cells abrogated MI-induced tumor growth. Furthermore, patients with early-stage breast cancer who experienced cardiovascular events after cancer diagnosis had increased risk of recurrence and cancer-specific death. These preclinical and clinical results demonstrate that MI induces alterations in systemic homeostasis, triggering cross-disease communication that accelerates breast cancer.

Despite advances in lipid-lowering therapies, people with diabetes continue to experience more limited cardiovascular benefits. In diabetes, hyperglycemia sustains inflammation and preempts vascular repair. We tested the hypothesis that the receptor for advanced glycation end-products (RAGE) contributes to these maladaptive processes. We report that transplantation of aortic arches from diabetic, Western diet-fed Ldlr-/- mice into diabetic Ager-/- (Ager, the gene encoding RAGE) versus WT diabetic recipient mice accelerated regression of atherosclerosis. RNA-sequencing experiments traced RAGE-dependent mechanisms principally to the recipient macrophages and linked RAGE to interferon signaling. Specifically, deletion of Ager in the regressing diabetic plaques downregulated interferon regulatory factor 7 (Irf7) in macrophages. Immunohistochemistry studies colocalized IRF7 and macrophages in both murine and human atherosclerotic plaques. In bone marrow-derived macrophages (BMDMs), RAGE ligands upregulated expression of Irf7, and in BMDMs immersed in a cholesterol-rich environment, knockdown of Irf7 triggered a switch from pro- to antiinflammatory gene expression and regulated a host of genes linked to cholesterol efflux and homeostasis. Collectively, this work adds a new dimension to the immunometabolic sphere of perturbations that impair regression of established diabetic atherosclerosis and suggests that targeting RAGE and IRF7 may facilitate vascular repair in diabetes.

Introduction & Objectives: AUA, EAU and NICE now recommend multiparametric (mp)MRI prior to all prostate biopsies, including initial. This creates a massive increase in demand on the limited resource of access to MRI. Biparametric (bp)MRI, without dynamic contrast enhancement (DCE) series, is rapidly gaining interest as a faster, cheaper, less invasive way of performing prostate MRI with the goal of maintaining diagnostic accuracy. Using an online training tool, the global BooMeR Study assesses accuracy and variation in reading of bpMRI.
Material(s) and Method(s): A free bpMRI version of the online mpMRI training program MRI PRO (prostatemristudy.com) was promoted via email, Twitter and LinkedIn from August to October 2019 to target radiologists and urologists around the world. MRI PRO is an interactive program which matches 300 prostate mpMRIs acquired and reported to PIRADS v.2 standard to wholemount radical prostatectomy in positive MRIs and template transperineal biopsy histology for negative MRIs. The bpMRI version matches 50 cases, without any DCE series. We designated true PIRADS 4 or 5 as true positive and true PIRADS 1 or 2 as true negative. True PIRADS 3 (equivocal) cases were excluded from analysis. MRI PRO's proprietary analysis tool was used to compare users' responses.
Result(s): 59 prostate MRI readers registered for the study. 33% were radiologists, 67% were urologists and all respondents were consultants or fellows. 59%, 12%, 9%, 20% were from Europe, Asia-Pacific, North America, or elsewhere, respectively. 33% had previously read over 100 prostate mpMRIs. A total of 1,090 cases were completed, for a mean of 18.4 cases per reader. 15 readers completed all 50 cases. The overall specificity and sensitivity was 78% and 71% respectively. Cohorts of experienced vs inexperienced readers were then compared where readers who had performed more than 4 cases were included. Readers with over 100 previous cases of experience [n=13] had specificity of 77.7% (95% CI 70.9 - 84.4) and sensitivity of 77.2% (95% CI 70.2, 84.2). Readers with less than 100 previous cases of experience [n=18] had a specificity of 53.8%, 95% CI [41.9%, 65.7%] and sensitivity of 64.4%, 95% CI [59.9%, 68.8%]. The difference in sensitivity (p=0.044) and specificity (p=0.003) between the two cohorts were statistically significant). PIRADS 3 reporting was 3.9% vs 8.2% in the experienced vs inexperienced groups.
Conclusion(s): Preliminary BooMeR Study results suggest variation in bpMRI reporting accuracy and likelihood of reporting PIRADS 3 are associated with reader experience. Adequate training and quality assurance in reporting bpMRI is essential.
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Background: Platelets are effector cells of the innate and adaptive immune system; however, understanding their role during inflammation can be challenging due to drawbacks associated with current platelet depletion methods. The generation of antisense oligonucleotides (ASO) directed to thrombopoietin (Thpo) mRNA represents a novel method to reduce platelet count to aid in elucidating the role of platelets during inflammation.
Aim(s): To understand if Thpo-targeted ASOs represent a viable strategy to reduce platelet count in mice, and to delineate the role of platelets to inflammation resolution during lower airway dysbiosis.
Method(s): Mice were treated with a Thpo-targeted ASO and the abundance of platelets, blood cells and bone marrow hematopoietic progenitor cells assessed. Additionally, platelet responsiveness to agonists and surface expression of P-selection and JON/A was measured. To assess the contribution of platelets to inflammation resolution Thpo-ASO and control-ASO treated mice were challenged with a bacteria cocktail to model lower airway dysbiosis. Thpo-ASO mediated platelet depletion was compared to anti-CD42b platelet depletion in the lung dysbiosis model.
Result(s): ASO-mediated silencing of hepatic Thpo reduces platelet, megakaryocyte, and megakaryocyte progenitor count by 50% relative to control ASO treated mice. Thpo-ASO treatment does not alter platelet reactivity to agonists, or platelet size. Following bacterial inoculation, we found a significant increase in lung platelet-leukocyte aggregates and consistent with a response to inflammation an increase in lung Ly6C^hi monocytes and macrophages in inoculated mice. Platelet depletion, by either Thpo-ASO or anti-CD42b treatment, reduces the accumulation of lung inflammatory immune cells, including monocytes (Ly6C^hi) and macrophages (CD45⁺CD11b⁺F4/80⁺). Furthermore, we found that in contrast to anti-CD42b platelet depletion, Thpo-ASO mediated depletion allows for introduction of new platelets.
Conclusion(s): Thpo ASO-mediated platelet depletion represents a viable approach to reduce platelet count. Platelet count directly impacts lung inflammation resolution during lower airway dysbiosis demonstrating the essential role of platelets in pulmonary immune defense

BACKGROUND:Platelets are effector cells of the innate and adaptive immune system, however understanding their role during inflammation-driven pathologies can be challenging due to several drawbacks associated with current platelet depletion methods. The generation of antisense oligonucleotides (ASO)s directed to thrombopoietin (Tpo) mRNA represents a novel method to reduce circulating platelet count. OBJECTIVE:To understand if Tpo-targeted ASO treatment represents a viable strategy to specifically reduce platelet count in mice. METHODS:Female and male mice were treated with TPO-targeted ASOs and platelet count and function assessed, in addition to circulating blood cell counts and hematopoietic stem and progenitor cells. The utility of the platelet-depletion strategy was assessed in a murine model of lower airway dysbiosis. RESULTS AND CONCLUSIONS/CONCLUSIONS:Herein, we describe how in mice, ASO-mediated silencing of hepatic TPO expression reduces platelet, megakaryocyte, and megakaryocyte progenitor count, without altering platelet activity. TPO ASO-mediated platelet depletion can be achieved acutely and sustained chronically in the absence of adverse bleeding. TPO ASO-mediated platelet depletion allows for the reintroduction of new platelets, an advantage over commonly used antibody-mediated depletion strategies. Using a murine model of lung inflammation, we demonstrate that platelet depletion, induced by either TPO ASO or anti-CD42b treatment, reduces the accumulation of inflammatory immune cells, including monocytes and macrophages, in the lung. Altogether, we characterize a new platelet depletion method that can be sustained chronically and allows for the reintroduction of new platelets highlighting the utility of the TPO ASO method to understand the role of platelets during chronic immune-driven pathologies.

Background: COVID-19 is a global pandemic with patients at increased risk for thrombosis. Platelets are central protagonists of thrombosis, and virus-platelet interactions are linked to viral pathogenesis and increased thrombotic risk.
Aim(s): To investigate the relationship between in vivo platelet activity markers, and thrombosis or death in hospitalized patients with COVID-19.
Method(s): Plasma samples were collected from 100 hospitalized patients on the day of PCR-confirmed COVID-19 diagnosis. Thromboxane B2 (TxB2), P-selectin (P-selectin), and soluble CD40 ligand (sCD40L) were measured in plasma, and mean platelet volume (MPV) assessed. Subjects were followed until discharge or death, and thrombotic events recorded.
Result(s): Among 100 patients, the median age was 65 years (IQR: 55, 75), 39% were female, and 32 died or experienced a thrombotic event. Baseline platelet activation markers were higher in patients who developed an adverse clinical event. After adjustment for age, sex, race/ethnicity, platelet count, antiplatelet therapy, and chronic obstructive pulmonary disease, TxB2 (p = 0.006), P-selectin (p = 0.005), sCD40L (p = 0.016), and MPV (p = 0.012) were independentlyassociated with thrombosis or death (Table 1). Correlation analysis between biomarkers identified that TxB2 is correlated with P-selectin (rho=0.42, p < 0.0001) and platelet count (rho=0.35, p = 0.0004), MPV is correlated with platelet count (rho=-0.31, p = 0.002), and P-selectin is correlated with sCD40L (rho=0.67, p < 0.0001).
Conclusion(s): Biomarkers of platelet activation are significantly associated with death or thrombosis in patients hospitalized with COVID-19. Our findings suggest multiple platelet activation mechanisms may contribute to adverse events. Further investigation into the mechanistic role of platelets in COVID-19 pathogenesis and the potential role of antiplatelet therapy is warranted