Faculty Bibliography

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

Neutrophil extracellular traps (NETs) promote inflammation and atherosclerosis progression. NETs are increased in diabetes and impair the resolution of inflammation during wound healing. Atherosclerosis resolution, a process resembling wound healing, is also impaired in diabetes. Thus, we hypothesized that NETs impede atherosclerosis resolution in diabetes by increasing plaque inflammation. Indeed, transcriptomic profiling of plaque macrophages from NET positive and negative areas in low-density lipoprotein receptor-deficient (Ldlr-/-) mice revealed inflammasome and glycolysis pathway upregulation, indicating a heightened inflammatory phenotype. We found that NETs decline during atherosclerosis resolution, which was induced by reducing hyperlipidemia in non-diabetic mice, but they persist in diabetes, exacerbating macrophage inflammation and impairing resolution. In diabetic mice deoxyribonuclease 1 (DNase1) treatment reduced plaque NETs content and macrophage inflammation, promoting atherosclerosis resolution after lipid-lowering. Given that humans with diabetes also exhibit impaired atherosclerosis resolution with lipid-lowering, these data suggest that NETs contribute to the increased cardiovascular disease risk in this population and are a potential therapeutic target.

Background Psoriasis is an inflammatory disease of the skin associated with heightened cardiovascular (CV) disease. Serum levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) associates with future CV risk and vascular dysfunction. We aimed to identify the relationship between pro-inflammatory pathways, circulating PCSK9, and vascular health in psoriasis. Methods Whole blood transcriptomics and serum proteomics was performed in 20 patients with psoriasis (mean age 42 +/- 14 years, 55% male, psoriasis area and severity index [PASI] 5 [3 - 11]) and 15 controls (mean age 41 +/- 14 years, 53% male) recruited into a clinical trial to assess vascular health in psoriasis (NCT03228017). Vascular health was assessed through flow mediated dilatation (FMD) and harvesting and analysis of brachial vein endothelial cells. Results Circulating PCSK9 was found to be 1.13-fold higher in psoriasis compared to controls (p=0.02) despite no difference in LDL-C (108 +/- 38 mg/dl vs. 90 +/- 25 mg/dl, respectively p=0.31). Circulating PCSK9 was correlated with psoriasis area severity index (PASI score, r=0.43, p=0.04) even after adjustment for age, gender, BMI and LDL-C (beta=0.02, p=0.03). Integration of the whole blood transcriptome yielded 322 transcripts which correlated with circulating PCSK9 (FDR<0.05). Network analysis of these transcripts highlighted interferon signaling (p=7.2x10^-6), a known pathogenic process in psoriasis, as a key regulator of PCSK9. Finally, circulating PCSK9 positively correlated with brachial vein endothelial expression of the pro-inflammatory transcripts CXCL10 (r=0.69, p<0.001), ICAM1 (r=0.49, p=0.02) and IL1beta (r=0.38, p<0.01) and inversely correlated with the functional measure of endothelial health, FMD (r=-0.52, p=0.03). Conclusion Circulating PCSK9 is elevated in psoriasis and associated with impaired vascular health. Analysis of the relationship between PCSK9 and systemic pathways revealed prominent interactions between PCSK9 and interferon signaling. Further research to better characterize these transcriptome and proteome variations and how it impacts vascular health in psoriasis may help elucidate new targets for therapeutic interventions.
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OBJECTIVE:=0.02). CONCLUSIONS:In patients with psoriasis, platelets are activated and induce endothelial cell inflammation. Low-dose aspirin improved endothelial cell health in psoriasis via platelet COX-1 inhibition. These data demonstrate a previously unappreciated role of platelets in psoriasis and endothelial cell inflammation, which suggests that aspirin may be effective in improving vascular health in patients with psoriasis. Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03228017.

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.