Faculty Bibliography

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.

PURPOSE OF REVIEW/OBJECTIVE:Monocytes and macrophages are key players in the pathogenesis of atherosclerosis and dictate atherogenesis growth and stability. The heterogeneous nature of myeloid cells concerning their metabolic and phenotypic function is increasingly appreciated. This review summarizes the recent monocyte and macrophage literature and highlights how differing subsets contribute to atherogenesis. RECENT FINDINGS/RESULTS:Monocytes are short-lived cells generated in the bone marrow and released to circulation where they can produce inflammatory cytokines and, importantly, differentiate into long-lived macrophages. In the context of cardiovascular disease, a myriad of subtypes, exist with each differentially contributing to plaque development. Herein we describe recent novel characterizations of monocyte and macrophage subtypes and summarize the recent literature on mediators of myelopoiesis. SUMMARY/CONCLUSIONS:An increased understanding of monocyte and macrophage phenotype and their molecular regulators is likely to translate to the development of new therapeutic targets to either stem the growth of existing plaques or promote plaque stabilization.

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.

Cardiovascular disease, with atherosclerosis as the major underlying factor, remains the leading cause of death worldwide. It is well established that cholesterol ester-enriched foam cells are the hallmark of atherosclerotic plaques. Multiple lines of evidence support that enhancing foam cell cholesterol efflux by HDL (high-density lipoprotein) particles, the first step of reverse cholesterol transport (RCT), is a promising antiatherogenic strategy. Yet, excitement towards the therapeutic potential of manipulating RCT for the treatment of cardiovascular disease has faded because of the lack of the association between cardiovascular disease risk and what was typically measured in intervention trials, namely HDL cholesterol, which has an inconsistent relationship to HDL function and RCT. In this review, we will summarize some of the potential reasons for this inconsistency, update the mechanisms of RCT, and highlight conditions in which impaired HDL function or RCT contributes to vascular disease. On balance, the evidence still argues for further research to better understand how HDL functionality contributes to RCT to develop prevention and treatment strategies to reduce the risk of cardiovascular disease.

Background: The mechanisms for increased cardiovascular risk in patients with Psoriasis (PsO) are unknown. Activated platelets adhere to damaged endothelium and secrete pro-inflammatory cytokines thus promoting atherosclerosis. The contribution of platelets to promote endothelial activation in PsO has not been established. Method(s): Patients with active PsO (n = 6, mean age 46 years, 50% male) were compared to age- and sex- matched controls. Result(s): Platelets were present in PsO lesional skin compared to non-lesional skin, and controls (Figure 1A). To investigate the clinical significance, isolated platelets from PsO and matched-controls demonstrated increased platelet adhesion to human aortic endothelial cells (HAECs) in both basal and activated (thrombin stimulated) states (Figure 1B). Platelets isolated from PsO subjects enhanced HAEC expression of pro-inflammatory transcripts IL-1B, IL-8 and COX-2 (Figure 1C) compared to controls. Next generation RNA sequencing of isolated platelets from PsO and controls revealed upregulation of transcripts indicative of platelet - endothelial interactions such as the pro-atherogenic mediators s100A8/A9 (p < 0.05). Conclusion(s): We describe for the first time platelet-endothelial interactions as a potential mechanism of early cardiovascular risk in patients with PsO. These findings have important clinical implications suggesting that targeting platelet specific pathways in PsO may reduce cardiovascular risk. [Figure presented]2019 American College of Cardiology Foundation. All rights reserved