Activated Platelets Induce Endothelial Cell Inflammatory Response in Psoriasis via COX-1

Abstract

Objective: Patients with psoriasis have impaired vascular health and increased cardiovascular disease (CVD). Platelets are key players in the pathogenesis of vascular dysfunction in cardiovascular disease and represent therapeutic targets in cardiovascular prevention. The object of this study was to define the platelet phenotype and effector cell properties on vascular health in psoriasis and evaluate whether aspirin modulates the platelet-induced phenotype. Approach and Results: Platelets from psoriasis patients (n=45) exhibited increased platelet activation (relative to age- and gender-matched controls, n=18), which correlated with psoriasis skin severity. Isolated platelets from psoriasis patients demonstrated a 2- to 3-fold (P<0.01) increased adhesion to human aortic endothelial cells and induced proinflammatory transcriptional changes, including upregulation of IL 8 (interleukin 8), IL1β, and Cox (cyclooxygenase)-2 Platelet RNA sequencing revealed an interferon signature and elevated expression of COX-1, which correlated with psoriasis disease severity (r=0.83, P=0.01). In a randomized trial of patients with psoriasis, 2 weeks of 81 mg low-dose aspirin, a COX-1 inhibitor, reduced serum thromboxane (Tx) B₂ and reduced brachial vein endothelial proinflammatory transcript expression >70% compared with the no-treatment group (P<0.01). Improvement in brachial vein endothelial cell inflammation significantly correlated with change in serum TxB₂ (r=0.48, P=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 and suggests that aspirin may be effective in improving vascular health in patients with psoriasis. Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03228017.

Keywords: aspirin; endothelium; inflammation; platelet aggregation; psoriasis.

Figure 1.

Platelet activation is present in psoriasis and associated with disease severity. A, Immunohistochemistry of platelets in normal skin from a healthy control and lesional (psoriasis) skin (arrows indicate platelet staining with anti-ITGA2B). B, Flow cytometry quantification of platelet P-selectin (CD62L) expression in basal or activated (thrombin stimulated) condition between psoriasis vs. control. Flow cytometry quantification of (C) Neutrophil CD45⁺CD61⁺ and (D) lymphocyte platelet aggregate CD45⁺CD61⁺ subtypes in psoriasis vs. control. E, Regression plot and the correlation coefficient between psoriasis area and severity index (PASI) and platelet P-selectin expression in basal or (F) activated (thrombin stimulated) condition. MFI, mean fluorescence units. N≈10 control/25 psoriasis patients per group. Bar graphs in Mean ± SEM unless otherwise specified. P<0.05*, p<0.01**

Figure 2.

Psoriasis platelets preferentially adhere to and promote endothelial inflammation. A, Isolated psoriasis vs. control platelets in basal and activated (thrombin stimulated) conditions (green) co-incubated with human aortic endothelial cells (HAECs, blue - DAPI stained, n≈6/group). B, Endothelial transcript expression after platelet co-incubation with HAECs in both basal and platelet activated conditions (n≈6/group, 4-hour co-incubation at 100:1 platelet to HAEC ratio). C, Scanning electron microscope images of a representative field depicting psoriasis platelets (dashed white arrow) interacting with HAECs (blue – superimposed immunofluorescence DAPI staining). DAPI, 4′,6-diamidino-2-phenylindole; IL, Interleukin. Bar graphs in Mean ± SEM unless otherwise specified. Green fluorescence in arbitrary units for quantification purposes. p<0.05*, p<0.01**

Figure 3.

Platelet RNA sequencing reveals biological pathways involved in cardiovascular disease. A, Heatmap and volcano plot (B) of platelet profiling by RNA sequencing in six psoriasis patients with active disease and six healthy age-, sex-matched controls (p<0.05). C, Biological pathway analysis (by Ingenuity Pathway Analysis) of platelet-derived pathways differentially expressed in psoriasis vs. control. D, Interferon-induced genes between psoriasis vs. controls. E, Experimental design of (F), MEG-01 cell line stimulated with interferon (IFN) Ɣ, interleukin (IL) – 17A or both (color indicates fold change, n=2–3 wells in 3 separate experiments). NCV, normalized count values. Bar graphs in Mean ± SEM. p<0.05*, p<0.01**

Figure 4.

Aspirin inhibition of platelets downregulates platelet-endothelial cell adhesion and activation. A, Resting or TNFα+IL-17A stimulated human aortic endothelial cells (HAECs, blue - DAPI stained) co-incubated with basal or aspirin pretreated platelets. B, Endothelial transcript expression after basal or aspirin pretreated platelets co-incubated with resting HAECs (n= 2–3 wells, 3 separate experiments). DAPI, 4′,6-diamidino-2-phenylindole; HAEC, human aortic endothelial cell; IL, Interleukin; TNF, tumor necrosis factor. Bar graphs in Mean ± SEM unless otherwise specified. p<0.05*, p<0.01**

Figure 5.

Aspirin decreases endothelial pro-inflammatory activation. A, Randomized clinical trial in psoriasis evaluating the efficacy of aspirin 81mg (n=15) vs. no treatment (n=15) to reduce endothelial pro-inflammatory activation. B - D, Two-week change in the composite brachial vein endothelial transcriptome, IL8 and IL1β after randomization to aspirin 81mg or no treatment. E - G, Regression plot and correlation coefficient between baseline serum thromboxane B2 (ng/ml) and brachial vein endothelial transcript expression. H - J, Regression plot and correlation coefficient between change in serum thromboxane B₂ and change in brachial vein endothelial transcript expression. Δ = relative change, (follow-up_{2 weeks} – baseline_{time 0})/baseline_{time 0}; IL, interleukin; Relative expression = Gene expression/hARP. Rx = no treatment group. Bar graphs in Mean ± SEM. p<0.05*, p<0.01**