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. 2021 May 4;10(9):e019413.
doi: 10.1161/JAHA.120.019413. Epub 2021 Apr 21.

Arterial Platelet Adhesion in Atherosclerosis-Prone Arteries of Obese, Insulin-Resistant Nonhuman Primates

Eran Brown  1 , Koya Ozawa  1 , Federico Moccetti  1 , Amanda Vinson  2 , James Hodovan  1 , The Anh Nguyen  1 , Lindsay Bader  2 , José A López  3 , Paul Kievit  2 , Gray D Shaw  4 , Dominic W Chung  3 , Warren Osborn  3 , Xiaoyun Fu  3 , Junmei Chen  3 , Jonathan R Lindner  1   2
Affiliations

Arterial Platelet Adhesion in Atherosclerosis-Prone Arteries of Obese, Insulin-Resistant Nonhuman Primates

Eran Brown et al. J Am Heart Assoc. .

Abstract

Background Platelet-endothelial interactions are thought to contribute to early atherogenesis. These interactions are potentiated by oxidative stress. We used in vivo molecular imaging to test the hypothesis that platelet-endothelial interactions occur at early stages of plaque development in obese, insulin-resistant nonhuman primates, and are suppressed by NADPH-oxidase-2 inhibition. Methods and Results Six adult rhesus macaques fed a Western-style diet for a median of 4.0 years were studied at baseline and after 8 weeks of therapy with the NADPH-oxidase-2-inhibitor apocynin (50 mg/kg per day). Six lean control animals were also studied. Measurements included intravenous glucose tolerance test, body composition by dual-energy X-ray absorptiometry, carotid intimal medial thickness, carotid artery contrast ultrasound molecular imaging for platelet GPIbα (glycoprotein- Ibα) and vascular cell adhesion molecule-1, and blood oxidative markers on mass spectrometry. Compared with lean controls, animals on a Western-style diet were obese (median body mass: 16.0 versus 8.7 kg, P=0.003; median truncal fat: 49% versus 20%, P=0.002), were insulin resistant (4-fold higher insulin-glucose area under the curve on intravenous glucose tolerance test, P=0.002), had 40% larger carotid intimal medial thickness (P=0.004), and exhibited oxidative signatures on proteomics. In obese but not lean animals, signal enhancement on molecular imaging was significantly elevated for GPIbα and vascular cell adhesion molecule-1. The signal correlated modestly with intimal medial thickness but not with the degree of insulin resistance. Apocynin significantly (P<0.01) reduced median signal for GPIbα by >80% and vascular cell adhesion molecule-1 signal by 75%, but did not affect intimal medial thickness, body mass, or intravenous glucose tolerance test results. Conclusion In nonhuman primates, diet-induced obesity and insulin resistance leads to platelet-endothelial adhesion at early atherosclerotic lesion sites, which is associated with the expression of pro-inflammatory adhesion molecules. These responses appear to be mediated, in part, through oxidative pathways.

Keywords: atherosclerosis; molecular imaging; platelets; von Willebrand factor.

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Conflict of interest statement

Gray D. Shaw is an inventor of US Patent 9 266 923 B2 and a founder of Quell Pharma, Inc. The remaining authors have no disclosures to report.

Figures

Figure 1
Figure 1. Indices of metabolic status in lean rhesus macaques, and obese animals on WSD at BL and after 8 weeks of apocynin treatment.
Bar‐whisker plots illustrate the median (bar), interquartile range (box), and range (whiskers) for (A) body mass, (B) truncal fat on DEXA, (C) product of insulin and glucose AUC on IVGTT, (D) HOMA‐IR index, (E) serum cholesterol, (F) LDL cholesterol, (G) HDL cholesterol, and (H) serum triglycerides. *P<0.05 vs lean. AUC indicates area‐under‐the‐curve; BL, baseline; DEXA, dual‐energy x‐ray absorptiometry; HDL, high‐density lipoprotein; HOMA‐IR, homeostatic model assessment for insulin resistance; IVGTT, intravenous glucose tolerance test; LDL, low‐density lipoprotein; and WSD, Western‐style diet.
Figure 2
Figure 2. Blood markers of oxidative stress in lean animals and in obese animals at BL study.
The markers include: (A) total thiol concentration in whole blood, (B) reduced thiols, (C) proportion of thiols in a reduced state, (D) total glutathione concentration normalized to hemoglobin, (E) reduced glutathione, and (F) proportion of glutathione in a reduced state. The thiol concentrations in panels A to F were normalized to hemoglobin concentration. Plasma markers included: (G) total cysteine in plasma, (H) protein‐bound cysteine, (I) total Cys‐Gly in plasma, (J) protein‐bound Cys‐Gly, and (K) plasma F2‐isoprostane concentrations by ELISA. The thiol concentrations in panels G to J were normalized to total protein concentration. *P<0.05 vs lean controls. BL indicates baseline.
Figure 3
Figure 3. Vascular morphology and functional analysis in lean rhesus macaques, and obese animals on WSD at BL and after 8 weeks of apocynin treatment.
A, Example of IMT thickening in the carotid bulb (arrow, top) and common carotid artery (arrow, bottom). Bar‐whisker plots illustrate the median (bar), interquartile range (box), and range (whiskers) for (B) carotid IMT, and (C) pulse wave velocity. *P<0.05 vs lean. BL indicates baseline; IMT, intima‐medial thickness; and WSD, Western‐style diet.
Figure 4
Figure 4. In vitro flow chamber data (shear rates of 1.0–8.0 dyne/cm2) for attachment of control and GPIbα‐targeted MB for (A) single platelets and small platelet aggregates (<50 µm2), and for (B) large platelet aggregates (>50 µm2).
Data are quantified as mean (±SEM) fluorescent area normalized to platelet area *P<0.05 vs control MB. C, Bar‐whisker plots illustrating the median (bar), interquartile range (box), and range (whiskers) for CEU molecular imaging for control (Ctrl) and targeted MB agents in lean and obese animals. *P<0.05 vs lean. D, Correlation between VCAM‐1 and GPIbα signal on CEU molecular imaging on a per‐artery basis. CEU indicates; GPIbα, glycoprotein Ibα; MB, microbubbles; VCAM‐1, vascular cell adhesion molecule‐1; and VIU, video intensity units.
Figure 5
Figure 5. Individual data for CEU molecular imaging signal in obese animals at BL and after 8 weeks of apocynin therapy for (A) control MB, (B) GPIbα‐targeted MB, and (C) VCAM‐1‐targeted MB.
D, Example carotid artery CEU molecular imaging of platelet GPIbα from an obese animal at BL and after apocynin therapy. Images show background‐subtracted and color‐coded (scale at right) CEU signal superimposed on a co‐registered 2‐dimensional B‐mode image. BL indicates baseline; CEU; GPIbα, glycoprotein Ibα; MB, microbubbles; VCAM‐1, vascular cell adhesion molecule‐1; and VIU, video intensity units.
Figure 6
Figure 6. Bar‐whisker plots illustrate the median (bar), interquartile range (box), and range (whiskers) for plasma (A) IL‐1β, (B) IL‐1RA, (C) CCL‐2, (D) IFN‐γ, (E) CCL‐5, (F) TNF‐α, (G) G‐CSF, and (H) VWF antigen.
*P<0.05 vs lean. BL indicates baseline; CCL‐2, chemokine c‐c motif ligand‐2; CCL‐5, chemokine c‐c motif ligand‐5; G‐CSF, granulocyte colony‐stimulating factor; IFN‐γ, interferon gamma; IL‐1β, interleukin‐1β; IL‐1RA, ingterleukin‐1 receptor antagonist; TNF‐α, tumor necrosis factor‐α; and VWF, von Willebrand factor.
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