Microvesicles in plasma reflect coronary flow reserve in patients with cardiovascular disease

Figures

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  Figure 1.. CFR negatively correlates with levels of platelet and endothelial MVs. A and C: scatter plots showing median ± 95% CI. Patients with high CFR have significantly lower concentration of platelet (A) and endothelial (C) MVs in plasma, compared with the low CFR group. Mann–Whitney U test for unpaired data. CD42a+ and CD62E+ MVs: n = 100 (low CFR group), n = 107 (high CFR group). *Statistically significant, P < 0.0001 for both platelet and endothelial MVs. B and D: CFR and MV correlation plots. Spearman’s rho for platelet MVs = −0.34, n = 207. Spearman’s rho for endothelial MVs = −0.24, n = 207. P < 0.0001 and P = 0.001 for platelet and endothelial MVs, respectively. The curves demonstrate the results of simple linear regression. CFR, coronary flow reserve; MVs, microvesicles.

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  Figure 2.. Endothelial MVs positively correlate with platelet MV levels in plasma. Scatter plot. Spearman’s rho = 0.52, P < 0.0001; n = 207 for both CD42a+ and CD62E+ MVs. MVs, microvesicles.

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  Figure 3.. CFR negatively correlates with seven cardiovascular disease-related proteins (A) that are lower in MVs from patients with high CFR (B). Olink proteomic CVD II and III panels were used to measure 180 cardiovascular biomarkers. A: correlation plots showing normalized protein expression (NPX) values of the significantly correlated proteins (x-axis), with corresponding CFR (y-axis), P < 0.05. AXL: n = 143, BAFF: n = 122, CD163: n = 137, IGFBP7: n = 166, NEMO: n = 130, perlecan: n = 145, resistin: n = 157. B: patients from the high CFR group have significantly lower biomarker concentration (NPX values) in plasma MVs than the low CFR group. Scatter plots presenting median ± 95% CI. Mann–Whitney U test for unpaired data. *Statistically significant, P < 0.05. AXL: n = 62 (low CFR), 81 (high CFR); BAFF: n = 52 (low CFR), 70 (high CFR); CD163: n = 58 (low CFR), 79 (high CFR); IGFBP7: n = 75 (low CFR), 91 (high CFR); NEMO: n = 62 (low CFR), 68 (high CFR); perlecan: n = 59 (low CFR), 86 (high CFR); resistin: n = 68 (low CFR), 89 (high CFR). BAFF, B cell activating factor; CFR, coronary flow reserve; IGFBP7, insulin-like growth factor-binding protein 7; MVs, microvesicles; NEMO, NF-κB…

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  Figure 4.. CFR negatively correlates to a parameter based on platelet MV concentration and protein NPX (A), and patients with high CFR have lower levels of this parameter. A: correlation plots showing normalized protein expression (NPX) values of the significantly correlated proteins, multiplied by platelet MV concentration (x-axis), with corresponding CFR (y-axis), P < 0.0001. AXL: n = 129, BAFF: n = 115, CD163: n = 132, IGFBP7: n = 151, NEMO: n = 121, perlecan: n = 133, resistin: n = 145. B: scatter plots showing median ± 95% CI. Mann–Whitney U test for unpaired data, *P < 0.05, statistically significant. AXL: n = 129, BAFF: n = 115, CD163: n = 132, IGFBP7: n = 151, NEMO: n = 121, perlecan: n = 133, resistin: n = 145. BAFF, B cell activating factor; CFR, coronary flow reserve; MV, microvesicle; IGFBP7, insulin-like growth factor-binding protein 7; MV, microvesicle; NEMO, NF-κB essential modulator.

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  Figure 5.. Tracking the origin of circulating BAFF+ MVs in plasma of patients with cardiovascular disease reveals its platelet origin. Platelet-free plasma was costained with BAFF and markers of platelets (top), leukocytes (middle), endothelial cells (bottom, left), and erythrocytes (bottom, right). There was a substantial positive costaining of BAFF with CD42b and CD31 platelet markers. Lack or very low costaining was observed for leukocytes, endothelial cells, or erythrocytes. This suggests that BAFF+ MVs in plasma represent a fraction of platelet MVs. BAFF, B cell activating factor; MVs, microvesicles.

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  Figure 6.. Additional staining performed to track BAFF and CD36 origin in plasma. Platelet-free plasma from patients with cardiovascular disease was costained with BAFF and CD36, an oxidized LDL receptor, present on different types of cells. There was a positive costaining for a substantial fraction of MVs (A). Establishing cellular origin of CD36 revealed that platelets (B) and not endothelial cells (C), leukocytes (D), or erythrocytes (E) were major sources of CD36. Thus, BAFF+ and CD36+ MVs in plasma represent a fraction of platelet MVs. BAFF, B cell activating factor; MVs, microvesicles.

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  Figure 7.. Additional staining performed to track CD31 origin in plasma, as it has been reported to be present on both platelets and endothelial cells. Platelet-free plasma from patients with cardiovascular was costained with CD31 and CD42b, a platelet MV marker (A) or CD144, an endothelial marker (B). The staining revealed that majority of CD31+ MVs came from platelets (A) and not endothelial cells (B), thus confirming our data that BAFF+ MVs represent a fraction of platelet MVs. BAFF, B cell activating factor; MVs, microvesicles.

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  Figure 8.. Release of BAFF+ MVs from MEG01 cells is increased upon TNFα stimulation. Megakaryocyte cell line was stimulated with pro-inflammatory conditions to measure release of BAFF+ MVs. The levels of BAFF-expressing MVs increased substantially (B) after applying TNFα, compared with the unstimulated MEG01 cells (A), pointing at the role of proatherosclerotic conditions in the formation of circulating BAFF+ MVs. BAFF, B cell activating factor; MVs, microvesicles.

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  Figure 9.. Microvesicles isolated from plasma of the low CFR group significantly increase ICAM-1 expression on endothelial cells. Bar graph showing means ± SD. One-way ANOVA, n = 3 experiments with replicates. *Statistically significant, P < 0.05. CFR, coronary flow reserve; ICAM-1, intercellular adhesion molecule 1.

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  Figure 10.. Proposed MV connection with decreased CFR. In physiological conditions, platelets and endothelial cells release low levels of platelet MVs (PMVs) and endothelial MVs (EMVs) (1). CVD risk factors, e.g., high LDL, contribute to platelet and endothelial activation (2) and a substantial increase in MV production, carrying an increased load of procoagulant and proinflammatory cargo. This proatherogenic status in the vessel leads to endothelial dysfunction (3) and atherosclerotic plaque progression (4) that further contribute to a decrease in the blood flow, finally manifested as low CFR, as well as a development of CVD. CFR, coronary flow reserve; MV, microvesicle.