Cause or effect of arteriogenesis: compositional alterations of microparticles from CAD patients undergoing external counterpulsation therapy

Abstract

Recently, a clinical study on patients with stable coronary artery disease (CAD) showed that external counterpulsation therapy (ECP) at high (300 mmHg) but not at low inflation pressure (80 mmHg) promoted coronary collateral growth, most likely due to shear stress-induced arteriogenesis. The exact molecular mechanisms behind shear stress-induced arteriogenesis are still obscure. We therefore characterized plasma levels of circulating microparticles (MPs) from these CAD patients because of their ambivalent nature as a known cardiovascular risk factor and as a promoter of neovascularization in the case of platelet-derived MPs. MPs positive for Annexin V and CD31CD41 were increased, albeit statistically significant (P<0.05, vs. baseline) only in patients receiving high inflation pressure ECP as determined by flow cytometry. MPs positive for CD62E, CD146, and CD14 were unaffected. In high, but not in low, inflation pressure treatment, change of CD31CD41 was inversely correlated to the change in collateral flow index (CFI), a measure for collateral growth. MPs from the high inflation pressure group had a more sustained pro-angiogenic effect than the ones from the low inflation pressure group, with the exception of one patient showing also an increased CFI after treatment. A total of 1005 proteins were identified by a label-free proteomics approach from MPs of three patients of each group applying stringent acceptance criteria. Based on semi-quantitative protein abundance measurements, MPs after ECP therapy contained more cellular proteins and increased CD31, corroborating the increase in MPs. Furthermore, we show that MP-associated factors of the innate immune system were decreased, many membrane-associated signaling proteins, and the known arteriogenesis stimulating protein transforming growth factor beta-1 were increased after ECP therapy. In conclusion, our data show that ECP therapy increases platelet-derived MPs in patients with CAD and that the change in protein cargo of MPs is likely in favor of a pro angiogenic/arteriogenic property.

Trial registration: ClinicalTrials.gov NCT00414297.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Characterization of circulating microparticles (MPs) by flow cytometry.

MPs were isolated from plasma of CAD patients before (BL) and after (Fup) ECP therapy using low (80 mmHg) or high inflation pressure (300 mmHg). A, number of MPs determined by Annexin V. B and C, CD31 and CD41 positive MPs. D, PMPs double-stained for CD31 and CD41. Whiskers indicate minimum and maximum values. *P<0.05.

Figure 2. Correlation of PMP and CFI changes.

Plotted values are the differences between follow-up and baseline measurements with platelet-derived microparticle positive for CD31CD41 on the horizontal axis and collateral flow index (CFI) on the vertical axis. Values are given for ECP therapy at high (300 mmHg) on top or low inflation pressure (80 mmHg) at bottom, respectively. Filled triangles represent CFI values from stenosed and open circles from normal vessels. The same correlations were detected with MPs stained positive with CD31 or CD41 alone (Figure S1). The values encircled in red in the 80 mmHg inflation pressure group belong to patient #14.

Figure 3. In vitro angiogenesis assay with isolated MPs.

Circulating MPs were isolated from patient's plasma before (BL) and after (Fup) ECP therapy at 80 mmHg (top row) or 300 mmHg inflation pressure (bottom row). Displayed are numbers of meshes formed by HUVECs relative to the four-hour time point. The horizontal bars represent the mean value at each time point. The dots in the BL80 and Fup80 plots are the triplicate values measured for patient #14. The mean values between the 80 mmHg and 300 mmHg inflation pressure were tested for their likelihood to be the same by Student's t-test (two-tailed, equal variance) and P values at each time point are given in the bottom row graphs.

Figure 4. MP Protein quantification by label-free proteomics.

Expression levels (LOG2 values of normalized protein abundance values) of cellular signaling proteins (left side) and proteins involved in the innate immune response (right side) are shown in boxplot format. RAS-related proteins RAB encompass RAB1B, RAB5C, RAB6B, RAB7A, RAB8A, RAB10, RAB11B, RAB15, RB27B, RAB35, RAP1B, and RAP2B, identified in all twelve analyzed MP samples. Guanine nucleotide binding factors encompass GNA13, GNAI2, GNAQ, GNAZ, GBB1, and GBB2, identified in at least eight MP samples. Complement factors considered were identified in at least eight MP samples: C1QA, C1QB, C1QC, C1R, C1S, CO2, CO3, CO4A, CO4B, CO5, CO6, CO7, CO8B, CO8G, CO9, and CFAB. All immunoglobulin heavy and light chains identified in at least 10 MP samples were considered. When one of the proteins was not identified its value was set at zero for statistical analysis of P values. P values are given below each box if the likelihood for a statistical difference between BL and Fup was at least 95%.

Figure 5. Label-free quantification of individual proteins.

Bars represent mean abundance values (LOG2 values of normalized PMSS) of individual proteins before (BL) or after (Fup) ECP therapy with 300 mmHg (top) or 80 mmHg inflation pressure (bottom). Whiskers represent one standard deviation. Proteins displayed are PECA1 (CD31), ITA2B (CD41), TSP1 (thrombospondin-1), and TGFB1 (transforming growth factor beta-1).

Figure 6. ECP therapy increases associations of Apo(a) and APOB to MPs.

The sum of Apo(a) and APOB (top panel) was increased after low and high inflation pressure treatment of CAD patients. Small molecular weight apolipoproteins (APOA1, APOA2, APOA4, APOC1, APOC2, APOC3, APOD, APOE, and APOL1) were not changed or slightly decreased (bottom panel) in case of 80 mmHg inflation pressure treatment (P = 0.039), respectively.