Lectin-Based Characterization of Vascular Cell Microparticle Glycocalyx

<div><p>Microparticles (MPs) are released constitutively and from activated cells. MPs play significant roles in vascular homeostasis, injury, and as biomarkers. The unique glycocalyx on the membrane of cells has frequently been exploited to identify specific cell types, however the glycocalyx of the MPs has yet to be defined. Thus, we sought to determine whether MPs, released both constitutively and during injury, from vascular cells have a glycocalyx matching those of the parental cell type to provide information on MP origin. For these studies we used rat pulmonary microvascular and artery endothelium, pulmonary smooth muscle, and aortic endothelial cells. MPs were collected from healthy or cigarette smoke injured cells and analyzed with a panel of lectins for specific glycocalyx linkages. Intriguingly, we determined that the MPs released either constitutively or stimulated by CSE injury did not express the same glycocalyx of the parent cells. Further, the glycocalyx was not unique to any of the specific cell types studied. These data suggest that MPs from both normal and healthy vascular cells do not share the parental cell glycocalyx makeup.</p></div

MPs released constitutively from vascular cells do not recapitulate the glycocalyx of the parent cells.

<p>All cell types were grown to confluence, media changed to serum free media for 1 hour. Media was then centrifuged as described to collect MPs. MPs were then stained with the lectin panel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135533#pone.0135533.t001" target="_blank">Table 1</a>. The MPs released from MVECs, PAECs, and AOECs (A, B, and C) do not show any significant positivity for any of the lectins studied (all ranging from 40–60% positive. P = not significant). The PASMC-MPs were significantly positive for SNA over MVEC-MPs and PAEC-MPs, but not AOEC-MPs (78.53 ± 4.5 vs. 57.66 ± 6.6 and 67.1 ± 9.06, respectively. P<0.05).</p

Lectin staining is inhibited by treatment with sugar.

<p>The lectins HP, SNA, MAA, and GS1 were presaturated with the sugar D(+)galactose. MPs isolated from MVECs were then stained with lectins or Dgal saturated lectins. Dgal significantly inhibited all lectin binding to the MPs. (HP 37 ± 5 vs. 13 ± 4; SNA 68 ± 2 vs. 51 ± 0.3; MAA 34 ± 0.6 vs. 18 ± 1; and GS1 54 ± 6 vs. 13 ±1; stained vs. Dgal saturated respectively, n = 3–5 per group P< 0.001).</p

Glycocalyx of cigarette smoke extract injured vascular cells.

<p>All cell types were grown to confluence, media changed to serum free media for 1 hour in the presence of 3% CSE, trypsinized, and stained for our lectin panel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135533#pone.0135533.t001" target="_blank">Table 1</a> as described in methods. (A) MVECs treated with 3% CSE have no significant changes in their glycocalyx profile compared to healthy cells. (B) Injury of PAECs with 3% CSE induces increased lectin binding of GS1 and HP (15.95 ±3.0 vs. 49.67 ±8.92 for GS and 15.58 ± 4.8 vs. 64.47 ± 8.2 for HP), but there was no significant change to SNA or MAA. (C) AOECs treated with 3% CSE had no change in GS1 or SNA1 binding, however both HP and MAA increased significantly (23.48 ± 4.4 vs. 45.20 ± 4.1 for HP and 36.9 ± 1.8 vs. 72.83 ± 14.09 for MAA. P<0.05). (D) There were no significant differences in lectin binding in CSE treated PASMCs.</p

Vascular cells from the macrocirculation have specific SNA lectin binding.

<p>All cell types were grown to confluence, media changed to serum free media for 1 hour, trypsinized, and stained for our lectin panel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135533#pone.0135533.t001" target="_blank">Table 1</a> as described in methods. (A) MVECs show preferential binding for <i>Griffonia simplicifolia</i> (GS1) lectin as previously reported (95.43 ± 1.9%). (B) PASMCs have significantly more binding to <i>Sambucus nigra</i> (SNA1) and <i>Maackia amurensis</i> (MAA) than GS or <i>Helix pomatia</i> (HP) (100 and 79.43 ± 1.2% vs. 15.95 ± 3.0 and 15.58 ± 4.8%, respectively; P<0.05). (C) AOECs preferentially bind GS1 and SNA1 compared to HP and MAA (88.7 ± 5.7 and 100% vs. 23.48 ± 4.4 and 36.9 ± 1.8%, respectively. *P<0.05). (D) PASMC bind GS1 and SNA1 preferentially (97.9 ± 1.5 and 100% vs. 16.6 ± 2 and 18.93 ± 3.0%, respectively. P<0.05).</p

Lectin panel used for all studies.

<p>¹ EY Laboratories; San Mateo, CA.</p><p>Lectin panel used for all studies.</p