Fig 1.
MVECs were seeded and grown to confluence on 100mm dishes. At confluence media was collected and centrifuged for MP collection and placement on filters as described in methods. TEM images of MPs were taken. These images illustrate that our collection protocol isolates MPs of the appropriate size.
Table 1.
Lectin panel used for all studies.
Fig 2.
Vascular cells from the macrocirculation have specific SNA lectin binding.
All cell types were grown to confluence, media changed to serum free media for 1 hour, trypsinized, and stained for our lectin panel in Table 1 as described in methods. (A) MVECs show preferential binding for Griffonia simplicifolia (GS1) lectin as previously reported (95.43 ± 1.9%). (B) PASMCs have significantly more binding to Sambucus nigra (SNA1) and Maackia amurensis (MAA) than GS or Helix pomatia (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).
Fig 3.
MPs released constitutively from vascular cells do not recapitulate the glycocalyx of the parent cells.
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 Table 1. 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).
Fig 4.
Lectin staining not affected by trypsin treatment.
MPs were collected from serum-free media of confluent cells as described in methods. Prior to formalin fixation MPs were re-suspended in trypsin for 5 minutes. A) Microparticles isolated from media of AOECs, MVECs, and PASMCs were untreated or treated with trypsin and stained with MAA. In Figs 2 and 4 MAA labels approximately 30% of the MPs and this was not altered by trypsin treatment. B) AOECs were manually dissociated using a cell scraper or exposed to trypsin followed by staining with MAA. There is no significant difference in staining between the two dissociation methods.
Fig 5.
Lectin staining is inhibited by treatment with sugar.
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).
Fig 6.
Glycocalyx of cigarette smoke extract injured vascular cells.
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 Table 1 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.
Fig 7.
Glycocalyx of MPs from cigarette smoke extract injured vascular cells.
All cell types were grown to confluence, media changed to serum free media for 1 hour in the presence of 3% CSE. Media was then centrifuged as described to collect MPs. MPs were then stained with the lectin panel in Table 1. The lectin staining of MPs isolated from MVECs, PAECs, AOECs, and PASMCs (A, B, C, and D) treated with 3% CSE were not significantly different from MPs isolated from the control cells. Thus, CSE injury to the parent cell does not seem to significantly alter the glycocalyx of the released MPs.