The calpain-myosin-9-Rab7b axis may regulate TLR4 containing α-granules trafficking in thrombin-stimulated platelets.

<p>Human platelets were pretreated with or without calpeptin at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes. (A) The total protein extracted from platelets was used for western blot analysis and immunoblotting with anti-myosin-9 antibody. The β-actin protein served as the loading control. (B) The morphology of platelets were observed using TEM. Control human washed platelets are showed in B-1 and B-2. The platelet with randomly distribution of α-granules (AG) containing immuno-gold conjugated TLR4 (white arrow). Platelets with thrombin treatment at 37°C for 20 minutes were showed in B-3 and B-4. Original magnification of B-1 and B-3 are 40000×, B-2 and B-4 are 100000×. (C) The total protein extracted from platelets was used for immunoprecipitation with anti-Rab7b antibody and immunoblotting with anti-myosin-9 antibody. A pre-immune control IgG was used to confirm the specificity of the Rab7b antibody. Immunoblotting with anti-Rab7b antibody was used as an IP control.</p

Thrombin induces TLR4 expression on human platelets via the PAR/PLC pathway.

<p>(A) Washed human platelets were treated with 0.4 U/mL thrombin at 37°C for 20 min, and the level of TLR4 on the surface of platelets was determined by flow cytometry. (B) Washed human platelets were treated with 0.1–0.4 U/mL thrombin at 37°C for 20 min and analyzed by flow cytometry for the surface level of TLR4 (n = 3). (C) The total and membrane protein fraction were extracted, and the TLR4 levels were further confirmed by western blot analysis and detected with anti-TLR4 antibody. α-tubulin served as the loading control in this assay. The bar graph showed the quantification of western blot analysis using densitometry. (D) Human platelets were directly treated with SFLLRN, AYPGKF, or SFLLRN plus AYPGKF at 37°C for 20 min. The level of surface TLR4 was determined by flow cytometry. (E) Washed human platelets were pretreated with U73122 or Y27632 at 28°C for 60 min followed by 0.4 U/mL thrombin treatments at 37°C for 20 min, and the surface level of TLR4 on the platelets was determined by flow cytometry. The m-3M3FBS was pretreated at 28°C for 60 min, than incubate at at 37°C for 20 min. The data represented the results of 5 independent experiments (mean ± SD; *<i>p</i><0.05).</p

Thrombin-mediated TLR4/myosin-9 interaction in human platelets is modulated by calpain activity.

<p>Platelets were treated with 5 µg/mL calpeptin followed by 4 U/mL thrombin for 20 minutes or 3 mM of CaCl2 for 30 minutes. (A and C) The interaction of myosin-9 with TLR4 was analyzed using immunoprecipitation. The pre-immune controls IgG were used to confirm the specificities of the TLR4 and myosin-9 antibodies. (B) The total protein extracted from treated platelets was used for immunoprecipitation with anti-myosin-9 antibody and immunoblotting with anti-TLR4 antibody. Immunoblotting with anti-myosin-9 antibody was used as an IP control. (D) The total protein extracted from treated platelets was used for immunoprecipitation with anti-TLR4 antibody and immunoblotting with anti-myosin-9 antibody. Immunoblotting with anti-TLR4 antibody was used as an IP control. The band density was determined using densitometry and was shown in the graph on the right. The data represented the results of three independent experiments (mean ± SD; *<i>p</i><0.05 was considered significant).</p

Mechanisms contributing to thrombin-mediated TLR4 expression in platelets.

<p>Thrombin may pass through the PAR1 and PAR4 receptors to activate downstream effectors for the PLC pathway but not the Rho pathway. The PLC pathway further activates calpain via calcium mobilization, and cleavages myosin-9, which decreases the interaction between myosin-9 and TLR4. In the other hand, myosin-9 does not coordinate with Rab7b to negatively regulate TLR4 containing α-granules trafficking in thrombin treated platelets, and leads to the increasing of TLR4 performance in thrombin-stimulated human platelets.</p

GroEL1 decreases integrin and E-selectin expression and induces adhesion molecule production in EPCs.

<p>(A) Late EPCs were treated with GroEL1 (100 ng/mL) for 4-24 hours. Quantitative real-time PCR was also performed for integrin α1, -α2, -β1, -β3, and E-selectin. (B) Late EPCs were treated for 4 hours with 25-100 ng/mL GroEL1. eNOS activation was analyzed by western blotting. The total eNOS and β-actin levels were used as loading controls. (C) EPCs were pretreated with 10 μM SNAP or SNOC for 1 hour followed by 100 ng/mL GroEL1 treatment. Quantitative real-time PCR was performed for integrin α1, -β1, -β3, and E-selectin. All data represent the results of three independent experiments and are expressed as the mean±SEM (*<i>p</i> < 0.05 compared with untreated group, †<i>p</i> < 0.05 compared with GroEL1-only-treated group).</p

TLR4 interacts with myosin-9.

<p>(A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer.</p

GroEL1 decreases EPC number and function.

<p>(A) The collected-human MNCs in growth medium were incubated with different concentrations of GroEL1 (1-100 ng/mL) for 4 days. Early EPCs are indicated with arrows. The numbers of EPCs were counted under microscope in a high-power field (HPF). (B) ECMatrix gel was used to assay the <i>in </i><i>vitro</i> angiogenesis capacity of late EPCs. Representative photos of <i>in </i><i>vitro</i> angiogenesis are shown. The mean total area of complete tubes formed by each GroEL1-treated group was calculated and compared with the non-GroEL1-treated group. (C) A modified Boyden chamber assay was used with VEGF as chemoattractive factor to evaluate late EPC migratory function. Representative photos are shown; the migrated cells were stained with hematoxylin and counted under microscope. (D) Late EPCs were treated with GroEL1 (1-100 ng/mL) for 24 hours. MTT assay was also performed to evaluate late EPC proliferation activity. (E and F) Late EPCs were preincubated for 24 hours with or without GroEL1, then labeled with BCECF/AM and attached to fibronectin/collagen-coated plates or HCAEC-cultured dishes for 1 hour. The attached EPCs were lysed using DMSO, and the fluorescence was quantified by fluorimetry. All data are expressed as the mean ± SEM (*<i>p</i> < 0.05 compared with untreated group). </p

Effect of GroEL1 on blood flow recovery after hind limb ischemia in C57BL/B6 and C57BL/10ScNJ

<p><b>mice</b>. (A) Upper, representative results of laser Doppler measurements before operation (control) and 1 day after hind limb ischemia surgery in C57BL/B6 mice. Lower, representative results of laser Doppler measurements 8 weeks after hind limb ischemia surgery in C57BL/B6 and C57BL/10ScNJ mice treated with 0-4 μg/kg BW of GroEL1. Color scale illustrates blood flow variations from minimal (dark blue) to maximal (red). Arrows indicate the ischemic (right) limb after hind limb ischemia surgery. (B) Doppler perfusion ratios (ischemic/non-ischemic hind limb) over time in the different groups. In C57BL/B6 mice, administration of 4 μg/kg BW (△) of GroEL1 or 2 μg/kg BW (▼) of GroEL1 impaired beneficial blood flow recovery compared with the non-administered group (●) 6 or 8 weeks after hind limb ischemia surgery. There was no significant difference in blood flow in the limb in the 1 μg/kg BW GroEL1-treated C57BL/B6 mice (○) or the 4 μg/kg BW GroEL1-treated C57BL/10ScNJ mice (█) compared with the non-administered C57BL/B6 mice (●). The results are expressed as the mean ± SEM (*<i>p</i> < 0.05 compared with non-GroEL1-treated C57BL/B6 mice at the same time point after ischemic surgery). (C) Eight weeks after ischemic surgery, the ischemia/normal perfusion ratio in the GroEL1-treated C57BL/B6 mice, but not GroEL1-treated C57BL/10ScNJ mice, was lower than that in the non-GroEL1-treated C57BL/B6 mice. The results are expressed as the mean ± SEM (n=6, *<i>p</i> < 0.05 compared with non-GroEL1-treated C57BL/B6 mice; †<i>p</i> < 0.05 compared with 4 μg/kg BW of GroEL1-treated C57BL/B6 mice). (D) Representative results of immunohistochemistry before operation (naïve) in C57BL/B6 mice. Mice were sacrificed 8 weeks after surgery, and capillaries (white arrow) in the ischemic muscles were visualized by anti-vWF immunostaining (original magnification x400). Hoechst dye (blue) was used to counterstain the nucleus. The graph shows the quantification of capillary density in hind limb-ischemic and GroEL1-administered C57BL/B6 and C57BL/10ScNJ mice. The results are expressed as the mean ± SEM (n=6, *<i>p</i> < 0.05 compared with hind limb ischemia+ non-GroEL1-treated C57BL/B6 mice; †<i>p</i> < 0.05 compared with hind limb ischemia+ 4 μg/kg BW of GroEL1-treated C57BL/B6 mice).</p

GroEL1 increases senescence, which is mediated by caspases and MAPK signaling in EPCs.

<p>(A) Late EPCs were treated with 1-100 ng/mL GroEL1 for 24 hours. Cell senescence was analyzed. The diagram shows the quantification of senescent EPCs. (B) Late EPCs were treated with 25-100 ng/mL GroEL1 for 12 hours. The activation of caspase-9, -8, and -3 were analyzed using western blotting. The β-actin level was used as loading control. (C) Late EPCs were pretreated with 10 μM Z-VAD-FMK, Ac-LEHD-CMK, or Ac-IETD-CHO for 1 hour followed by 100 ng/mL GroEL1 treatment for 24 hours. The cell senescence assay was performed, and the quantified results are shown as a diagram. (D) After treatment of EPCs with 25-100 ng/mL of GroEL1 for 12 hours, the phosphorylation of p38 MAPK, ERK1/2, and JNK/SAPK were analyzed by western blotting. The total p38 MAPK, ERK1/2, and JNK/SAPK levels were used as loading controls. (E) EPCs were pretreated with 10 μM SB203580, PD98059, or SP600125 for 1 hour followed by 100 ng/mL GroEL1 treatment. The cell senescence assay was performed, and the quantified results are shown as a diagram. All data represent the results of three independent experiments and are expressed as the mean±SEM (*<i>p</i> < 0.05 compared with untreated group; †<i>p</i> < 0.05 compared with GroEL1-only-treated group).</p

Effects of GroEL1 on EPC mobilization and eNOS expression at ischemic sites in hind limb-ischemic mice.

<p>(A) EPC was determined using flow cytometry. Forware SCatter (FSC) and Side SCatter (SSC) were used to gate the monocytes (R1). Hematopoietic stem cells were defined as Sca-1⁺/CD34⁺ cells (R2). EPCs were defined as Sca-1⁺/CD34⁺/Flk-1⁺ cells (R3). (B) The Sca-1⁺/CD34⁺/Flk-1⁺ cells in control (bar 1), non-GroEL1 (bar 2), 1 μg/kg BW GroEL1 (bar 3), 2 μg/kg BW GroEL1 (bar 4), and 4 μg/kg BW GroEL1 (bar 5)-administered mice 2 to 6 weeks after surgery. The results are expressed as the mean ± SEM (n=6, *<i>p</i> < 0.05 compared with naive C57BL/B6 mice at the same time point; †<i>p</i> < 0.05 compared with hind limb ischemia+non-GroEL1-treated C57BL/B6 mice at the same time point). (C) Immunostaining of ischemic hind limb muscle with anti-CD34 antibody conjugated to Alexa Fluor 633 (red) and anti-eNOS antibody conjugated to Alexa Fluor 488 (green) in C57BL/B6 mice treated with 1-4 μg/kg BW of GroEL1. The CD34-positive homed hematopoietic stem precursor cells were indicated with white arrows. By immunofluorescence staining, 2 μg/kg BW GroEL1 moderately decreased and 4 μg/kg BW GroEL1 severely decreased the number of eNOS/CD34-double-positive cells (arrow) in ischemic muscle compared with the non-GroEL1-treated group. Hoechst dye was used to counterstain the nucleus. The ischemic hind limb tissue was evaluated by fluorescence microscopy at a magnification of 400x. The bar graph shows the CD34 positivity/myofiber ratio. The results are expressed as the mean ± SEM (*<i>p</i> < 0.05 compared with hind limb ischemia+ non-GroEL1-treated C57BL/B6 mice).</p