1. Introduction
2. Materials and methods
2.1. Platelet isolation, purification and activation
Fig. 1. Platelet microvesicles (PMVs) drive VSMCs migration. The platelets were collected and activated, VSMCs were isolated, identified, and treated with PMVs. (A) The flow chart showed the processes of platelet isolation, purification, and activation. (B) Representative immunofluorescent image of VSMCs. VSMCs were identified by SMA (red). Nuclei (blue) were stained with DAPI. Scale bar = 50 μm. (C) Schematic diagram of VSMCs co-cultures with platelets. (D) Representative images of VSMC migration. VSMCs were co-cultured with platelets or HEPES/Tyrode's buffer for 24 h and images were captured at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h respectively. The migration rate was detected by the wound healing assay. Scale bar = 200 μm. (E) The line chart showed the wound closure percentage of VSMCs after co-culture with platelets (n = 4). Data are presented as Mean ± SEM, ∗∗P < 0.01. |
2.2. Cell culture
2.3. Platelets and VSMCs co-culture system
2.4. Wound healing assay
2.5. GO analysis
2.6. Immunofluorescent staining
2.7. Gelatin zymography
2.8. Gelatin degradation assay
2.9. Statistical analysis
3. Results
3.1. Platelets promote VSMC migration in a contactless way
3.2. PMVs promote VSMC migration via inducing podosomes formation
Fig. 2. PMVs drive VSMC migration via inducing podosome formation. Human platelet protein composition was obtained from the primary source [25] and used for GO analysis. (A) The top 300 proteins expressed in platelets were used for GO analysis by DAVID (https://david.ncifcrf.gov/tools.jsp). Nine biological processes associated with VSMC phenotype were elected, platelets were found to participate in the positive regulation of podosome assembly and 60 proteins were found to involve in these processes. (B) Sixty proteins included in 9 biological processes were categorized by Cellular Component analysis, 51 of them were found in extracellular exosomes. (C) Representative immunofluorescent image of podosomes in VSMCs. Podosomes were identified based on the co-localization of cortactin (green) with F-actin (red). Nuclei (blue) were stained with DAPI. Scale bar = 50 μm. The platelets were collected and activated, and VSMCs were isolated, identified, and treated with PMVs. (D) Representative immunofluorescent image of podosomes in VSMCs, indicating podosome formation after PMV treatment. Podosomes were identified based on the co-localization of cortactin (green) with F-actin (red). Nuclei (blue) were stained with DAPI. Arrows indicate the podosomes in the PMVs treating group compared to the control group. Scale bar = 50 μm. (E) Quantitative analysis of podosome number. Platelets increased the number of podosomes in VSMC. Data are presented as Mean ± SEM, ∗∗∗P < 0.001. |
3.3. PMVs promote VSMC migration via elevating MMP-9 activity
Fig. 3. PMVs drive VSMC migration via inducing MMP-9 activity in podosomes. The platelets were collected and activated, and VSMCs were isolated, identified, and treated with PMVs. (A) The MMP activity in VSMCs co-cultured with platelets or HEPES/Tyrode's buffer for 24 h was detected by gelatin zymography. (B) Quantitative analysis of MMP-9 activity. Platelets increased the activity of MMP-9 in VSMC. (C) Gelatin degradation was detected using a gelatin degradation assay. VSMCs were stained with DAPI (blue) and rhodamine-phalloidin (red). Oregon Green 488 gelatin was green. Arrows indicate the degradation areas (dark). Scale bar = 30 μm. (D) Quantitative analysis of gelatin degradation. Data are presented as Mean±SEM, ∗∗P<0.01, ∗∗∗P<0.001. |
Fig. 4. PMVs drive VSMC migration by forming podosomes and promoting MMP-9 activity. The number of podosomes was increased, MMP-9 activity was elevated and the VSMC migration rate was promoted after PMV treatment. |