Platelet-derived microvesicles drive vascular smooth muscle cell migration via forming podosomes and promoting matrix metalloproteinase-9 activity

Abstract

We have shown that platelet-derived microvesicles (PMVs) induce abnormal proliferation, migration, and energy metabolism of vascular smooth muscle cells (VSMCs) after vascular intimal injury. Here, we examined a novel role of podosome in mediating matrix metalloproteinase-9 (MMP-9) dependent VSMC migration induced by platelet-derived microvesicles (PMVs). VSMCs were isolated from the thoracic aortas of male Sprague Dawley (SD) rats and identified with immunofluorescent staining. Blood samples were collected from SD Rats, the platelets were isolated with density gradient centrifugation from the blood samples and activated by collagen I. Intriguingly, proteins expressed in platelets were found to participate in the positive regulation of podosome assembly using GO analysis by DAVID, and most of the proteins were found in extracellular exosomes. Of note, activated platelets indirectly induced VSMC migration via releasing PMVs which was verified using platelets and VSMCs transwell co-culture system. Besides, podosome, an invasive protrusion to mediate extracellular matrix (ECM) remodeling, was formed in VSMCs to induce cell migration. Furthermore, MMP-9 activity detected by gelatin zymography was used to verify the function of the podosome in ECM remodeling. The result indicated that MMP-9 activity was robustly activated in VSMCs to implement the function of the podosome. In addition, gelatin degradation was detected in intact VSMCs using a gelatin degradation assay after co-culture with platelets. Taken together, our data reveal a novel mechanism that PMVs promote VSMC migration via forming podosomes and inducing MMP-9 activity.

Keywords: Matrix metalloproteinase-9 (MMP-9); Migration; Platelet-derived microvesicles (PMVs); Podosome; Vascular smooth muscle cells (VSMCs).

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.

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.

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.