Endothelial cell microparticles act as centers of matrix metalloproteinsase-2 (MMP-2) activation and vascular matrix remodeling - PubMed (original) (raw)

Endothelial cell microparticles act as centers of matrix metalloproteinsase-2 (MMP-2) activation and vascular matrix remodeling

Thomas P Lozito et al. J Cell Physiol. 2012 Feb.

Abstract

Endothelial cell (EC)-derived microparticles (MPs) are small membrane vesicles associated with various vascular pathologies. Here, we investigated the role of MPs in matrix remodeling by analyzing their interactions with the extracellular matrix. MPs were shown to bind preferentially to surfaces coated with matrix molecules, and MPs bound fibronectin via integrin α(V) . MPs isolated from EC-conditioned medium (Sup) were significantly enriched for matrix-altering proteases, including matrix metalloproteinases (MMPs). MPs lacked the MMP inhibitors TIMP-1 and TIMP-2 found in the Sup and, while Sup strongly inhibited MMP activities but MPs did not. In fact, MPs were shown to bind and activate both endogenous and exogenous proMMP-2. Taken together, these results indicate that MPs interact with extracellular matrices, where they localize and activate MMP-2 to modify the surrounding matrix molecules. These findings provide insights into the cellular mechanisms of vascular matrix remodeling and identify new targets of vascular pathologies.

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Figures

Figure 1

MicroEC MPs are plasma membrane derived and bind to extracellular matrix substrates. (A) MPs isolated from microECs were observed by negative staining and transmission electron microscopy as intact membranous structures. (B) Size profile of isolated MPs. (C) Fluorescence photometry analysis of membranes (Mem), MP, and Sup samples (1, 5, 10, or 20 μg) isolated from DiO-labeled microECs. MPs and Mem samples were enriched for DiO. *** p < 0.001 compared to Sup values of corresponding protein amounts (D) Western blot analysis comparing localization of membrane proteins (N-cadherin, VE-cadherin, E-cadherin, Integrin αV) among microEC total protein (TP), MPs, and Sup fractions. Blots of actin and GAPDH were included as loading controls. (E) Comparison of the amount of MPs produced under control conditions or in the presence of IL-1β, TNF-α, or hypoxia. ** p < 0.01 (F) DiO-labeled MPs bound to surfaces coated with Matrigel, fibronectin, or gelatin. * p < 0.05, *** p < 0.001 compared to BSA controls (G) Higher percentages of DiO-labeled MPs produced in the presence of IL-1β, TNF-α, or hypoxia bound fibronectin-coated surfaces than those produced under control conditions. ** p < 0.01 Results are means ± SD.

Figure 2

MicroEC MPs bind endogenous fibronectin. (A) Fibronectin ELISA analysis of TP, MP, and Sup samples. *** p < 0.001 (B) Fibronectin and integrin αVβ3 western blots of TP, MP, and Sup samples collected from microECs cultured under control conditions or in the presence of IL-1β, TNF-α, or hypoxia. (C) High levels of EDTA during isolation reduced fibronectin associated with MPs. (D) Treatment with 2.5 mM RGD peptide, but not RAD, during isolation reduced fibronectin content of MPs. Results are means ± SD.

Figure 3

MicroEC MPs bind exogenous fibronectin with integrin αV. (A) FITC-labeled fibronectin (Fn-FITC) incubated with microEC-CM co-precipitated with MP samples. Ultracentrifugation pellets (UCP) and supernatants (UCS) collected from samples of Fn-FITC incubated without MPs were included as controls. All samples were analyzed with fluorescent scans of SDS-PAGE protein gels and integrin αV western blots. (B) UCP and MPs incubated with or without Fn-FITC were also analyzed via fluorescence photometry for Fn-FITC content. *** p < 0.001 (C) Incubating EC-CM with Fn-FITC in the presence of 10 mM EDTA, or RGD (2.5 mM), but not RAD (2.5 mM), interfered with exogenous fibronectin localization to MP fractions. MP and Sup samples were analyzed for FN-FITC content via fluorescence scans of protein gels, and (D) MP samples were also analyzed by fluorescence photometry. *** p < 0.001 (E) “Bait and fish” method involving the biotin transfer reagent sulfo-SBED in identifying fibronectin receptors. Exogenous fibronectin was pre-labeled with sulfo-SBED and incubated with MP proteins, including fibronectin receptors. Upon activation with UV light, the unreacted arm of sulfo-SBED was covalently crosslinked to fibronectin receptors. The disulfide-linkage between fibronectin and the fibronectin receptors was cleaved, and the samples were subjected to SDS-PAGE. Upon transfer to a PVDF membrane, fibronectin receptors were detected with fluorescent neutravidin probes. (F) 120 kDa fibronectin receptors were detected in MP samples crosslinked with sulfo-SBED-labeled fibronectin. Bands corresponding to fibronectin receptors were also detected in integrin αV western blots. Biotin was not transferred to fibronectin receptors in the presence of EDTA (10mM), or RGD (2.5 mM). (G) Fibronectin and integrin αV western blot analyses of samples of sulfo-SBED-labeled fibronectin, MP protein alone, or sulfo-SBED fibronectin incubated with MP proteins precipitated by monomeric avidin. Results are means ± SD.

Figure 4

MPs contain multiple MMPs. (A) Western blot detection of MMP-2, MMP-13, MMP-7, MMP-1 and plasminogen in MP and Sup protein samples treated with APMA or vehicle control. (B) MMP-2 ELISA analysis of MP and Sup samples. *** p < 0.001 (C) Fibrin zymography of and plasminogen western blot of MP and Sup fractions. Samples of plasmin (10 ng) were included in fibrin zymograms as positive controls. (D) Samples of microEC TP, MP, and Sup were analyzed by MMP-14, MMP-15, and MMP-16 western blots. Results are means ± SD.

Figure 5

MMPs are enriched in MMP- activities. (A) MicroEC Mem and MP samples, either treated with APMA or vehicle control, exhibited higher MMP activities than Sup samples in assays using 3 different fluorogenic MMP substrates. Asterisks above APMA values denote comparisons to corresponding vehicle control samples, while asterisks above Mem and MP vehicle control samples denote comparisons to Sup vehicle control samples. ** p < 0.01, *** p < 0.001 Results are means ± SD. (B) Gelatin zymography analysis of Mem, MP, and Sup fractions. (C) MMP-14 and pan-cadherin western blot analysis of microEC TP, Mem, MP, and Sup fractions.

Figure 6

MPs lack the MMP-inhibition activity found associated with the rest of microEC-secreted factors. (A) Samples of microEC MP and Sup fractions were analyzed via reverse zymography and by TIMP-1, TIMP-2, and TIMP-4 western blots. (B) Sup samples inhibited MMP-2, MMP-9, MMP-1, MMP-13, and MMP-14 activities more than Mem and MP samples. *** p< 0.001 Results are means ± SD.

Figure 7

MPs remain functional units of MMP activity in pathological conditions simulated in vitro with cytokines and hypoxia. (A) MMP-2 and MMP-1 western blot of MP and Sup fractions collected from microECs cultured under simulated pathological conditions (exposure to IL-1β, TNF-α, or hypoxia). (B) MMP-14 western blot of microEC MP and TP samples from control and pathological conditions. (C) MMP activity assays with MMP substrates I and II. ***p < 0.001 (D) Reverse zymography and TIMP-1, 2, and 4 western blots of MP and Sup produced in the presence of IL-1β, TNF-α, or hypoxia. (E) MMP-2 and MMP-9 inhibitor assays. ***p < 0.001 Results are means ± SD.

Figure 8

MPs activate endogenous MMP-2. (A) Gelatin zymography and MMP-2, -1, and -13 western blot analyses of MP and Sup fractions incubated for 0 or 20 hours at 37°C. Active MMP-2 was detected in incubated MP samples. (B) MMP activities of MP, but not Sup, samples increased after 20 hours of incubation at 37°C. *** p < 0.001 (C) Time-dependence of MMP-2 activation in MP fractions over 20 hours. (D) MMP-2 western blot of MP samples incubated for 20 hours under control conditions or in the presence of inhibitors: 10 mM EDTA, 10 mM 6-aminocaproic acid (6-ACA), GM6001 vehicle control (VC-G), 20 μM GM6001, MMP-2/9 Inhibitor IV vehicle control (VC-IV), 2 μM MMP-2/9 inhibitor IV (2/9 IV), TIMP-1 (1 nM (+), 5 nM (++), 10 nM (+++)) or TIMP-2 (+, ++, +++). (E). MMP-14 and MMP-1 activity assays of inhibitor effects. *** p < 0.001 (F) Gelatin zymography and (G) MMP activity assays comparing MPs and Sup isolated from microECs treated with IL-1β, TNF-α, or hypoxia following incubation for 0 or 20 at 37°C. *** p < 0.001 Results are means ± SD.

Figure 9

MPs bind and activate exogenous MMP-2. (A) MMP-2 western blot analysis of MP fractions pre-incubated with or without MSC-CM. Ultracentrifugation pellets (UCP) collected from samples of MSC-CM incubated without MPs were included as negative controls, and blots of actin and GAPDH served as loading controls. MSC-secreted MMP-2 localized to MP fractions. (B) UCP and MPs incubated with or without MSC-CM were analyzed via MMP activity assays. *** p < 0.001 (C) MSC-CM samples were incubated with MP or Sup fractions for 0 or 20 hours and analyzed via MMP-2 western blots. Active MMP-2 was detected primarily in MSC-CM samples incubated with MPs. (D) MSC-CM samples incubated with MPs exhibited higher increases in MMP activity than those incubated with Sup fractions. *** p < 0.001 (E) Time-dependence of MMP-2 activation in MSC-CM samples incubated with MP fractions over 20 hours. Results are means ± SD.

Figure 10

MPs degrade bound matrix molecules with MMPs. (A) Fn-FITC samples co-isolated with MP or Sup were incubated for 0 or 3 days and analyzed with fluorescent scans of SDS-PAGE protein gels and integrin αV western blots. Fn-FITC incubated with MPs was degraded. (B) Samples of Fn-FITC co-precipitated with MPs and incubated with GM6001 (20 μM) exhibited less degradation than samples incubated with vehicle controls (VC). (C) EDTA (10 mM) and RGD (2.5 mM), but not RAD (2.5 mM), interfered with degradation of Fn-FITC by MPs.

References

1. Abid Hussein MN, Meesters EW, Osmanovic N, Romijn FP, Nieuwland R, Sturk A. Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo. J Thromb Haemost. 2003;1:2434–2443. - PubMed
1. Ades EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC, Lawley TJ. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol. 1992;99:683–690. - PubMed
1. Anderson HC. Matrix vesicles and calcification. Curr Rheumatol Rep. 2003;5:222–226. - PubMed
1. Berckmans RJ, Neiuwland R, Boing AN, Romijn FP, Hack CE, Sturk A. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost. 2001;85:639–646. - PubMed
1. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta. 2000;1477:267–283. - PubMed

Endothelial cell microparticles act as centers of matrix metalloproteinsase-2 (MMP-2) activation and vascular matrix remodeling - PubMed (original) (raw)