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First published online 3 May 2005
doi: 10.1242/jcs.02360

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MMP-1 activation by serine proteases and MMP-10 induces human capillary tubular network collapse and regression in 3D collagen matrices

Department of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, 208 Reynolds Medical Building, College Station, TX 77843-1114, USA

View larger version (88K): [in a new window]   Fig. 1. Plasma kallikrein induced MMP-1 activation and contraction of endothelial tube networks. ECs were suspended in collagen matrices and plated in 96-well tissue culture plates (4.5 mm diameter) (A) or 384 micro-well plates (3 mm square) (B). Culture media contained plasma kallikrein at 1 µg/ml. The same culture well was photographed at the indicated times (in hours) during the capillary tube regression and collagen gel contraction process. (C) Conditioned media were collected and triplicate cultures were examined for evidence of capillary tube regression and collagen gel contraction (+ indicates occurrence of gel contraction). Samples were run on SDS-PAGE gels, blotted to PVDF membranes and probed with anti-MMP-1 antibodies. Arrows indicate the position of MMP-1 zymogen and arrowheads indicate activated MMP-1. Note the absence of activated MMP-1 (arrowhead) in the absence of kallikrein. Bar, 500 µm.

View larger version (108K): [in a new window]   Fig. 2. Time-lapse photography of plasma kallikrein-induced MMP-1 activation and capillary tube regression. ECs were suspended in collagen matrices and cultured in the absence or presence of 1 µg/ml kallikrein. Sequential time-lapse photographs were taken of individual EC fields at the time in hours denoted in each panel. (A) In the absence of kallikrein, ECs form sprouts, vacuoles and tubular networks, a process known as morphogenesis. (B) In the presence of kallikrein, morphogenesis is initiated (arrows); however, capillary tubes and vacuoles collapse (arrowheads), eventually resulting in individual EC rounding, aggregation and death. (C) ECs were allowed to undergo morphogenesis and establish EC networks for 48 hours prior to the addition of kallikrein. An EC-lined tube (arrows) is visible undergoing tube regression (arrowheads) prior to the onset of gel contraction. Bar, 100 µm.

View larger version (36K): [in a new window]   Fig. 3. Quantification of plasma kallikrein- or prekallikrein-mediated MMP-1 activation and capillary tube regression in 3D collagen matrices. ECs were suspended in collagen matrices and placed in a quantifiable 384 micro-well regression assay (15 µl gels, n=8 gels/condition) as shown in Fig. 1B. At time zero, media containing varying doses of active plasma kallikrein (A) or 2 µg/ml plasma prekallikrein and varying doses of Factor XII (B) were added to cultures. Collagen gels were monitored for tube regression and gel contraction over time and percentage of gel contraction was calculated (see Materials and Methods for details). Conditioned media were collected at 50 hours and analyzed for MMP-1 expression and activation. (C) ECs were suspended in collagen matrices. Culture media contained prekallikrein at 2 µg/ml, along with varying concentrations of HMW kininogen as indicated and either no cations or 100 µM ZnCl2 or MnCl2. Triplicate cultures were examined after 24 hours of culture for evidence of capillary tube regression and gel contraction (indicated by +) and analyzed for MMP-1. For all western blot analyses, arrows indicate the position of MMP-1 zymogen and arrowheads indicate activated MMP-1.

View larger version (33K): [in a new window]   Fig. 4. Activation of MMP-1 correlates with increased collagenase activity in ECs during plasma kallikrein- and plasmin-induced capillary tube regression. ECs were suspended in collagen matrices and cultured in the absence (Control) or presence of active plasmin (PL) or kallikrein (Kal) at two doses (0.5 µg/ml, 2.5 µg/ml). Gels were monitored for tube regression and gel contraction via the 384 micro-well regression assay (as in Fig. 3). At the indicated time points, conditioned media were collected and MMP-1 levels were analyzed by western blotting. In addition, conditioned media were analyzed for collagenase activity via the DQ collagenase assay. Triplicate wells of conditioned media from the above time points were incubated with 25 µg/ml of DQ collagen overnight at room temperature. Absorbance was measured at 528±20 nm via a fluorescent microplate reader. Background fluorescence was subtracted prior to reporting of fluorescence in arbitrary units as mean±s.d. (n=3). Time to 50% gel contraction is reported above each data set. In each case, fluorescence corresponded with the degree of MMP-1 activation on the western blot and the time to 50% collagen gel contraction. Arrows and arrowheads indicate the position of MMP-1 in latent or activated forms, respectively.

View larger version (67K): [in a new window]   Fig. 5. Treatment of ECs with MMP-1 siRNA decreases MMP-1 protein expression and delays capillary tube regression and collagen gel contraction. ECs were transfected as described with MMP-1, 2 macroglobulin (A2Mac), or luciferase control siRNA duplexes at a final concentration of 200 nM, suspended in collagen matrices and placed in the quantitative 384 micro-well regression assay (as in Fig. 3). At time zero, culture media was added with or without serine proteases at the doses indicated and the percentage of gel contraction was calculated over time. (A) Culture media contained control media, plasma kallikrein, or prekallikrein and Factor XII at the indicated doses. Upon completion of gel contraction, conditioned media were collected and analyzed for MMP-1 expression and activation. (B) ECs transfected with the indicated siRNAs were established in collagen matrices for 46 hours in the absence of serine proteases and were fixed, stained and photographed. Arrows denote newly established EC vascular networks and open lumenal structures. Bar, 100 µm.

View larger version (35K): [in a new window]   Fig. 6. MMP-10 is induced during EC morphogenesis, is activated by serine proteases and treatment of ECs with siRNA targeting MMP-10 delays capillary tube regression and collagen gel contraction induced by multiple serine proteases. ECs were suspended in collagen matrices as described in the absence (A) or presence (B) of varying doses of plasma kallikrein. Gels were monitored for contraction (indicated by +) and conditioned media were collected for western blot analysis of MMP-10 at the indicated time points (A) or at 72 hours (B). Note that latent MMP-10 (arrows) is visible in each condition; however, active MMP-10 (arrowheads) is visible only in the presence of kallikrein. (C) ECs were transfected as described with MMP-1, MMP-10, MMP-2, MMP-9 or luciferase control siRNA and suspended in collagen matrices. At time zero, culture media was added with or without prekallikrein (2 µg/ml) and Factor XII (1 µg/ml) and percentage gel contraction was calculated over time. Conditioned media were collected and analyzed for MMP-10 or MMP-1 expression and activation, as well as for MMP-2 and MMP-9 (data not shown). Arrows and arrowheads indicate the latent or activated forms of MMPs, respectively.

View larger version (39K): [in a new window]   Fig. 7. Increased expression of stromelysin-2 (MMP-10) and stromelysin-1 (MMP-3) induce rapid capillary tube regression and collagen gel contraction via activation of MMP-1 zymogen. ECs were infected with GFP, MMP-10, or MMP-3 adenoviruses 24 hours prior to suspension in collagen gels. Gels were monitored over time for contraction and conditioned media were collected at the completion of the contraction response. (A) ECs infected with GFP Ad or MMP-10 Ad were cultured in control media or media containing active kallikrein (0.5 µg/ml), prekallikrein (2 µg/ml) and Factor XII (1 µg/ml) (Prekal, XII), or plasminogen (2 µg/ml) (Plg). Note that low levels of MMP-10 were present in GFP infected cells, whereas cells infected with MMP-10 Ad had increased levels of latent (arrows) and active (arrowheads) forms of MMP-10, regardless of the absence or presence of serine proteases. Addition of serine proteases accelerated capillary tube regression and gel contraction. (B) ECs infected with GFP Ad or MMP-3 Ad were cultured in control media or media containing kallikrein, prekallikrein and Factor XII, or plasminogen at the concentrations listed in panel A. No detectable MMP-3 was present in GFP infected cells, while cells infected with MMP-3 Ad had increased levels of latent (arrows) and active (arrowheads) forms of MMP-3, regardless of the absence or presence of serine proteases. In all cases, the addition of serine proteases led to more rapid capillary tube regression and gel contraction. (C) ECs infected with GFP Ad or MMP-10 Ad were cultured in the absence or presence of 0.5 µg/ml kallikrein. Gels were monitored for contraction (indicated by +) and conditioned media were collected at the indicated time points. Under control conditions, latent MMP-1 (arrows) was present in both GFP and MMP-10 expressing cells, however, active MMP-1 (arrowheads) was present only in cells expressing increased levels of MMP-10. In the presence of kallikrein, a small amount of active MMP-1 was present in the GFP-expressing cells, however, the presence of both kallikrein and MMP-10 substantially increased the amount of active MMP-1, which directly correlated with the time to tube regression and gel contraction.

View larger version (32K): [in a new window]   Fig. 8. MMP-1 and MMP-10 regulate EC tube regression in three dimensional collagen matrices. (A-C) ECs were transfected with siRNA against MMP-1 or luciferase control. Recombinant adenoviruses were used 24 hours later to deliver GFP or MMP-10. Cultures were established in the absence (A) or presence of 0.5 µg/ml kallikrein (B) or 2 µg/ml plasminogen (C). Gels were monitored every 4 hours for tube regression and percent gel contraction was recorded. At 56 hours, cultures were fixed and conditioned media were collected for western blot analysis of MMP-1 levels (A-C). Note that in all panels a decrease in latent MMP-1 was present in cells treated with MMP-1 siRNA compared to luciferase control. MMP-1 activation occurred due to an increase in MMP-10 levels and also in the presence of the serine proteases plasma kallikrein and plasminogen. (D) ECs were transfected with siRNA against MMP-10 or luciferase control as described. 24 hours later, ECs were infected with GFP-Ad or MMP-1 Ad. Cultures were established in the absence or presence of 2 µg/ml plasminogen. Cultures were monitored every 4 hours for tube regression and percentage gel contraction was recorded. At 56 hours, cultures were fixed and conditioned media were collected for western blot analysis of MMP-10 levels. Note that in all cases, a decrease in latent MMP-10 was present in cells treated with MMP-10 siRNA compared to controls. Arrows and arrowheads indicate the position of MMP-10 in latent or activated forms, respectively.

View larger version (26K): [in a new window]   Fig. 9. Schematic diagram illustrating the ability of multiple serine proteases to activate MMP-1 and MMP-10 zymogens to control capillary tube regression in 3D collagen matrices. MMP-1 is activated directly by either serine proteases or activated MMP-10 (bold arrow). MMP-10 and serine proteases act synergistically to super-activate MMP-1, leading to type I collagen degradation, capillary tube regression and EC apoptosis. MMP-10 contributes to capillary tube regression by activating MMP-1 zymogen (bold arrows) and may contribute to degradation of basement membrane matrix leading also to tube regression and apoptosis (dashed arrow).

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