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Originally published online as doi:10.1189/jlb.1106687 on March 29, 2007

Published online before print March 29, 2007
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(Journal of Leukocyte Biology. 2007;81:1496-1503.)
© 2007 by Society for Leukocyte Biology

Ang-2 and PDGF-BB cooperatively stimulate human peripheral blood monocyte fibrinolysis

Louise Bezuidenhout*, Mona Bracher*, Glenda Davison{dagger}, Peter Zilla* and Neil Davies*,1

* Cardiovascular Research Unit and
{dagger} Department of Hematology, University of Cape Town, Cape Town, South Africa

1 Correspondence: Cardiovascular Research Unit, Chris Barnard Building, University of Cape Town Medical School, Anzio Road, Observatory, 7925, Cape Town, South Africa. E-mail: neil.davies{at}uct.ac.za


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ABSTRACT
 
Angiopoietin-2 (Ang-2) is a growth factor, which was identified originally as playing a critical role in vessel remodeling during angiogenesis. More recent evidence has indicated additional involvement in vascular homeostatic responses such as coagulation and inflammation, which are central to wound healing. We therefore determined whether a relationship existed between Ang-2 and monocytes, one of the initial cell types to be recruited to a wound, in the context of fibrin clot invasion. Ang-2 significantly increased monocyte invasion of fibrin in the presence of serum. In the absence of serum, it required a combination of Ang-2 and platelet-derived growth factor BB (PDGF-BB) to increase invasion by threefold. Furthermore, it was shown that the heightened invasion was dependent on serine proteases and matrix metalloproteinases (MMPs) and that the combination of Ang-2 and PDGF-BB increased urokinase plasminogen-activator receptor expression, as well as MMP-9 and membrane type 1 MMP expression. These data give further credence to the concept of Ang-2 as a key regulator of several essential phases of wound healing.

Key Words: wound healing • proteases • matrix metalloproteinases • invasion


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INTRODUCTION
 
The angiopoietins are a family of endothelial growth factors known to play an important role in blood vessel stability [1 ]. Angiopoietin-1 (Ang-1) induces Tie-2 receptor phosphorylation, which results in the maintenance of vessel stability and the reduction of inflammation [2 ]. In contrast, Ang-2 acts as a structural antagonist to Ang-1 and initiates vessel destabilization [1 ]. Ang-2 expression has been shown to be highly up-regulated at sites of vascular remodeling, where its levels are found to be elevated consistently relative to Ang-1 [3 , 4 ], implicating it as a critical factor in the initiation of angiogenesis [5 ]. This is supported by the fact that binding of Ang-2 to the Tie-2 receptor results in sprouting of vessels in the presence of vascular endothelial growth factor (VEGF) and vessel regression in its absence [6 , 7 ].

Adult angiogenesis is restricted to the female reproductive cycle, tissue repair, and pathological remodeling. The initial stage of angiogenesis is characterized by hyperpermeability of the vasculature [8 , 9 ], which leads to plasma exudation and deposition of plasma proteins. Fibrinogen, a major component of plasma, polymerizes in the presence of tissue factor and/or platelet degranulation, leading to formation of a fibrin clot [10 ], which is believed to serve as an extracellular matrix enabling migration of cells involved in the formation of a new vessel sprout [9 , 11 ].

In almost all of these instances of adult angiogenesis, the growth of new vessels is associated with inflammation [9 ]. During tissue repair, pathological remodeling or tumoral growth, an inflammatory infiltrate composed mainly of neutrophils and monocytes, often precedes or accompanies angiogenesis [8 , 12 ]. Conversely, monocyte depletion from the angiogenic environment has been shown to lead to impaired angiogenesis [12 ], suggesting that inflammation and angiogenesis are interdependent processes [13 ].

It is interesting that in addition to its role in angiogenesis, recent evidence has strongly suggested that Ang-2 plays a role in rapid endothelial cell-related responses such as hemostasis and inflammation [2 , 14 ]. Ang-2 was shown to be stored within the endothelial cell in Weibel-Palade bodies [2 ]. These intracellular storage pools allow for immediate response to vascular perturbations through release of preformed, bioactive factors, avoiding the time-consuming need for gene up-regulation, transcription, and translation [15 ]. Ang-2 is released in conjunction with the von Willebrand factor, a critical factor in hemostasis and the major constituent of Weibel-Palade bodies.

Ang-2 has also been found to regulate the adhesion of rolling leukocytes to the endothelium through sensitizing the endothelial cell to the proinflammatory cytokine TNF-{alpha} and increasing expression of TNF-{alpha}-induced adhesion proteins ICAM-1 and VCAM-1 [14 ]. It is interesting that in addition to this indirect recruitment of leukocytes, Ang-2 has been shown to directly stimulate adhesion and migration of neutrophils [16 , 17 ].

As mentioned previously, sites of adult angiogenesis, such as wound healing, encompass hemostasis, inflammation, and angiogenesis. An obvious and necessary step for the participation of monocytes in the wound-healing process is their invasion of the fibrin clot through proteolytic mechanisms. Ang-2 has already been shown to mediate heightened invasiveness of glioma tumors and was linked to the up-regulation of proteolytic elements matrix metalloproteinase 2 (MMP-2), and membrane type 1 (MT1)-MMP [18 ]. A further indication of the involvement of Ang-2 in the regulation of proteolysis is its induction of MMP-1, MMP-9, and urokinase plasminogen activator (uPA) in endothelial cells [19 ].

The discovery of Ang-2 as a Weibel-Palade body molecule, its interaction with inflammatory cells, as well the recent identification of its involvement in cellular invasiveness via increased proteolysis led us to investigate the potential influence of Ang-2 on the invasion of monocytes into fibrin clots. We demonstrated that Ang-2 does indeed up-regulate monocyte fibrinolysis via a proteolytic mechanism. It is intriguing that we also found that the Ang-2-induced invasiveness was dependent on the presence of platelet-derived growth factor BB (PDGF-BB), another early-release cytokine, and operated via serine protease and MMP fibrinolytic pathways.


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MATERIALS AND METHODS
 
Isolation of human monocytes
Human monocytes were obtained from peripheral blood of healthy, medication-free donors and isolated by gradient centrifugation using Histopaque-1077 (Sigma, Steinheim, Germany). The monocytes were then purified by adherence for 3 h to cell culture flasks (Corning Inc., Corning, NY, USA) pre-treated with 5 mg/ml fibronectin (Roche, Mannheim, Germany). Non-adherent cells were washed off with room temperature RPMI-1640 (Sigma). Monocytes were then lifted with 5 nM EDTA (Sigma) in PBS and pelleted down in human pooled serum. The cells were resuspended in RPMI-1640 (Sigma), and cell number and viability were assessed using 0.1% Trypan blue (Sigma). The purity of the isolations, assessed using flow cytometry with PE-conjugated, antihuman CD14 (Becton Dickinson, Franklin Lakes, NJ, USA), was above 95%. Cells were used for in vitro assays immediately after counting. All experiments involving human materials were performed after approval from the University of Cape Town (Cape Town, South Africa) Ethical Committee, in accordance with the Declaration of Helsinki. Informed consent was received from all donors participating in the study.

Transwell invasion assays
Purified human plasma fibrinogen stock (Fluka, Switzerland) was dialyzed against two changes of PBS (pH 7.5) and one change of HEPES-buffered saline (pH 7.5), and the concentration was determined by spectrophotometer readings at OD280. The upper side of Transwell-permeable support chamber membranes (8 µm pores, Corning) was coated with fibrin gel, using bovine thrombin (1.4 u/ml, Sigma) and the fibrinogen stock at 3 mg/ml. The upper compartment was seeded with monocytes in RPMI-1640 medium containing 20% human pooled serum (Sigma) and the lower compartment filled with serum-containing RPMI-1640 medium. Recombinant human Ang-2 (carrier-free, R&D Systems, Minneapolis, MN, USA) activity was tested by dilution curve analysis (100, 250, 500, 1000 ng/ml). In all further experiments, Ang-2 was added at 500 ng/ml to the upper or lower compartment. The Transwell chambers were incubated in 5% CO2 and 20% O2 overnight at 37°C.

Following incubation, the level of fibrin invasion was determined by the number of cells present in the bottom compartment. Transwell inserts were removed, and the cells in the bottom compartment were fixed with 2.5% gluteraldehyde (Sigma). Cells were visualized at 50x magnification (Leica DM IRBE, Solms, Germany), and all cells in a well were counted using the automated counting function on Leica QWin image analysis software (Leica, Solms, Germany).

In certain experiments, inhibitors were added to the top and bottom compartments, as well as to the fibrin. The inhibitors were GM6001 (20 µM, Chemicon International, Temecula, CA, USA), aprotinin (20 µg/ml, Bayer, Germany), and anti-uPA receptor (uPAR; 4 µg/ml, Abcam, Cambridge, UK).

Human pooled serum was analyzed for PDGF-BB using an ELISA kit (R&D Systems) according to the manufacturer’s specifications. Certain experiments were repeated using serum-free RPMI 1640 (0.1% BSA, Sigma), 0.01% insulin, transferrin, selenium (ITS) liquid medium supplement (Sigma), Ang-2 (500 ng/ml), and/or PDGF-BB (15 ng/ml, PeproTech Inc., Rocky Hill, NJ, USA) added to the lower compartment. PDGF-BB concentration is described previously in in vitro monocyte studies [20 , 21 ].

Analysis of monocyte cell lysates
Monocytes were seeded in serum-free RPMI 1640 containing Ang-2 (500 ng/ml) and PDGF-BB (15 ng/ml) at 37°C with 5% carbon dioxide for 15 h. The conditioned media were drawn off for further analysis. Cells were washed thoroughly in PBS and lysed for 2 h at 4°C using radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 5 mM iodoacetamide, 2 mM PMSF, Sigma) [22 ]. The lysates were spun down at 10,000 RPM for 5 min and resolved using 10% SDS-PAGE under reducing conditions.

Briefly, after resolving cell lysates using 10% reducing SDS-PAGE, the proteins were transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech), which was then blocked with 5% nonfat milk powder in TBS-T. Detection was performed using goat polyclonal uPAR antibody (Abcam) or rabbit polyclonal MT1-MMP antibody (Chemicon International) with HRP-conjugated rabbit polyclonal to goat IgG antibody and donkey polyclonal to rabbit antibody (Abcam), respectively.

The protein bands were visualized using ECL (Amersham Pharmacia Biotech) and quantified using the densometric analysis software, NIH-1.62.

Following detection, the membrane was washed thoroughly three times with TBS-T, and the detection procedure was repeated using rabbit polyclonal to ß-actin-loading control antibody (Abcam) and donkey polyclonal to rabbit IgG antibody (Abcam).

The relative quantity of uPAR or MT1-MMP in each sample was determined by normalizing the densometric values according to the ß-actin values.

Analysis of monocyte-conditioned medium
Conditioned medium was also analyzed for proteolytic activity using gelatin zymography. Conditioned medium samples were resolved using 10% SDS-PAGE containing 0.1% gelatin (Sigma) under nonreducing conditions.

Gels were treated as described previously [23 ] by washing thoroughly with 2.5% Triton X-100 to remove SDS and then incubating in substrate buffer (50 mM Tris, 5 mM calcium chloride, pH 8) for 18 h at 37°C with agitation. Following that, the gel was stained in Coomassie blue for 30 min and destained for 2 h using 15% methanol and 7.5% glacial acetic acid. The gels were scanned, and zones of lyses were analyzed using the densometric analysis software, NIH-1.62.

Determination of Tie-2 expression in human monocytes
Semiquantitative RT-PCR
Monocytes were incubated in serum-free RPMI 1640 containing Ang-2 (500 ng/ml) and/or PDGF-BB (15 ng/ml) at 37°C with 5% carbon dioxide for 6 h. Human saphenous vein endothelial cells (HSVEC; Passage 3) and human dermal fibroblasts (Passage 3) were used as positive and negative controls, respectively. RNA was isolated from all cell preparations using the RNeasy Micro RNA isolation kit (Qiagen GmbH, Hilden, Germany) and quantified using the Ribogreen RNA quantification kit (Molecular Probes, Leiden, The Netherlands) according to the manufacturer’s specifications.

cDNA was made by RT using the Impromp-II RT kit (Promega, Madison, WI, USA) and 100 ng RNA sample, according to the manufacturer’s specifications. Semiquantitative PCR was performed using primers to Tie-2 (left 5' TCGAGGAGAGGCAATCAGG 3', right 5' CTGAGCATGAGGCAGGTGT 3') and hypoxanthine guanine phosphoribosyl transferase (HPRT; left 5' CCCTGGCGTCGTGATTAGT 3', right 5' GCCTCCCATCTCCTTCATC 3') on an automated PCR machine (PCR Express, Hybaid, Chile) with 30 cycles of 60°C/1 min annealing, 72ºC/2.5 min extension, and 94°C/30 s denaturation.

PCR products were analyzed on a 1% agarose gel containing 0.1% ethidium bromide and photographed using the Biorad GelDoc system.

Tie-2 flow cytometry
The expression of the Tie-2 (R&D Systems) antibody was analyzed using direct immunofluorescent techniques. The monocytes were separated from peripheral blood using standard histopaque centrifugation and resuspended in PBS with 0.5% BSA. HSVEC (Passage 3) were used as a positive control and human dermal fibroblasts (Passage 3), as a negative control.

To prevent any nonspecific binding of the antibody, equal volumes of the cells and AB serum were incubated at room temperature for 10 min prior to the addition of the antibody. Thereafter, 10 µl PE-conjugated Tie-2 antibody was added. PE-conjugated anti-IgG1 (Becton Dickinson) was used as a negative, isotypic control. The cells were incubated at room temperature for 30 min. After washing the cells with PBS, analysis took place on a FACSCalibur flow cytometer (Becton Dickinson). A minimum of 2000 cells was counted, and the population analyzed was gated using forward- and side-scatter. The percentage positivity was assessed using single color histograms.

Statistical analysis
One-way ANOVA was performed when more than two groups were compared. A two-tailed Student t test was used to assess differences between two groups. P < 0.05 was considered significant. Data are expressed as mean values ± SEM.


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RESULTS
 
Ang-2 induces monocyte invasion of a fibrin clot in the presence of serum
The effect of Ang-2 on the movement of monocytes through a fibrin clot was investigated using a modified Transwell invasion assay. Dose response analysis (Fig. 1A ) indicated that concentrations of Ang-2 greater than 250 ng/ml caused an increase in the number of monocytes present in the bottom chamber of the invasion assay. Therefore, 500 ng/ml was decided on as the concentration for Ang-2 to be used in all further experiments.


Figure 1
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  		Figure 1. Relative increase in monocyte fibrinolysis as a result of the addition of Ang-2 to the invasion assay. (A) A dose response curve for Ang-2 showing the increase in the number of cells in the bottom compartment when cells are exposed to increasing amounts of Ang-2. *, P < 0.05 (compared with concentrations ≤250 ng/ml). Experiment in triplicate (one donor). (B) Summary of the increase in the number of cells in the bottom compartment when Ang-2 (500 ng/ml) is added to the bottom compartment. *, P < 0.001. Nine experiments in triplicate (five donors). (C) Summary of the increased number of cells in the bottom compartment when Ang-2 (500 ng/ml) is added to the top compartment. *, P < 0.01. Four experiments in triplicate (three donors).

The presence of Ang-2 (500 ng/ml) in the bottom chamber resulted in a 60% increase in monocytes migrating through the fibrin clot relative to media, which contained serum alone (Fig. 1B , P<0.001). Although clear evidence of fibrinolysis could be visually discerned (data not shown), it was necessary to confirm that the increase in monocyte migration was not a result of a chemotactic response, an augmented proteolytic response, or a combination of both. Were it solely a chemotactic response, the presence of Ang-2 in the upper chamber should not result in increased monocyte movement through the fibrin clot. This negation was not observed, and the resultant increase in monocyte movement in response to Ang-2 present in the upper compartment differed nonsignificantly from that elicited by Ang-2 in the bottom compartment (Fig. 1C , P<0.01). When fibrin was omitted from the Transwell assay, monocytes did not migrate through the etched polycarbonate membrane (5 µm pores) at a faster rate in the presence of an Ang-2 concentration gradient (data not shown), further confirming that the response of the monocytes to Ang-2 was not a result of chemotaxis.

Ang-2-induced monocyte migration through fibrin is dependent on MMPs and serine proteases
As monocytes did not migrate toward Ang-2, this indicated that the observed invasion was at least, in part, a result of a proteolytic mechanism. GM6001, a MMP inhibitor, and aprotinin, a serine protease inhibitor, repressed the invasive response to Ang-2 by four- and 24-fold, respectively (Fig. 2 , P<0.05), demonstrating the involvement of both types of proteases.


Figure 2
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  		Figure 2. GM6001 and aprotinin decrease Ang-2-induced monocyte fibrinolysis. Columns 2 and 3 reflect the decrease in cells in the bottom compartment in the presence of GM6001 plus Ang-2 (fourfold, Column 2) or aprotinin plus Ang-2 (24-fold, Column 3) relative to cells with Ang-2 alone. Results are displayed as a percentage of untreated wells. *, P < 0.05. Five experiments in triplicate (four donors).

Expression of Tie-2 in monocytes
Ang-2 is known to bind with high affinity to the Tie-2 receptor expressed on endothelial cells, and there have also been reports of Tie-2 being expressed on another leukocyte, namely the neutrophil [16 ]. The expression of Tie-2 on monocytes was therefore analyzed at mRNA and protein level. RT-PCR showed no expression of the receptor mRNA in monocytes under any of the conditions used in these studies, and HSVEC had a strong signal as expected (Fig. 3A ). However, as it is possible that mRNA expression may not accurately reflect the steady-state protein levels, FACS analysis of Tie-2 expression in monocytes was also conducted using anti-Tie-2 and isotype IgG. HSVEC were used as a positive control, and human dermal fibroblasts were used as a negative control [24 ]. Ninety-five percent of endothelial cells were positive for Tie-2, and monocytes and fibroblasts displayed no discernable expression (Fig. 3B) .


Figure 3
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  		Figure 3. Neither Ang-2 nor PDGF-BB elicits Tie-2 expression in monocytes. (A) Tie-2 was not detected in monocytes (Lane 1) nor if they were treated with PDGF-BB (Lane 2), Ang-2 (Lane 3), or both (Lane 4). Fibroblasts were used as a negative control (Lane 5) and HSVEC as a positive control (Lane 6). All samples were run in conjunction with HRPT as a control, housekeeping gene. (B) Using flow cytometry, Tie-2 expression was not detected in monocytes (dashed line) or fibroblasts (gray line) but was in HSVEC (black line). Representative graph of three independent experiments.

Ang-2-induced monocyte invasion requires PDGF-BB under serum-free conditions
It is interesting that when the Transwell invasion assay was carried out on monocytes under serum-free conditions, the response to Ang-2 was lost (Fig. 4 , Column 3). This appeared to indicate that monocytes require the presence of one or more cofactors to allow interaction with Ang-2. As Ang-2 is an initiating factor in angiogenesis and elevated during wound healing [1 ], we speculated that a potential cofactor could be PDGF-BB, a critical factor in wound healing. PDGF-BB was present in the human pooled serum-containing medium at levels of 850 pg/ml, as determined by ELISA. Although PDGF-BB alone did not induce increased invasion by monocytes, in conjunction with Ang-2, it was observed to induce a threefold increase in invasion above that in the untreated wells (Fig. 4 , P<0.01 compared with all other conditions).


Figure 4
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  		Figure 4. Ang-2 and PDGF-BB act cooperatively to induce monocyte fibrinolysis in serum-free conditions. PDGF-BB (20 ng/ml) and Ang-2 (500 ng/ml) did not alter monocyte fibrinolysis significantly (Columns 2 and 3, respectively); however, Ang-2 and PDGF-BB added together resulted in a threefold increase in the number of cells in the bottom well (Column 4). *, P < 0.01 (compared with all other conditions). Three experiments in triplicate (three donors).

Ang-2/PDGF-BB-mediated invasion involves uPA
When monocyte cell lysates were examined using immunoblotting, it was observed that Ang-2 and PDGF-BB together caused an 87% increase in uPAR expression, and individually, neither had an effect on uPAR levels (Fig. 5A , P<0.05).


Figure 5
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  		Figure 5. Ang-2 and PDGF-BB interact with uPAR in monocytes. (A) Inset contains representative immunoblots for uPAR (upper) and ß-actin (lower). The addition of Ang-2 (500 ng/ml) and PDGF-BB (20 ng/ml) to monocytes in culture did not increase uPAR expression in cell lysates (Columns/Lane 2 and 3, respectively). When added together, however, they elicited an 87% increase in uPAR expression (Column/Lane 4). *, P < 0.05 (compared with all other conditions). Four experiments in triplicate (four donors). (B) The addition of anti-uPAR antibody did not affect baseline cell invasion (Columns 1 and 2) but removed the invasion stimulated by Ang-2/PDGF-BB (Columns 3 and 4). *, P < 0.05 (compared with all other conditions). Four experiments in triplicate (four donors).

When a blocking antibody to uPAR was added to the Transwell invasion assay, the migration of untreated monocytes was not altered significantly, but the effect of Ang-2 and PDGF-BB on monocyte invasion was lost (Fig. 5B , P<0.05). It is interesting, however, that a degree of fibrinolysis similar to the control remained, which indicated that uPAR was important in the Ang-2/PDGF-BB-mediated aspect of fibrinolysis.

Ang-2 and PDGF-BB together up-regulate MT1-MMP and MMP9
The immunoblotting analysis of monocyte cell lysates indicated that Ang-2 and PDGF-BB in combination caused a 59% up-regulation in MT1-MMP (Fig. 6A , P<0.05), and individually, they had no significant effect on MT1-MMP expression.


Figure 6
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  		Figure 6. Ang-2 and PDGF-BB in combination mediate the up-regulation of MMP9 and MT1-MMP. (A) Summary of immunoblot analysis of MT1-MMP expression. Inset contains representative immunoblots for MT1-MMP (upper) and ß-actin (lower). Although treatment with neither PDGF-BB (20 ng/ml, Column/Lane 2) nor Ang-2 (500 ng/ml, Column/Lane 3) altered MT1-MMP expression significantly, the combination of both growth factors increased average MT1-MMP expression by 59%. *, P < 0.05 (compared with all other conditions). Four experiments in triplicate (four donors). (B) Summary of zymographic analysis of MMP9 expression. Inset is the inverse of a representative zymogram. Cells treated with PDGF-BB (20 ng/ml, Column/Lane 2), Ang-2 (500 ng/ml, Column/Lane 3), or both (Column/Lane 4). In the cells treated with both, an average increase of 54% was observed. *, P < 0.05 (compared with all other conditions). Four experiments in triplicate (four donors).

Similarly, Ang-2 and PDGF-BB individually did not alter the expression of MMP9 in monocytes, but zymographic analysis of the conditioned medium revealed that monocytes treated with a combination of the two growth factors had a 54% increase in MMP9 expression (Fig. 6B , P<0.05).


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DISCUSSION
 
We have shown that Ang-2 in concert with PDGF-BB can up-regulate key components of the serine protease and MMP pathways in human peripheral blood monocytes, resulting in them assuming a more invasive phenotype. The cooperation between these two factors appears pertinent to wound healing, as Ang-2 is released from Weibel-Palade bodies by various stimulants, amongst which is the key coagulant factor, thrombin [25 ], and during clot formation, PDGF-BB is released from the {alpha} granules of activated platelets [26 ]. This suggests a clear conjunction of their delivery during initiation of repair. In addition to wound healing, this congruence of events occurs during pathological remodeling situations such as myocardial infarction [27 ].

The data presented in this study further support the perception of Ang-2 as a factor involved in inflammatory responses, as this was proposed initially as a result of its presence in Weibel-Palade bodies [2 ]. In addition, it has been observed recently that Ang-2 indirectly increases neutrophil and monocyte adhesion and extravasation to and through the endothelium via Tie-2-induced endothelial cell stimulation [14 ]. Furthermore, Ang-2 was seen to increase platelet-activating factor and ß2-integrin synthesis directly in neutrophils via Tie-2 and also increased their adhesion to endothelial cells [16 ].

The receptor for PDGF-BB has previously been reported to be expressed on human monocytes [28 ]. Although recent work has divided monocytes into various subpopulations [29 ], some of which, such as endothelial precursor cells (EPCs), have shown Tie-2 expression [30 31 32 ], we found no evidence for Tie-2 expression in monocytes under any of the conditions studied in these experiments. As EPCs are present in extremely low numbers in circulating blood [33 ], this anomaly may be a result of differing flow cytometry conditions, isolation procedures, or idiosyncrasies of donor conditions.

Although the mechanism through which Ang-2 interacts with monocytes was not investigated, it has previously been found to interact with a number of other cell types via mechanisms separate from Tie-2 binding. Fibroblasts as well as skeletal myocytes were found to bind to surfaces coated with Ang-1 and Ang-2 [34 , 35 ]. This adhesion was mediated by integrins {alpha}v, {alpha}6, ß1, and ß3 with concomitant activation through MAPKp42/44 and Akt pathways. Alternative, persuasive evidence that Ang-2 is able to elicit a response in Tie2-deficient cells comes from a series of investigations into the interactions between Ang-2 and glioma cells. Ang-2 expression in vivo was linked with a more invasive tumor profile, and this invasiveness was shown to be, in part, a result of Ang-2-induced expression of MMP-2, MT1-MMP, and laminin-V [36 ]. Subsequently, MMP-2 induction in gliomas was shown to be mediated by Ang-2 via the {alpha}vß1 integrin through a focal adhesion kinase signaling pathway [37 ]. It is of interest that PDGF-BB has been found to influence the clustering of integrin ß1 [38 ] and induced MMP-9 production in newborn vascular smooth muscle cells simultaneously exposed to an integrin {alpha}vß3 ligand [39 ].

As the increased invasiveness of the monocytes could be inhibited by protease inhibitors, we have begun to determine which proteolytic components play a role. The monocytes migrated through the fibrin prior to completing lysis of the clot, suggesting that pericellular lysis was central to the increased fibrinolysis. Two membrane-bound proteins involved in pericellular fibrinolysis [11 , 40 ], uPAR and MT1-MMP, were up-regulated by a combination of Ang-2 and PDGF-BB. uPAR and MT1-MMP have been shown to be involved in the recruitment of monocytes to sites of inflammation [41 , 42 ]. It is intriguing that MT1-MMP was found to be necessary for proper extravasation of monocytes through a TNF-{alpha}-activated endothelium [42 ]. This suggests that Ang-2 plays a central role in directing the recruitment of monocytes to a site of vascular disturbance through not only regulating adhesion to the endothelium [14 ] but also enabling the cell to move through the endothelial layer and invade the underlying matrix.

The evidence for MMP-9 having fibrinolytic activity is not as compelling as for the above proteins. MMP-9 null mice have increased fibrin deposits, and MMP-9 was found to degrade fibrin in vitro [43 ]; however, the transfection of fibroblasts with the MMP-9 gene did not elevate their ability to invade a fibrin clot [44 ]. It is possible, though, that Ang-2-increased expression of MMP-9 in monocytes may have an impact on the ability of monocytes to induce vessel ingrowth during wound healing, as MMP-9 has been proposed as an angiogenic switch for tumors by increasing the availability of VEGF-A [45 , 46 ].

Although the mechanism by which these two growth factors act on the monocyte still needs to be elucidated further, it may differ from that functioning in glioma cells, as MMP-9 was not found to be up-regulated in these cells [37 ], and we did not observe an increase in MMP-2 in monocytes. Furthermore, all of the above proteolytic elements have been implicated previously as markers of activation of monocytes to macrophages [47 ]. It therefore requires further investigation into whether Ang-2/PDGF-BB up-regulates a specific subset of polypeptides, which facilitate invasion, or acts more broadly during monocyte activation.

It is worth reiterating that Ang-2 appears to function in an interconnected manner [14 , 48 ] with other growth factors. It has been shown previously to stimulate angiogenesis in conjunction with VEGF [1 , 7 ], inflammatory cell adhesion via TNF-{alpha} [14 ], and now, increased proteolytic invasion through PDGF-BB. This suggests that Ang-2 is a key regulator of wound healing, enabling other necessary growth factors to elicit their required responses.

The identification of Ang-2 and PDGF-BB as factors that may be involved in the initial recruitment of monocytes to a wound site suggests that they could prove to be useful, clinical targets in the drive toward reduction of scarring during tissue repair. Given the increasing importance of scar prevention in regenerative medicine, together with the recent realization that the presence of macrophages throughout the duration of the wound-healing response may not be required [49 , 50 ], suggests that manipulation of their recruitment may prove increasingly important for engineered tissue regeneration.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant from the South African Medical Research Council.

Received November 21, 2006; revised February 23, 2007; accepted February 26, 2007.


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