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Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:97-104
Published online before print November 8, 2007, doi: 10.1161/ATVBAHA.107.157537
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:97.)
© 2008 American Heart Association, Inc.

Cell Biology/Signaling

Regulation of Endothelial Cell Proliferation by Primary Monocytes

Shai Y. Schubert; Alejandro Benarroch; Janne Ostvang; Elazer R. Edelman

From the Harvard-MIT Division of Health Sciences and Technology (S.Y.S., A.B., J.O., E.R.E.), Massachusetts Institute of Technology, Cambridge, Mass; IQS, Institut Químic de Sarrià (A.B.), Barcelona, Spain; and the Cardiovascular Division, Department of Medicine (E.R.E.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.

Correspondence to Shai Y. Schubert, Massachusetts Institute of Technology, Division of Health Sciences and Technology, 77 Massachusetts Avenue, room E25-438, Cambridge, Massachusetts 02139. E-mail shai{at}mit.edu

Abstract

Objective— Endothelial cell–monocyte cross talk is essential for vascular repair. Monocytes colocalize with endothelial cells forming a complex set of interactions distinct from the growth promoting cytokines secreted by differentiated macrophages. In the present work we examined the growth regulation and in vitro wound repair early after binding of monocytes to endothelial cells.

Methods and Results— After direct contact with primary unactivated monocytes, endothelial cells enter S-phase through a mechanism mediated in part by contact-dependent activation of endothelial Met as demonstrated by siRNA silencing of Met, neutralizing antibodies for hepatocyte growth factor and Met as well as by specific inhibition of Met by the Met kinase inhibitor SU11274. Monocytes robustly promote endothelial cell proliferation and migration into a wounded endothelial monolayer. Monocyte-induced endothelial cell proliferation is accompanied by prolonged extracellular signal-regulated kinase (ERK) activation and is inhibited by the specific ERK inhibitor PD98059. The contact-mediated effect of monocytes is specific to endothelial cells and does not occur with vascular smooth muscle cells. Interestingly, although Flk1 is activated by monocytes, the proliferative effect of monocytes reported here is minimally mediated by Flk1 signaling.

Conclusions— These results suggest that the early interaction between endothelial cells and monocytes is critical for the regulation of endothelial cell proliferation. This complex regulation is mediated in part by contact-dependent Met and ERK phosphorylation. These findings add to a broader set of leukocyte-endothelial contact mediated signals that together regulate endothelial function in health and disease.

After direct contact with primary unactivated monocytes, endothelial cells enter S-phase through a mechanism mediated in part by contact-dependent activation of endothelial Met. Monocyte-induced endothelial cell proliferation is accompanied by prolonged ERK activation, it is specific to endothelial cells, and does not occur with vascular smooth muscle cells.


Key Words: endothelial cell proliferation • monocytes • vascular remodeling • angiogenesis • endothelium

Endothelial cell (EC) proliferation is tightly regulated by a diverse set of growth-inducing and growth-inhibiting factors that drive formation of the intact endothelial monolayer to a specific morphology at specific sites and vascular beds. Cytokines and chemokines secreted from leukocytes, and adhesion molecules expressed on the surface of activated ECs, recruit and adsorb circulating leukocytes.1 Monocytes are recruited to sites of vascular remodeling in atherosclerotic lesions, after angioplasty or stent implantation,2,3 and in tissues made ischemic and after reperfusion injury.4,5 Monocyte recruitment and tissue accumulation are associated with EC proliferation in atherosclerotic lesions.6 Collateral arteries that develop after rabbit femoral artery occlusion express adhesion molecules and are replete with adherent monocytes as early as 12 hours after occlusion7; these 2 events are closely associated with induced endothelial cell proliferation (ECP).4 Intraarterial infusion of MCP-1 significantly increased resting blood flow in a porcine hindlimb ligation model,8 and local infusion attracted monocytes and increased collateral vessel formation and peripheral conductance.9 Circulating monocyte number and induction of angiogenesis correlate after manipulation of blood monocyte number with injection of the antimetabolite 5-fluorouacil.10 Taken together these data suggest that monocytes play an active role in ECP.

Activated monocytes and macrophages synthesize and secrete multiple growth promoting cytokines and growth factors11,12 such as interleukin (IL)-8, VEGF, and PDGF.13–17 Yet, depending on their activation state monocytes can potently inhibit EC proliferation.18 Given the range of vascular modulating factors potentially produced by monocytes, and the apparent contradictory effects reported on the regulation of ECs in response to monocyte binding, we studied the early response of ECs after interaction with unactivated primary monocytes. We hypothesize that the early interaction of ECs with unactivated peripheral blood monocytes, unlike the postinvasive chronic stage, is critical for the maintenance of functional and intact endothelium by regulation of endothelial proliferation and function.

Others have shown that confluent ECs in the presence of MCSF can stimulate the proliferation of monocytes after 6 days in culture.19 We now demonstrate that direct monocyte contact can induce proliferation in human ECs, but not in vascular smooth muscle cells or fibroblasts. ECs entered S-phase 12 to 16 hours after the addition of primary monocytes in a binding-dependent manner mediated in part by endothelial Met activation. Direct contact induction of ECP is more rapid than systemic cytokine mediated effects and remarkably was minimally mediated by Flk1 signaling.

Methods

Cells
Primary human umbilical vein (HUVECs), aortic endothelial cells (HAECs), and smooth muscle cells (HASMCs) were from Cambrex (Walkersville, Md). Human fetal lung 1 fibroblasts (HFL1) cells were from ATCC (Manassas, Va). Primary monocytes were separated before each assay from blood drawn from healthy male subjects under formal protocols sanctioned by the MIT Committee on Use of Humans in Experimental Sciences, and separated using a negative isolation method (Miltenyi Biotec). Monocyte separation yield was over 90% and validated using fluorescence-activated cell sorter (FACS) analysis with anti–CD14-RPE antibodies (DakoCytomation). Monocyte viability after negative selection separation was over 98% as measured by trypan blue staining.

Coculture Proliferation Assays With Monocytes
ECs cultures (passage 4 to 6) were grown in DMEM with 10% FBS in 24-well plates to subconfluence (5x104 cells per cm2) followed by serum starvation (DMEM, 5% heat inactivated FBS, 24 hours). Primary monocytes were prepared before EC contact and added in proportional number. Assays were performed in DMEM with 5% heat inactivated FBS. In assays where neutralizing antibodies or inhibitors were used, antibodies/inhibitors were preincubated with ECs, monocytes, or both, for 30 to 60 minutes. Cultures were incubated (37°C, 5% CO2), and 20 to 22 hours after the addition of monocyte cultures were pulsed with 3H-thymidine (1 µCi/mL, 2 hours, Perkin Elmer Life Sciences). Cultures were washed twice with 2 mL of ice cold PBS followed by 30 minutes incubation in 5% wt/vol trichloroacetic acid (TCA). TCA was washed twice with cold PBS, followed by lysis with 0.4 mL (0.5% SDS, 0.5 N NaOH). The TCA-insoluble radioactivity was counted in a liquid scintillation counter, Packard 25000-TR.

The amount of thymidine incorporation reported in the study may vary significantly between figures. The unique growth kinetics measured during monocyte induced-endothelial cell proliferation (MIECP), which is the subject of a separate study, together with the use of primary endothelial cells and monocytes taken from different donors, resulted in high variability in the measured thymidine incorporation during the time window of thymidine pulsing. Therefore each assay is performed in triplicates and should be evaluated by the controls used in the specific assay.

In Vitro Endothelial Wound Assay
The ability of monocytes to accelerate growth into denuded areas of cultured EC monolayer was evaluated using the endothelial wound repair model as previously described.20 ECs were grown to confluence in 3.5-cm tissue culture plates. Confluent cultures were serum-starved in 2 stages (DMEM 10% inactivated FBS, 24 hours followed by DMEM 5% inactivated FBS, 24 hours) Cultures were mechanically wounded by removing cells on a path 2 mm wide with the exposed side of sterile wooden cotton tipped applicator. Cultures were washed once in assay medium (DMEM 5% inactivated FBS), and quantification of cell migration and proliferation was performed under phase contrast and inverted fluorescent microscopy. In some plates monocyte were added 1 hour after injury in direct contact or in transwell inserts residing above EC cultures, in ratio of 3 monocytes to 4 ECs. The injured area was photographed using phase contrast microscopy 10 minutes and 30 hours after injury.

Antibodies and Inhibitors
Neutralizing antibodies for VEGF165 were from US Biological. Neutralizing antibodies for VEGF receptor 2 and HGF from ABcam. Neutralizing antibodies for HGF receptor from Chemical Co. p-ERK42/44, p-Flk1, p-Met and β-actin antibodies were obtained from Cell Signaling Technology. Normal mouse IgG and goat IgG obtained from Santa Cruz Biotechnology. Alexa Fluor 594 conjugate, BrdU, and YOYO1 were obtained from Molecular Probes. Dynabeads CD14 obtained from Dynal Biotech. PD98059, SU11274 and TRITC-conjugated Phalloidin were obtained from Calbiochem.

BrdU Labeling
To study the role of endothelial cell proliferation in monocyte-induced endothelial wound closure, HUVECs were grown on glass coverslips coated with gelatin or on 3.5-cm tissue culture plates in DMEM, 10% heat inactivated FBS to confluence followed by serum starvation (DMEM, 5% heat inactivated FBS, 24 hours). Primary monocytes were added in proportional numbers, and after 18 hour cultures were pulsed with BrdU (10 µmol/L, 6 hours). Cultures were washed 3 times in ice cold PBS followed by 20 minutes incubation in 1 mL of Carnoy fixative (4°C) and acid DNA denaturation (HCL 2 mol/L, 37°C, 1 hour). BrdU was immunostained using Alexa Fluor 594 conjugate anti-BrdU antibody. Where BrdU labeling was done in conjugation with surface cell immunostaining, cultures were fixed with 2% paraformaldehyde, pH 7.2, 30 minutes and DNA digested by DNAas (Amersham Pharmacia).

siRNA Silencing
The role of endothelial Met in MIECP was studied by blocking Met with the specific Met kinase inhibitor SU11274, neutralizing antibodies for Met and HGF, or by downregulation of Met expression by siRNA silencing. For downregulation of Met expression ECs were seeded in 6 cm plates (2x105 cells per plate) or in 24-well plates (4x104 cells per well) in DMEM 10% inactivated FBS without antibiotics. Before transfection cells were washed with transfection medium (Santa Cruz Biotechnology) and transfected with 60-pmol Met siRNA or control siRNA in transfection reagent (Santa Cruz Biotechnology) following the manufacturer protocol. Assays using monocyte interaction were performed 48 hours after transfection. For specific Met inhibition by SU11274, ECs were preincubated with the inhibitor (2.5 µmol/L) or Met neutralizing antibodies 1 hour before the addition of monocytes. EC proliferation was determined by thymidine incorporation assay as described in section "Coculture Proliferation Assays With Monocytes".

FACS and Western Blot Analysis
For FACS analysis cells were treated with BrdU labeling reagent (Invitrogen) for 12 hours. Cultures were harvested (PBS, EDTA 5 mmol/L, 15 minutes) followed by costaining with anti–BrdU-fluorescein isothiocyanate (FITC) with DNase. Cells were analyzed by flow cytometry (FACS, Becton Dickinson Immunocytometry Systems), and at least 10 000 positive events were acquired. Listmode files were analyzed using FlowJo software (TreeStar). Whole cell extracts of HUVECs and monocytes were harvested at different times of coculture by separation of monocytes with repeated washes (PBS, 2.5 mmol/L EDTA, 2 minutes) as previously described.21 Protein samples were separated on glycin-SDS gels, transferred to polyvinylidene fluoride (PVDF) membranes, and immunobloted with the indicated antibodies.

Data Analysis
All assays were performed in triplicate and repeated at least 3 times. Data are presented as mean±SD. When applicable, values were compared by Student t test or ANOVA. P<0.05 was considered to be statistically significant.

Results

Monocytes Induce Endothelial Cell Proliferation
Direct contact of monocytes increased thymidine uptake by HUVECs and HAECs 22.2±2.7 (P<0.0001) and 20.2±2 (P<0.0001)-fold, respectively (Figure 1a). In contrast monocytes had an order of magnitude lower effect on HASMCs and HFL1 cells, increasing proliferation by only 1.9±0.23 (P<0.05) and 1.2±0.14-fold, respectively. To study the role of direct contact, monocytes were cultured on polycarbonate membrane transwell inserts with a cutoff of 0.4 µm residing above assayed cells. When monocytes were added in these transwell inserts only the potentiated proliferation of HUVECs and HAECs was affected; no effect was noted on other cell lines. Thymidine incorporation decreased by 90% to 2.3±0.28 (P<0.05) and 2.0±0.25-fold (P<0.05) above controls in endothelial cells exposed to monocytes in coculture inserts, but virtually not at all in HASMCs and HFL1 cells under the same conditions. Addition of monocytes in coculture inserts above HASMCs or HFL1 cells increased proliferation 1.9±0.2- and 1.1±0.14-fold above control (P<0.05) but statistically indistinguishable from the effect of direct monocyte contact with these cells.


Figure 1
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  		Figure 1. Primary monocyte-induced cell–cell contact-dependent thymidine incorporation and proliferation of ECs. Direct cell contact of monocytes induced proliferation of subconfluent HUVECs or HAECs, but not HASMCs or HFL1 cells (a). EC proliferation was unaffected by monocyte-depleted PBMCs, platelets, or conditioned medium (CM) from HUVEC–monocytes cocultures (EC-M), Monocytes only (M) (b). Cyclin D1 expression is restricted to HUVECs when cocultured with primary monocytes (c). FACS analysis of subconfluent HUVECs cocultured with monocytes for 24 hours and treated with BrdU. Cultures were costained for BrdU and CD14 (d). n ≥3, *significantly higher than untreated control, P<0.001.

Specific Induction of Proliferation
Coculture of HUVECs with monocyte-depleted peripheral blood mononuclear cells (PBMCs) did not induce thymidine incorporation, emphasizing the specific role of monocytes. In addition, incubation of HUVECs with platelet-rich cell free fraction did not induce thymidine incorporation, excluding a possible role for platelets in MIECP (Figure 1b). Conditioned medium (CM) from HUVECs cocultured with monocytes for 20 hours showed no proliferation-inducing effect on HUVECs, supporting the dependency of MIECP on direct cell contact (Figure 1b). Monocytes (M) shows only trace levels of thymidine incorporation when isolated from HUVEC–monocyte cocultures by magnetic separation employing Dynabeads CD14 antibodies (Figure 1b).

Increased Cyclin D1 expression was evident in HUVECs 12 hours after the addition of monocytes, but was not evident in the EC-bound monocyte cell population (Figure 1c). To confirm the selective proliferation of ECs in coculture with monocytes, cocultures were treated with BrdU-labeling reagent for 2 hours, 20 hours after monocyte addition. Cultures were harvested (PBS, EDTA 5 mmol/L, 15 minutes) and costained with anti–BrdU-FITC with DNAse and anti–CD14-RPE. FACS analysis demonstrated that BrdU incorporation was restricted to CD14-negative cells (Figure 1d, P<0.0001, and supplemental Figure I, available online at http://atvb. ahajournals.org). The amplification of BrdU incorporation (10 µmol/L, 6 hours) in HUVECs by direct contact with primary monocytes was visualized using Alexa Fluor 594 conjugate anti BrdU antibody (Figure 2a and 2b). The increase in EC density was readily evident under phase contrast microscopy (Figure 2c and 2d). The binding and spreading of primary monocytes (M) on these subconfluent ECs (Figure 2e) was visualized by confocal microscopy. HUVECs were cocultured with primary monocytes for 4 hours followed by fixation (4% paraformaldehyde, 30 minutes) and costaining with TRITC-conjugated Phalloidin and YOYO1.


Figure 2
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  		Figure 2. Direct contact of HUVECs with monocytes increase BrdU incorporation (a, b) and cell density (c, d). A confocal image of primary monocytes (M) bound to subconfluent HUVECs after costaining with TRITC-conjugated Phalloidin and YOYO1 illustrates the spreading of monocytes over subconfluent ECs 4 hours after coculture (e).

Endothelial Wound Repair In Vitro
The addition of monocytes in direct contact resulted in nearly complete closure of the denuded area after 30 hours (Figure 3b). In contrast, both the untreated control and addition of monocytes in transwell inserts produced minimal injury closure (Figure 3c and 3d). Wound closure is well demonstrated in nuclear staining of monocyte-treated ECs (Figure 3e) compared with untreated control (Figure 3f) 30 hours after injury. BrdU staining clearly demonstrates that wound closure in monocyte-treated cultures arises from massive EC proliferation in the denuded area (Figure 3g) as compared with untreated control (Figure 3h). Selective staining for HAECs, monocytes, and BrdU in the injury front of cultured HAECs treated and untreated with monocytes clearly demonstrate the substantial role of proliferation in the closure of denuded endothelium. Serum-starved monolayers of HAECs were left untreated (Figure 3i) or treated with primary monocytes (Figure 3j) as described for Figure 3a through 3h. Twenty-two hours after the injury cultures were pulsed with BrdU for 2 hours followed by 30 minutes incubation with FITC conjugated anti CD14 antibodies. Cultures were fixed and stained for BrdU and DAPI nuclear stain. The increase in the number of proliferating ECs is well visualized in the ratio of blue (BrdU) to green (DAPI) in untreated (Figure 3i) and monocyte treated (Figure 3j) endothelial injury front.


Figure 3
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  		Figure 3. Direct contact of HUVECs with monocytes accelerates wound reendothelization: 10 minutes after wounding (a). Wounded HUVECs with monocytes 30 hours (b). Wounded HUVECs untreated, 30 hours (c). Wounded HUVECs with monocytes in transwell inserts, 30 hours (d). Nuclear staining of HUVECs with monocytes 30 hours (e). Nuclear staining of untreated injured HUVECs, 30 hours (f). BrdU immunostaining of HUVECs with monocytes and BrdU, 30 hours (g). BrdU immunostaining of injured HUVECs with BrdU, 30 hours (h). Injury front of untreated HAECs with BrdU stained for BrdU and DAPI (i). Injury front of HAECs with monocytes, DAPI (green), BrdU (blue), monocytes (red) (j). n ≥3, *significantly higher than untreated control, P<0.001.

VEGF in MIECP
The role of VEGF in ECP is well characterized and its potential contribution to contact-mediated ECP was evaluated using neutralizing antibodies for VEGF165 (10 µg/mL, 1 hour preincubation) resulting in up to 12.4±3.6% (P<0.001) inhibition of MIECP (Figure 4a). Abrogation of VR2 signaling with either a neutralizing antibody (25 µg/mL, 1 hour preincubation) or a VR2-specific inhibitor ZM323881 (0.1 µmol/L, 1 hour preincubation) virtually eliminated HUVEC stimulation by VEGF165, but had no effect on monocyte induced-proliferation (Figure 4b).


Figure 4
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  		Figure 4. Role of VEGF in MIECP. Pretreatment of HUVECs with neutralizing antibodies for VEGF165 (10 µg/mL) before the addition of monocytes failed to inhibit MIECP. Isotope control total mouse IgG (a). VEGF stimulation of HUVECs is eliminated by blockade of VEGF binding to VEGF receptor 2 (VR2/Flk1) with neutralizing antibody (25 µg/mL), or inhibition of VR2 kinase activity by ZM323881 (0.1 µmol/L) (b). n ≥3, *significantly lower than VEGF-treated control, P<0.001.

Met Mediates MIECP
Met or hepatocyte growth factor receptor is a potent regulator of endothelial cell proliferation, migration, and angiogenesis.22–25 Recently the targeting of Met has been considered as a novel approach to antiangiogenic therapy.26 To study the potential activation of Met during early endothelial–monocyte interaction, ECs were cultured in 6-cm plates and grown to subconfluence (5x104 cells per cm2) followed by starvation (DMEM, 1% heat inactivated FBS, 24 hours). Primary monocytes were added in proportional numbers in starvation medium, and 5 minutes before each collection time point sodium orthovanadate (SOV) was added to a final concentration of 500 µmol/L. Monocytes were removed from cocultures as previously described21 by short incubation in PBS–EDTA 5 mmol/L with 500 µmol/L SOV. ECs were then harvested in TGP buffer (1% Triton X-100, 10% glycerol, 50 mmol/L Hepes pH. 7.4, 1 mmol/L SOV, and protease inhibitor cocktail). Phosphorylation of Met, Flk1, and ERK42/44 was determined by Western blot with antibodies for the phosphorylated forms of the 3 target proteins (Figure 5a). Interestingly, endothelial-monocyte interaction resulted in a biphasic phosphorylation of Met. An early phosphorylation of Met is apparent on contact of ECs with monocytes and a delayed, second activation is apparent 60 minutes after the addition of monocytes. If considering ERK phosphorylation as a marker for active binding between ECs and monocytes then the second phosphorylation of Met follows shortly after the binding of monocytes to ECs. To study the possible role of HGF and its receptor in MIECP we used multiple methods to inhibit Met mediated signaling. Inhibition of HGF by neutralizing antibodies resulted in a dose-dependent inhibition of MIECP. Two (2) µg/mL of antibody inhibited 45.2±4.7% (P<0.0001), whereas 5 µg/mL of antibody completely inhibited MIECP as well as thymidine incorporation induced by treatment with HGF (25 µg/mL; Figure 5b and supplemental Figure III). A similar effect was measured when Met was targeted by neutralizing antibodies, with maximum inhibition measured with antibody concentration of 2 µg/mL (Figure 5b). Moreover, both SU11274 and si-Met silencing resulted in up to 35%±5.5% inhibition of MIECP (Figure 5c). Together, the early activation of endothelial Met after interaction with monocytes and the responsiveness of MIECP to inhibition of Met signaling indicates an active role for HGF and Met in early endothelial-monocyte interaction and MIECP.


Figure 5
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  		Figure 5. Role of Met signaling in MIECP. Phosphorylation kinetics of endothelial Met, Flk1, and ERK after interaction with monocytes (a). Inhibition of HGF or Met by neutralizing antibodies inhibit MIECP- and HGF-induced EC proliferation (b). Met inhibitor SU1172 and Met siRNA transfection partially inhibit MIECP (c). (n ≥3, *significantly lower than MIECP control, {lozenge} significantly lower than HGF control, P<0.001.

ERK Is Activated Independently From Met and Is Important for MIECP
As ERK phosphorylation appears before Met phosphorylation in the second activation phase after EC-monocyte interaction (Figure 5a, 45 minutes), we hypothesized that ERK activation is involved in MIECP. To test the effect of the ERK inhibitor PD98059 on MIECP we measured thymidine incorporation in EC cultures pretreated with PD98059 (10 µmol/L) for 30 minutes. MIECP was inhibited up to 48%±7% in the presence of PD98059 indicating that ERK signaling is actively involved in MIECP (Figure 6a). To study whether a dependency occurs between ERK phosphorylation and monocyte-mediated Met phosphorylation, EC cultures were kept untreated or transfected with si-Met as described for Figure 6b. No dependency between Met signaling and ERK phosphorylation was detected under these conditions, indicating that ERK phosphorylation is independent of Met signaling and is apparent in untreated as well as in Met silenced ECs after interaction with monocytes (Figure 6b).


Figure 6
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  		Figure 6. Role of ERK signaling in MIECP. Inhibition of ERK42/44 with the specific ERK inhibitor PD98059 resulted in up to 2-fold inhibition of MIECP (a). Downregulation of Met expression with Met siRNA did not inhibit the phosphorylation of ERK42/44 initiated by contact with monocytes. n≥3, *significantly lower than MIECP control, P<0.001.

Discussion

It has long been demonstrated that monocytes and ECs interact in vascular repair and that the one cell type can induce proliferative effects and phenotypic changes in the other.19 The process we identify now of contact-mediated induction of EC proliferation by monocytes has not been reported to the best of our knowledge. Its mechanism and time course make it a unique aspect of vascular repair.

Monocyte diapedesis is initiated as signals mediated by integrin bound adhesion molecules on ECs inducing structural changes in ECs. Monocyte density within the vessel wall is a determinant of subsequent vascular repair and angiogenesis.4,8–10,27 Monocytes secrete matrix metalloproteinases (MMPs) and cytokines, including interleukin 8 (IL-8), whose role in angiogenesis is suggested to involve regulation of matrix metalloproteinase (MMP) activation and ECP. Nonetheless, the MIECP that we observe is mediated primarily by direct contact and is distinct from IL-8 as neutralization of IL-8 with antibodies did not inhibit MIECP (supplemental Figure II). Early studies of EC–monocyte interaction failed to demonstrate the proliferative effect we report here. On the contrary, a study by Liote et al18 reported an inhibitory effect of monocytes on EC proliferation. This observation, however, does not contradict our data as monocyte separation methods used at the time resulted in activated monocytes. Indeed, activation state of monocytes determined the inhibition of EC proliferation, indicating the critical importance of monocyte activation state for their ability to induce a specific EC response. In contrast we used negatively selected monocytes which are not activated during the separation process to not shift EC response to monocyte interaction by preactivated monocytes.

In our assay system unactivated monocytes induce a single cell cycle in ECs (Schubert et al, unpublished data, 2004). When monocytes were activated with tumor necrosis factor (TNF){alpha} before their interaction with ECs we observed the inhibitory effect on EC proliferation as reported by Liot et al,18 indicating the importance of monocytes being unactivated before the interaction with ECs. The role of monocyte activation state on EC proliferation kinetics adds to the complex regulation over EC proliferation and is currently a part of an ongoing study.

Soluble growth factors are important EC stimuli that have long been demonstrated to restore the integrity of damaged endothelium and to induce vascular remodeling.28 Initial release of growth factors in ischemic tissue is critical for the survival and proliferation of ECs.29 Activated monocytes release both VEGF and PDGF.14,15 However, neutralization of PDGF and FGF (Schubert et al, unpublished data, 2005) as well as VEGF165 and the critical vascular VEGF receptor Flk1, offers minimal inhibition of MIECP (Figure 4a and 4b). The lack of correlation between Flk1 phosphorylation and EC proliferation in response to interaction with monocytes was surprising. Unpublished data from our study of EC Flk1 receptor expression levels after interaction with monocytes revealed a sharp decrease in Flk1 receptor levels 3 to 5 hours after the addition of monocytes. This could be part of an autoregulatory loop mediated by the transient activation of Flk1 during the early stages of EC–monocyte interaction, resulting in the receptor internalization and degradation. The reason for the unique Flk1 response and expression during EC–monocyte interaction are not yet clear and are part of an ongoing study.

Hepatocyte growth factor (HGF) is a mesenchyme-derived pleiotropic factor that regulates the growth, motility, and morphogenesis of various cell types. HGF was demonstrated to be a potent mediator of angiogenesis through its receptor Met30 and is attracting increasing interest as a potential target for anti- as well as proangiogenic therapy.31–33 In ECs HGF induced proliferation, migration, and angiogenesis through the activation of Met22,23,34 and indirectly through activation of the angiogenic transcription factor est1,35 as well as through the augmentation of VEGF-induced angiogenesis.36 The expression of HGF by monocytes during direct cell–cell interaction was previously reported for the interaction of monocytes with smooth muscle cells,37 human mesangial cells,38 and uveal melanoma cells.39

The effect of monocytes on Met phosphorylation in ECs described here is particularly interesting as it highlights the occurrence of 2 sequential phases of Met activation. The first phase is transient, appearing with the initial contact of ECs and monocytes and is followed by a gradual decay in the phosphorylation (Figure 5a). The second activation of Met follows shortly after the appearance of ERK phosphorylation, a possible indication of the initiation of firm binding mediated through adhesion molecules and integrins before the second activation of Met. Indeed when monocytes where added in transwell inserts to block direct contact they did not induce phosphorylation of Met (data not shown) or proliferation (Figure 1a). Targeting of Met signaling by neutralizing antibodies for HGF and Met (Figure 5b) as well as the use of siRNA silencing of Met or specific Met kinase inhibitor (Figure 5c) all resulted in the inhibition of MIECP. Previous studies demonstrated a correlation between Met signaling and Flk1 signaling. The activation of Met by HGF increased VEGF-mediated angiogenesis36 whereas inhibition of Met signaling by HGF/NK4 inhibited VEGF-mediated EC proliferation and migration as well as in vivo angiogenesis in a rabbit corneal micropocket assay.40 The existence of a connection between Met and Flk1 signaling may be a possible explanation to the minimal effect of Flk1 and VEGF inhibition on MIECP. The potent effect of monocytes on EC wound closure demonstrated by increased migration and proliferation into a denuded area (Figure 3) is supported by several studies which demonstrated that HGF added into injured EC monolayers enhanced EC migration and wound closure.22,35 Monocyte induced EC migration mediated through the activation of Met may also be pivotal for the induction of vascular permeability following monocyte binding during diapedesis.

The contact-dependent proliferative effect of monocytes is specific to ECs alone, and does not involve a transformation of monocytes to ECs. Though monocytes might serve as endothelial cell precursors such endothelial regeneration is distinct from the process we now report. Flk1+ monocytes that can be identified later as ECs, represent a limited subpopulation of mononuclear cells (0.08±0.04%). These cells require several days to differentiate and do not proliferate in culture.41 The proliferation we observed after monocyte contact is restricted to cells of a size compatible with ECs and completely lacking CD14 expression (Figure 1d). The end effect is similarly restricted to ECs. Monocytes do release soluble growth inducing factors when in coculture, independent of direct cell contact. The proliferation of ECs and vascular smooth muscle cells increased approximately 2-fold when cocultured with monocytes in transwell inserts or with EC–monocyte conditioned medium (Figure 1a and 1b). Only ECs, however, respond with added proliferative potential to direct monocyte contact (Figure 1a). Indeed, this observation coincides well with the MIECP dependence on Met signaling and EC–monocyte binding as Met was demonstrated to induce proliferation in ECs but not in vascular smooth muscle cells.42 Interestingly HGF was reported to be released by both SMCs and monocytes during direct cell–cell interaction43 indicating a possible role for this interaction in vascular repair. One might well envision that such specificity aids in the healing of damaged blood vessels. We and others for example demonstrated that endothelial denudation is of the first events after endovascular stent placement and that endothelial recovery coincides with adhesion of monocytes to the denuded arterial surface.44 The endothelial wound assays presented here confirmed the contribution of monocytes to vascular reendothelization. Normally, vascular reendothelization is the result of the combination of cell migration, cell proliferation, and cell spreading. BrdU labeling and immunostaining of denuded EC monolayers untreated or treated with monocytes (Figure 3) clearly demonstrate the key role of monocyte induced proliferation in the reendothelization process. Stimulation of endothelial repair early after vascular injury and without promoting smooth muscle cell growth may well restore vascular homeostasis while limiting potential luminal obstruction and intimal hyperplasia.

This study offers new insight into the complex regulation of ECP and endothelial regeneration. The active involvement of leukocytes in endothelial proliferation and the role of Met signaling in this process may be important in the initiation and progression of vascular pathologies. Further research is needed to elucidate the precise role of monocyte mediated Met signaling in vascular physiology. Better understanding of MIECP offers an opportunity to control and possibly treat vascular diseases.

Acknowledgments

Sources of Funding

This work was supported by grants from the National Institutes of Health (to E.R.E.; HL 49309, and HL67246), the National Institute on Aging (T32-AG023480) and the Philip Morris External Research Program Postdoctoral Fellowship (to S.Y.S.).

Disclosures

None.

Footnotes

Original received October 11, 2006; final version accepted November 1, 2007.

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