Published online before print December 6, 2007, doi: 10.1161/ATVBAHA.107.150664
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© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Forkhead Factor, FOXO3a, Induces Apoptosis of Endothelial Cells Through Activation of Matrix Metalloproteinases
From the Innovative Research Institute for Cell Therapy (H.Y.L., H.J.Y., J.Y.W., S.W.Y., H.J.C., K.W.P., Y.B.P., H.S.K.), Seoul National University Hospital, and the Department of Internal Medicine (H.Y.L., H.J.C., K.W.P., Y.B.P., B.H.O., H.S.K.) and the Department of Biochemistry and Molecular Biology (W.-Y.P., J.-S.S.), Seoul National University College of Medicine, Seoul, Korea; and the Whitaker Cardiovascular Institute (K.W.), Boston University School of Medicine, Boston, Mass.
Correspondence to Hyo-Soo Kim, MD, PhD, Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu Seoul 110-744 Korea. E-mail hyosoo{at}snu.ac.kr
Abstract
Background— The forkhead factor, FOXO3a, is known to induce apoptosis in endothelial cells (ECs). However, its effects on extracellular matrices (ECM), which are important in EC survival, remained unknown. Here, we evaluated the role of FOXO3a on EC-ECM interaction.
Methods and Results— Constitutively active FOXO3a was transduced to human umbilical vein endothelial cells by adenoviral vector (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection led to dehiscence of ECs from fibronectin-coated plates, resulting in anoikis, which was significantly reversed by matrix metalloproteinase (MMP) inhibitor, GM6001. FOXO3a increased the expression of MMP-3 (stromelysin-1) but decreased the expression of tissue inhibitors of metalloproteinases-1 (TIMP-1), which was associated with increased MMP enzymatic activity in zymography. Pathophysiologic conditions such as serum starvation or heat shock also induced activation of endogenous FOXO3a, leading to activation of MMP-3 and apoptosis, which was reversed by GM6001. Delivery of Ad-TM-FOXO3a to the intraluminal surface in vivo led to EC denudation, disrupted vascular integrity, and impaired endothelium-dependent vasorelaxation.
Conclusion— Activation of MMPs and possible ECM disruption represent novel mechanisms of FOXO3a-mediated apoptosis in ECs.
The forkhead factor, FOXO3a, is known to induce apoptosis in endothelial cells. Here, we evaluated the role of FOXO3a on endothelial cells-extracellular matrix interactions. We found activation of MMPs and possible ECM disruption represent novel mechanisms of FOXO3a-mediated apoptosis in endothelial cells.
Key Words: FOXO3a • MMP • endothelial cell • apoptosis
Cell-to-cell and cell-to-matrix contacts are pivotal for the maintenance of survival in anchorage-dependent cells such as endothelial cells (ECs).1 Withdrawal of anchorage-dependent cells from their association with the ECM is known to result in anoikis, a type of apoptosis that is induced by inadequate cell-to-matrix interactions,2,3 and proteolysis of adherens junctions disrupting endothelial monolayer integrity is known to induce apoptosis.4,5
The forkhead factors, which were identified as important downstream molecules of phosphoinositide-3-OH kinase (PI3K)/Akt pathway,6,7 are known to play important roles in the regulation of apoptosis,6,8 cell cycle arrest,8,9 and adaptation to cellular stress.10,11 The forkhead factors have been also reported to serve essential roles in the maintenance of vascular stability.12 Various pathophysiologic conditions including hyperglycemia13 have been shown to activate forkhead factors, whereas vascular protecting factors such as shear stress14,15 and statins16 are reported to inhibit forkhead factors in blood vessels. FOXO3a, a member of the forkhead factor family, has been reported to induce apoptosis in vascular smooth muscle cells,6,17,18 as well as in endothelial cells.7,19 However, the mechanism of FOXO3a-mediated apoptosis in ECs is still unclear and has not yet been fully defined.
In the present study, we identify a novel action of FOXO3a in the regulation of EC-ECM interaction. We found that the activation of FOXO3a induced detachment of endothelial cells from ECM through the activation of matrix metalloproteinase-3 (MMP-3, also called as stromelysin-1) and the suppression of tissue inhibitor of metalloproteinase-1 (TIMP-1). Here, we specify the functional significance of increased MMP activity by FOXO3a signaling in regulating EC survival and vascular integrity.
Materials and Methods
For expanded methods, please see the supplemental materials, available online at http://atvb.ahajournals.org.
Cell Culture, Adenoviral Vectors, and siRNA
Four to 6 passage human umbilical vein endothelial cells (HUVECs; Clonetics) were seeded on 2% gelatin-coated (Sigma) culture plates and incubated in endothelial growth medium (EGM bullet kit, Clonetics) with 10% fetal bovine serum.
Cell Viability and Apoptosis Assay
Viability of HUVECs was quantified using tetrazolium salt, WST-1, as instructed by the manufacturer (Roche). Apoptosis after 24 hours of adenoviral vector transfection was determined by measuring the hypodiploid fragmented DNA content using fluorescence-activated-cell sorter (FACS) analysis.20
Real-Time Polymerase Chain Reaction Analysis
Changes in RNA-expression of MMP-3 was determined by quantitative real-time polymerase chain reaction (PCR) as previously described.7
Immunoblot Analysis
Immunoblot analysis was performed by modification of the procedures described previously.21
Casein and Gelatin Zymography
Zymography was performed using a previously described method.22
Immunofluorescent Staining
For immunofluorescent staining, HUVECs were cultured on fibronectin coated dishes.
Fibronectin Degradation Assay
The degradation of fibronectin by the supernatant from Ad-TM-FOXO3a–transfected HUVEC cultured dish was evaluated.
Cell Detachment Assay
HUVECs (1x104 cells per well) were seeded on each well of the fibronectin (5 µg/cm2) precoated 96-well plates and incubated at 37°C for 6 hours to allow adhesion.
In Vivo Gene Delivery in Rabbit Carotid Arteries
In vivo gene delivery was performed in carotid arteries of New Zealand White rabbits as previously described23 to evaluate whether FOXO3a transduction induced EC denudation and functional derangement.
Histological Analysis of Endothelial Denudation
The harvested arterial segments were stained with Evans blue to identify the endothelium-denuded luminal surface.
Organ Chamber Analysis for Vascular Reactivity
Rings (4 mm long) from each carotid artery were used to assess vascular reactivity. Rings were connected to isometric force displacement transducers (Grass Instruments) and suspended in organ chambers as previously described.23
Statistical Analysis
All data are expressed as mean ± SD
Results
FOXO3a Induces Detachment of HUVECs from Matrix, Leading to Anoikis
HUVECs were infected by either Ad-GFP or Ad-TM-FOXO3a in the presence of serum and were harvested 24 hours thereafter. We found that Ad-TM-FOXO3a-transfected HUVECs showed significant detachment from the culture dish (Figure 1A). Survival of HUVECs was also found to decrease using a WST-1 assay (Figure 1B). Interestingly, Ad-TM-FOXO3a-transfected HUVECs were found to lose contact with plates as well as with neighboring cells at early stages after adenoviral transfection and before the appearance of other morphological changes that are associated with apoptosis (Figure 1C).
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From these observations, we presumed that the activation of FOXO3a might disrupt cell-to-cell and cell-to-ECM adhesive interactions, leading to apoptosis. We also presumed that MMP activation might play a role in this process, because in microarray experiments comparing Ad-TM-FOXO3a–transfected HUVECs with Ad-GFP, the MMP-3 gene was one of the most robustly upregulated genes after FOXO3a-transduction, whereas TIMP-1 gene expression was found suppressed (data not shown).
To confirm whether MMPs play a main role in EC detachment after FOXO3a-transduction, we pretreated HUVECs with GM6001, a general MMP inhibitor, which markedly decreased EC detachment from the plate as well as from the neighboring cells (Figure 1D and 1E). Furthermore, FACS analysis for hypodiploid DNA showed about 50% reduction of apoptotic fraction in the presence of GM6001 (Figure 1F), suggesting that MMP activation might induce anoikis in FOXO3a-transduced HUVECs.
FOXO3a Induces MMP-3 in Endothelial Cells
To confirm whether the endothelial cell detachment after FOXO3a overexpression was mediated by MMP activation, we evaluated the regulation of MMP-3 after FOXO3a transduction. mRNA levels of MMP-3 was found to be significantly upregulated by real-time PCR assay. The relative fold elevation of MMP-3 transcript compared with GAPDH was 1±0.3 at 16 hours, 137±40 at 24 hours, and 690±250 at 40 hours (Figure 2A). MMP-3 protein synthesis also increased in Ad-TM-FOXO3a–transfected HUVECs and decreased in Ad-DN-FOXO3a–transfected HUVECs. In contrast, TIMP-1 protein expression was found to be modestly decreased in Ad-TM-FOXO3a–transfected HUVECs and increased in Ad-DN-FOXO3a–transfected HUVECs. (Figure 2B).
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Next, we performed casein and gelatin zymography to study whether increased MMP-3 protein retained enzymatic activity. In the casein zymography analysis, a major band corresponding to 57 kDa, and caused by the protease activity of MMP-3 was increased in media from Ad-TM-FOXO3a–transfected cells compared with media from Ad-GFP–transfected cells. In contrast, transduction of dominant-negative FOXO3a decreased MMP-3 activity in this assay (Figure 2C, upper panel). Of note, although mRNA or protein expression of MMP-2 or MMP-9 did not increase (data not shown), gelatin zymography showed an increased enzymatic activity of MMP-9 (92kDa) and MMP-2 (72kDa; Figure 2C, lower panel). Because MMP-3 is reported to activate other MMPs including MMP-2/MMP-9,24,25 we performed MMP-3 specific knockdown experiments using MMP-3 siRNA. We found MMP-2 and MMP-9 enzymatic activity decreased with MMP-3 siRNA, suggesting that MMP-3 activation contributed to increased enzymatic activity of MMP-2 or MMP-9 after FOXO3a activation. Moreover, we also found that MMP-3 specific knockdown showed similar degree of reduction of apoptosis to that of GM6001 (supplemental Figure IIA through IID).
Stress to Endothelial Cells Induces Endogenous FOXO3a-MMP-3 Activation
To examine the role of the endogenous FOXO3a-MMP regulatory axis during the stress response, HUVECs were subjected to serum starvation or to heat shock without adenoviral transfection. An increase in active FOXO3a in nuclear fraction was detected at 6 hours after the initiation of serum starvation (supplemental Figure IIIA), which correlated with the time course of MMP activity increase in the zymography assay (supplemental Figure IIIB). In the nonstressed state, endogenous FOXO3a is located in cytoplasm and where it is inactive (supplemental Figure IIIC, left upper panel). In addition, heat shock rapidly induced intranuclear translocation and activation of endogenous FOXO3a by 30 minutes (supplemental Figure IIIC, left lower panel), which can be confirmed by the increased FOXO3a amount in nuclear fraction (supplemental Figure IIID). Enzymatic activity of MMP-3 was also found to be increased under these conditions (supplemental Figure IIIE). Heat shock induced cell death, which was significantly reduced by GM6001 treatment, suggesting that FOXO3a-MMP signaling contributes to the cytotoxicity of ECs during stress (supplemental Figure IIIF).
FOXO3a Reduces Cell-to-ECM Interaction
To investigate whether FOXO3a activation leads to ECM degradation, we evaluated the degradation of the matrix protein fibronectin after FOXO3a-transduction. After 12 hours of Ad-TM-FOXO3a transfection, fibronectin levels decreased significantly and this downregulation was effectively reversed by treatment with GM6001, suggesting that the enhanced MMP activity might contribute to the degradation of fibronectin (supplemental Figure IVA). To examine this regulation further, the supernatant from each culture dish was mixed with 5 µg of fibronectin and the degree of protein degradation was measured. The supernatant from the Ad-TM-FOXO3a–transfected culture dish displayed significantly increased fibronectin-proteolytic activity. Furthermore, the enhanced degradation was reduced by treatment with GM6001, suggesting these responses were mediated by a secreted MMP (supplemental Figures IVB and V). Next, to evaluate the functional significance of FOXO3a activation on adhesive capacity of HUVECs to ECM, we tested the adhesive capacity of HUVECs on fibronectin coated plates. FOXO3a activation significantly decreased the adhesion of HUVECs to the plates, which was reversed in the presence of GM6001 (supplemental Figure IVC).
FOXO3a Reduces Cell-to-Cell Adhesion
Next, we examined whether FOXO3a affected cell-to-cell adhesion systems maintained by the major adhesion molecules, VE-cadherin and β-catenin. Figure 3A shows uniform VE-cadherin staining over the entire cell margin, and Figure 3B shows the staining pattern of its intracellular partner β-catenin, that is present at adherens junction as well as in the cytoplasm of HUVECs (Figure 3A and 3B, left). At 24 hours after Ad-TM-FOXO3a transfection, the expression of both of these molecules was decreased at the cell-cell junctions (Figure 3A and 3B, middle). The degradation of both VE-cadherin and β-catenin was almost completely reversed in the presence GM6001, suggesting that MMP might mediate these response (Figure 3A and 3B, right). These morphological changes were corroborated by immunoblot analysis of cell lysates, which showed decreased VE-cadherin and β-catenin protein in TM-FOXO3a-transduced HUVECs and partial reversal by GM6001 (Figure 3C).
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FOXO3a Activation in the Vessel Wall Induces Endothelial Denudation and Loss of Barrier Function
We evaluated vascular integrity after FOXO3a activation in rabbit carotid arteries after FOXO3a transduction. At 24 hours after Ad-TM-FOXO3a transfer, arterial segments showed significant endothelial denudation, which was markedly reversed by GM6001 (supplemental Figure VIA and VIB). Immunohistochemical staining of PECAM-1 revealed greater endothelial denudation after FOXO3a gene transfer compared with the control. Endothelial denudation was significantly reversed by GM6001, suggesting that these findings were mediated by MMPs (Figure 4A, supplemental Figure VIC). Scanning electron microscopy showed a regular and smooth-surfaced endothelial lining in the Ad-GFP group (Figure 4B, upper middle), similar to the endothelial lining of a normal, uninjured carotid artery (Figure 4B, upper left). In contrast, the luminal surface of segments harvested from the Ad-TM-FOXO3a–transfected group demonstrated an irregular endothelial lining, exposing subendothelial tissue and various stages of endothelial cells detached from the subcellular matrix (Figure 4B, lower panels). GM6001, again, significantly reversed these phenomena (Figure 4B, upper right).
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FOXO3a Activation Impairs Endothelial Function
Vascular rings from each carotid artery were applied to an organ chamber system to assess both endothelium-dependent (Figure 4C) and endothelium-independent vasorelaxation (Figure 4D). Both endothelium-dependent and endothelium-independent vasorelaxation were significantly impaired in the TM-FOXO3a–transduced vessels. However, the FOXO3a-induced impairment of endothelium-dependent vasorelaxation in response to acetylcholine was much more profound compared with the relatively mild impairment of endothelium-independent vasorelaxation in response to sodium nitroprusside. In addition, the MMP blocker, GM6001, which would in theory prevent cellular detachment or anoikis, significantly reversed only the deficit in endothelium-dependent vasorelaxation (Figure 4C) and not endothelium-independent vasorelaxation (Figure 4D). These data suggest that FOXO3a activation in the vessel results in a greater impairment of endothelial-dependent vasorelaxation, and that the profound impairment of endothelium-dependent vasorelaxation may be related to anoikis.
Discussion
The most important finding of our study is that FOXO3a, a major forkhead transcription factor expressed in endothelial cells, suppressed cell-to-cell and cell-to-matrix interaction in ECs. These data suggest a novel mechanism of FOXO3a-induced apoptosis in endothelial cells. First, FOXO3a induced detachment of ECs from ECM or adjacent ECs leading to anoikis through degradation of major adherens junctional proteins such as fibronectin, VE-cadherin, and β-catenin. Second, FOXO3a induced MMP-3 and suppressed TIMP-1 expression in ECs. The finding that EC detachment, disruption of ECM and resulting anoikis were significantly reversed by GM6001, a MMP inhibitor, suggests the increased MMP activity mediates these responses. Third, these phenomena were also reproduced under pathophysiologic conditions. For example, endogenous FOXO3a induction and MMP activation were observed in ECs under stressful conditions such as serum starvation or exposure to heat shock. Finally, we showed both in vivo and ex vivo, that FOXO3a induced endothelial denudation and endothelial dysfunction in blood vessels.
FOXO3a, ECM, and EC Survival
Control of apoptosis in the endothelium is a critical issue during pathologic processes such as inflammation, vascular remodeling, and allograft vasculopathy.26 The binding to ECM is an important survival signal to ECs. The degradation of the ECM proteins such as fibronectin has been shown to affect the apoptotic program of ECs and epithelial cells.27–30 Another important element in EC survival is the adherens junction, in which VE-cadherin anchors the cytoskeleton of neighboring cells via β- or 
-catenin.31–33 Both the degradation of VE-cadherin and β-catenin have been observed during apoptosis, implicating their role in apoptosis.34,35 In the present study, we found FOXO3a activation decreased fibronectin, VE-cadherin, and β-catenin levels. This decrease could be the result of the reduced synthesis of ECM, rather than the increased degradation. In this regard, Daly et al showed that FOXO1, another forkhead transcription factor, regulated genes involving in the remodeling of ECM such as decorin, lumican, and collagen type III.36 However, this hypothesis does not explain both the rapid decrease of ECM and adherens junction proteins after FOXO3a transduction, nor does it explain the ability of the MMP inhibitor to neutralize the effect of FOXO3a on cytotoxicity. Thus, we think that degradation by MMPs plays a major role in matrix regulation by FOXO3a.
FOXO3a and MMP3 Activation in ECs
MMPs constitute a family of extracellular proteases that are involved both in normal physiological remodeling and in pathologic degradation of the ECM.37 ECM degradation by MMPs has been shown to be a signal that can induce apoptosis in several studies.27,38 Moreover, exogenous TIMP-1, the major negative regulator of MMPs, has been shown to exert a potent antiapoptotic effect on ECs.39 Among various MMPs, we suggest MMP-3 might mediate FOXO3a-induced EC anoikis for 3 reasons. First, MMP-3 was highly upregulated in the microarray analysis of FOXO3a-induced genes, and this upregulation was confirmed at both mRNA and protein levels in the present study. Second, MMP-3 has a consensus binding site for the forkhead factors in its promoter sequences40 suggesting transcriptional regulation by FOXO3a. Third, we found MMP-3 levels increased after FOXO3a activation. Other studies evaluating promoter sequence or microarray analysis have suggested possible regulation of some MMPs by forkhead factors,40,41 and though PI3K/Akt pathway was suggested to increase enzymatic activity of MMPs.42,43 However, to the best of our knowledge, this is the first report documenting a direct regulatory connection between MMPs and forkhead factors.
FOXO3a and Other MMPs Activation in ECs
Interestingly, enzymatic activities of MMP-2 and MMP-9 also increased after FOXO3a activation. However, we could not find any evidence of direct regulation of MMP-2 or MMP-9 by FOXO3a. First, neither mRNA nor the protein amount of MMP-2/MMP-9 changed after FOXO3a activation. Second, in the microarray data comparing Ad-TM-FOXO3a–transfected HUVECs with either Ad-GFP or Ad-DN-FOXO3a–transfected HUVECs, the expression pattern of MMP-2/MMP-9 did not show any significant change (data not shown). Third, the promoter sequences of MMP-2 or MMP-9 do not contain the consensus binding site for the forkhead transcription factors. Therefore, we hypothesize that the increased enzymatic activity of MMP-2 or MMP-9 observed after FOXO3a activation involves indirect regulation through changes in the expression of other molecules. We suspected that MMP-3 activation may be responsible for the activation of MMP-2/MMP-9, consistent with previous findings.24,25 Indeed, we found that MMP-3 knockdown with siRNA led to significant reduction in the enzymatic activity of MMP-2 and MMP-9, supporting the hypothesis that MMP-3 activation contributes to increased enzymatic activity of MMP-2 or MMP-9 after FOXO3a activation. Moreover, we also found that MMP-3 knockdown revealed a significant reduction of apoptosis compared with that of GM6001, suggesting further that MMP-3 plays a key role in FOXO3a-induced ECM disruption. However, because MMP activity is also regulated by other molecules, such as TIMP-1,25 we do not exclude the possibility that the suppression of TIMP-1 or the activation of other MMPs might result in global MMP activation. For this reason, we decided to use the general MMP inhibitor GM6001 to investigate the association between FOXO3a, MMP activity, and endothelial cell apoptosis. Finally, we cannot ascribe FOXO3a-induced apoptosis solely to MMP activation, because blocking experiment with GM6001 failed to completely reverse the effects of FOXO3a on apoptosis and anoikis. However, these findings offer new insights to the multifaceted role of FOXO3a in ECs.
In Vivo Significance of MMP Activation by FOXO3a in ECs
We found that FOXO3a activation induced endothelial denudation in vessels through in vivo gene delivery. We also found FOXO3a activation impaired both endothelium-dependent and endothelium-independent vasorelaxation through ex vivo evaluation of vasoreactivity. These effects may be caused by the proapoptotic actions of FOXO3a on ECs7,19 as well as smooth muscle cells.17,44,45 However, the degree of vasorelaxation impairment had a significantly greater impact on endothelium dependent function. Furthermore, GM6001 significantly reversed endothelium-dependent vasorelaxation, but had little or no effects on endothelium-independent vasorelaxation, suggesting that MMP activation may play a role in endothelial dysfunction following FOXO3a activation in vivo.
In summary, suppression of EC-ECM or EC-EC interaction represents a novel mechanism of FOXO3a-mediated EC apoptosis. Our results also suggest that MMP activation caused by deregulated FOXO3a expression could contribute to endothelial dysfunction of blood vessel.
Acknowledgments
Sources of Funding
This study was supported by the grants from the National Research Laboratory for Cardiovascular Stem Cell, Ministry of Science & Technology, and from the Innovative Research Institute for Cell Therapy (IRICT: A062260), Ministry of Health & Welfare, Republic of Korea.
Disclosures
None.
Footnotes
H.-Y.L. and H.-J.Y. contributed equally to this study.
Original received June 28, 2007; final version accepted November 21, 2007.
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