Cardiovascular Research

Cardiovascular Research 2001 49(4):872-881; doi:10.1016/S0008-6363(00)00295-9
© 2001 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

The angiopoietin–tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis

Injune Kim^a, Sang-Ok Moon^a, Chang-Yeop Han^b, Youngmi Kim Pak^b, Sung Ki Moon^c, Jong Jun Kim^c and Gou Young Koh^a,*

^aNational Creative Research Initiatives Center for Cardiac Regeneration and Institute of Cardiovascular Research, Chonbuk University School of Medicine, Chonju, South Korea
^bDepartment of Biomedical Sciences, Division of Metabolic Disease Research, National Institute of Health, Seoul, South Korea
^cDepartment of Internal Medicine, Chonju Hospital, Chonju, South Korea

^* Corresponding author. Tel.: +82-63-270-3080; fax: +82-63-270-4071 gykoh{at}moak.chonbuk.ac.kr

Received 2 August 2000; accepted 7 November 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 
Objectives: A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin–Tie2 system in porcine coronary artery. Methods: Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used. Results: Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery. Conclusion: These results suggest that adult coronary artery contains Ang1–Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.

KEYWORDS Arteries; Endothelial receptors; Gene expression; Growth factors

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 
The coronary artery endothelium, like other endothelia, consists of a mono-layer of endothelial cells and is involved in a variety of functions, including maintaining normal vascular tone, prevention of thrombosis, and pathologic remodeling [1,2]. Damage to the coronary endothelium can initiate thrombosis formation, neointimal hyperplasia, and atherogenesis. Therefore, maintaining a normal integrity of coronary endothelium in response to physical, biochemical and immune-mediated damages is important to prevent coronary artery diseases [1–3]. Of these biochemical damages, oxidized low-density lipoprotein (OxLDL) is one of the most threatening metabolites to endothelial cells [4,5]. It is firmly established that elevated plasma concentration of oxidized low-density lipoprotein (OxLDL) is associated with coronary artery diseases. OxLDL produces numerous detrimental effects on endothelial cell function including the induction of apoptosis [6–8].

Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have been identified as ligands of the endothelial cell-specific Tie2 receptor [9,10]. In vivo analyses by targeted gene inactivation and transgenic overexpression suggest that Ang1 recruits and sustains periendothelial support cells while Ang2 disrupts blood vessel formation in the developing embryo by antagonizing the effects of Ang1 on Tie2 [10,11]. Interestingly, transgenic overexpression or gene transfer of Ang1 increases vascularization [12,13] and decreases vascular leakage in vivo [14,15]. In vitro experiments have shown that Ang1 has specific effects on endothelial cells: it potently induces chemotactic response [16], network formation [17], sprouting [18,19] and survival from apoptosis [20,21]. However, the role of the angiopoietin–Tie2 system in normal adult blood vessels is not known. Recently we found that Ang1 is constitutively expressed by the periendothelial cells of several arteries, including vascular smooth muscle cells of the adult coronary artery [21]. Interestingly, previous reports indicated that Tie2 is selectively expressed in the endothelial cells of normal adult vessels, in which vasculogenesis and angiogenesis do not normally occur [22]. More surprisingly, it is present in these cells primarily in its phosphorylated (activated) form. Given that Ang1 is a strong apoptosis survival factor without mitotic effect, we suspect that constitutive expression of Ang1 may activate the Tie2 receptor, thereby acting as a paracrine factor that maintains normal integrity in non-proliferating endothelial cells.

Considering the importance of normal coronary endothelial integrity for prevention and recovery from coronary artery diseases, activity of the angiopoietin–Tie2 system in the coronary artery should be carefully analyzed. Here, we examined the angiopoietin–Tie2 system in intact porcine coronary artery, primary cultured porcine coronary artery endothelial cells (PCAECs), and porcine coronary artery vascular smooth muscle cells (PCAVSMCs). Our results suggest that Ang1 produced by the periendothelial cells may continuously activate the endothelial Tie2 receptor and thus enhance endothelial cell survival in the coronary artery. In addition, we demonstrate that Ang1 increases survival of these cells in response to oxidized low-density lipoprotein (OxLDL). Therefore, we conclude that the angiopoietin–Tie2 system is essential to the maintenance of normal coronary artery endothelial survival and integrity.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 
2.1 Materials
Ang1* was obtained from Regeneron Pharmaceuticals, Inc. Ang1* is a recombinant version of Ang1 that is easier to produce and purify [10]. Ang1* contains a modified NH₂-terminus and mutated Cys²⁴⁵. The Cys²⁴⁵ residue is not shared between Ang1 and Ang2, and its mutation does not alter Ang1's agonistic properties. The biological activity of recombinant Ang1 and Ang1* is similar, confirmed by their high-affinity binding to and stimulation of the Tie2 receptor in vitro. Oxidized low-density lipoprotein (OxLDL) was generated according to the method described previously [23,24]. Briefly, low-density lipoprotein (LDL) was isolated by sequential ultracentrifugation (d = 1.019–1.063) from freshly drawn, citrated normolipidemic human plasma to which EDTA was added. LDL was oxidized in the presence of 5 µM CuSO₄ for 24 h at 37°C to make a OxLDL. The degree of oxidation was assessed by the increase of mobility on 1% agarose gel (3.13 versus native LDL). Polyclonal anti-Ang1 and anti-Ang2 antibodies were produced by immunization of rabbits with a recombinant NH₂-terminal portion of human Ang1 and a recombinant mid-portion of human Ang2, respectively, as described previously [21,25]. Anti–Tie2 antibody was obtained from Santa Cruz Biotech (Santa Cruz, CA). Most other biochemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise specified. Media and sera were obtained from Gibco BRL (Gaithersburg, MD).

2.2 Immunohistochemistry in porcine coronary artery
Pieces of external cardiac wall including coronary artery were dissected from adult pigs and immediately fixed with 10% (v/v) neutral buffered formalin. Paraffin tissue blocks were sectioned at 4 µm. Immunohistochemistry was performed as described previously [26]. Sections were incubated with the preimmune serum or primary antibody at 4°C overnight, and signals were visualized with the EnVison system (DAKO, Copenhagen, Denmark). Sections were counterstained with Meyer's hematoxylin.

2.3 Cell culture
Primary cultured PCAECs and PCAVSMCs were prepared as previously described [27]. The endothelial or muscle origin of the cultures was confirmed by immunofluorescent staining with an anti-von Willebrand factor antibody or anti-smooth muscle actin antibody. Acceptable cultures had <97% fluorescent cells with the corresponding antibody. These cells were maintained in Dulbecco's modified Eagle's (DME) medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HIFBS) at 37°C in 5% CO₂. The primary cultured cells used in this study were between passage 2 and 5.

2.4 Molecular cloning of porcine Ang1 and Ang2
Partial cDNAs of porcine Ang1 and Ang2 were amplified using PCAEC and PCAVSMC cDNAs as PCR templates. PCR was performed for 30 cycles at an annealing temperature of 52°C using the following conserved peptides of human Ang1 and Ang2: GEYWLG (Ang1, amino acids 353–358; Ang2, amino acids 351–356) and SNLNGM (Ang1, amino acids 455–460; Ang2, amino acids 453–458) [9,10]. A DNA band of the expected size (300 base pairs) was amplified from both templates. The amplified DNA was sequenced by cycle sequencing (AmpliCycle, Perkin Elmer, Foster City, CA). The product from PCAECs contained a partial porcine Ang2 cDNA, and the product from PCAVSMCs contained a partial porcine Ang1 cDNA. To clone the remaining coding regions, rapid amplification of cDNA ends PCR (RACE-PCR) was performed with PCAEC and PCAVSMC cDNAs that were prepared by the Marathon cDNA amplification kit (Clontech, Palo Alto, CA).

2.5 RNase protection assay for expression analysis of Ang1, Ang2, and Tie2 mRNA transcripts
Fragments of porcine Ang1 (bp 439–964), porcine Ang2 (bp 1984–2265), porcine Tie2 (bp 1415–1812) and porcine glyceraldehyde 3-phosphate dehydrogenase (GAPDH, bp 323–578) were used as templates for RNA probe synthesis. The PCR products from PCAECs and PCAVSMCs were subcloned into pBluescript II KS+ as DNA template (Stratagene, La Jolla, CA) for RNA synthesis. After linearizing with XhoI, ³²P-labeled antisense RNA probes were synthesized by in vitro transcription using T7 polymerase (Maxiscript kit, Ambion, Austin, TX). After gel purification of the probes, RPA was performed with total RNAs using the Ambion RPA kit. An RPA using GAPDH as probe was carried out for RNA quantification.

2.6 Phosphorylation assays of Tie2
PCAECs were seeded to 6-well plates at a density of 5x10⁴ cells/cm² and were grown in DME medium with 10% HIFBS for 24 h. After 24 h of serum deprivation, Ang1 (200 ng/ml) or Ang2 (200 ng/ml) was added to the cells at the indicated amounts, and the cells were incubated for 10 min at 37°C in 5% CO₂. The phosphorylation assay of Tie2 was performed as previously described [21].

2.7 Induction of apoptosis in endothelial cells and quantification of apoptosis
Endothelial cells were plated onto gelatinized 24-well plates (5x10⁴ cells per well) in medium containing 10% HIFBS and incubated for 24 h. Then, control buffer, LDL (50 µg) or OxLDL (50 µg) was added to the growth medium containing 5% HIFBS and various amounts of Ang1 and the cells were incubated for 12 h. In some cases, soluble Tie2 receptor, rTie2–Fc (2 µg/ml; a five-fold molar excess), PI 3'-kinase inhibitors, wortmanin (30 nM; RBI, Natick, MA) or LY294002 (100 nM; RBI) was added 1 h before Ang1* (200 ng/ml) treatment. Quantification of apoptosis was performed as described previously [21]. Briefly, floating cells were collected with two PBS washes; adherent cells were collected by trypsinization. The numbers and size distributions of the floating and adherent cells were determined with a Coulter Model Z1 Dual Counter System. More than 95% of the floating cells were apoptotic cells, as confirmed by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit (Oncor, Gaithersburg, MD). To detect the apoptotic cells among the adherent cells, the cells in parallel wells were washed with PBS, fixed for 5 min with 1.0% (w/v) paraformaldehyde, and subjected to a TUNEL assay. The adherent cells were examined at 400x magnification, and the stained cells were counted in four different random locations, each containing approximately 250 cells. The percentage of apoptotic cells is based on the sum of the floating cells plus the apoptotic adherent cells in a given cell population.

2.8 Explant culture and detection of apoptotic cells in the endothelium of porcine coronary artery
Explant culture of porcine coronary artery was performed according to the method described by Merrick et al. [28]. Fresh porcine coronary arteries were washed with PBS three times and were cut into 5-mm ring segments. The rings were cultured in DME medium containing 5% HIFBS, treated with OxLDL (50 µg/ml) in the absence or presence of Ang1 (200 ng/ml) for 12 h at 37°C, 5% CO₂. The rings were washed in PBS, fixed in 10% neutral formalin, and embedded in paraffin. Tissue blocks were sectioned at 8 µm. An anti-CD31 monoclonal antibody (clone JC70A, DACO) was used for endothelial staining and the TUNEL method was used for calculation of survival cells. The endothelial cells and apoptotic cells in the endothelium of the aortic rings were viewed, counted, and photographed with a microscope equipped with color CCD camera and monitor. Seven to eight arterial rings from different coronary arteries were used for each group. Approximately 370–380 endothelial cells were counted from one section of each coronary arterial ring. For quantitative analysis of survival of endothelial cells in coronary arterial rings, the total number of endothelial cells per ring section of non-incubated coronary arterial rings was counted after immunostaining with anti-CD31 antibody. In porcine coronary arteries, each ring section has a 375±4 (n = 22) endothelial cells. Therefore, the percent survival was calculated by (No. of CD31 positive cells–No. of TUNEL positive cells/375)x100.

2.9 Statistics
Data are expressed as mean±standard deviation. Statistical significance was tested using one-way ANOVA followed by the Student–Newman–Keuls test. Statistical significance was set at P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 
3.1 Ang1, Ang2, and Tie2 are selectively localized and expressed in the porcine coronary artery
Immunohistochemical staining shows that the Ang1 protein was mainly located in muscular areas of coronary artery (Fig. 1). In contrast to Ang1 protein, Ang2 and Tie2 proteins were selectively located in endothelial cells (Fig. 1). However, Ang2 immunoreactivity was present, but at a relatively low level, in the endothelial cells and sub-endothelial internal layer of coronary artery. In contrast, Ang2 strongly stained in endothelial cells of small blood vessels of breast adenocarcinoma used as a positive control (Fig. 1, inset).

View larger version (90K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Immunohistochemistry of normal adult porcine coronary artery. Immunoperoxidase staining of Ang1, Ang2, and Tie2. The slides were incubated with post-immune polyclonal antibodies (upper panels) or the corresponding pre-immune sera (lower panels). As a positive control for immunostaining of Ang2, small blood vessels of breast adenocarcinoma are shown (inset). Arrowheads indicate reddish–brown immuno-positive signals. Bar=25 µm.

 
To examine the expression of Ang1, Ang2, and Tie2 at the cellular level, the PCAECs and PCAVSMCs were isolated from coronary artery and cultured. Primary cultured PCAECs show typical cobblestone appearance, whereas the primary cultured PCAVSMCs show typical spindle-shaped morphology with a hill-and-valley pattern at confluence (Fig. 2). In agreement with the immunohistochemical analyses, an RNase protection assay showed that Ang1 mRNA was detected mainly in the cultured PCAVSMCs, whereas Tie2 mRNA was detected mainly in the cultured PCAECs (Fig. 3). Interestingly, a relatively high level of Ang2 mRNA was detected in the cultured PCAECs, whereas only a low level of Ang2 mRNA was detected in PCAVSMCs (Fig. 3). Thus, expression of Ang2 may be up-regulated in culture.

View larger version (115K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Phase-contrast microscopy of primary cultured PCAECs and PCAVSMCs. After second passages, PCAECs and PCAVSMCs were grown to confluence in the presence of 10% serum. The PCAECs show typical cobblestone appearance, whereas the PCAVSMCs show typical spindle-shaped morphology with a hill-and-valley pattern at confluence. Magnifications are x200.

 

View larger version (46K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 RNase protection assay of Ang1, Ang2, and Tie2. After third passage, PCAECs and PCAVSMCs were grown to subconfluence in the presence of 10% serum. Each lane contains 10 µg total RNA. The RNAs were subjected to RNase protection assay using a corresponding antisense probe. The integrity of the probes is shown (first lane). Total yeast RNA (10 µg) was similarly assayed in the presence of RNase (second lane). Ang1, Ang2, and Tie2 transcripts generated protected bands of 440, 281, and 397 bp, respectively. Equivalent loading was confirmed by RPA using GAPDH as probe (lowest panel). Results were similar in two independent experiments.

 
3.2 High degrees of identity are found among porcine, human, and mouse Ang1 and Ang2 sequences
Using homology-based PCR and RACE-PCR from PCAVSMC and PCAEC cDNAs, we obtained full sequences of porcine Ang1 and Ang2. The porcine Ang1 and Ang2 cDNAs encode 498-amino acid and 496-amino acid polypeptides, respectively (GenBank AF233227 [GenBank] and AF233228 [GenBank] ). Both have coiled-coil domains in the NH₂-terminal portion and conserved fibrinogen-like domains in the COOH-terminal portion, as do angiopoietins from other species [9,10]. Porcine Ang1 shares 97 and 95% amino acid identity with human and mouse Ang1, whereas porcine Ang2 shares 92 and 84% amino acid identity with human and mouse Ang2 (Table 1). Nine or eight cysteines found in mouse and human Ang1 or Ang2 are conserved in porcine Ang1 or Ang2.

View this table: [in this window] [in a new window]   Table 1 Percent identity among porcine, human, and mouse Ang1 or Ang2 full nucleotide and amino acid sequencesa

 
3.3 Ang1, but not Ang2, phosphorylates the Tie2 receptor in PCAECs
Previous experiments indicated that in HUVECs maximal Tie2 phosphorylation occurred 10 min after addition of Ang1 [21]. In the present study, Ang1 (200 ng/ml) induced approximately a 16-fold increase in Tie2 phosphorylation in PCAECs (Fig. 4). As expected, however, Ang2 (200 ng/ml) did not produce significant changes in Tie2 phosphorylation (Fig. 4). These results suggest that Ang1 acts as a functional agonist to the Tie2 receptor in coronary artery endothelial cells.

View larger version (90K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effect of Ang1 and Ang2 on Tie2 phosphorylation in PCAECs. Cells were incubated for 24 h in serum-free medium, then incubated for 10 min in the presence of control buffer (CB), Ang1 (200 ng/ml), or Ang2 (200 ng/ml). The cell lysates were immunoprecipitated with anti-Tie2 antibody, and separated by SDS–PAGE. After western blotting with anti-phosphotyrosine antibody (upper), the membranes were stripped and re-incubated with corresponding antibodies (middle) for detection of total Tie2. Lower panels, densitometric analysis shows the relative ratio of phosphorylated Tie2/total Tie2. Each control is arbitrarily estimated as one. Data are presented as mean±S.E. of the ratios from three experiments.

 
3.4 Ang1 inhibits OxLDL-induced apoptosis
OxLDL treatment (50 µg/ml) caused apoptosis in PCAECs, evidenced by floating cells seen with phase-contrast microscopy and positively stained nuclei seen in cells assayed with TUNEL (Fig. 5A). OxLDL treatment (50 µg/ml) for 12 h increased apoptosis from 2.7% under control condition to approximately 25.4% in treated cells (Fig. 5B). In contrast, treatment with LDL (50 µg/ml) for 12 h did not produce significant apoptosis. In all experiments using OxLDL treatment, the proportion of floating apoptotic cells to total apoptotic cells was 33–39% (data not shown).

View larger version (90K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of Ang1 on OxLDL (50 µg/ml)-induced apoptosis in cultured PCAECs. (A) Representative phase-contrast microscopy (Phase) and light microscopy of TUNEL assay (TUNEL). Note that there are fewer adherent cells and more floating dead cells 12 h after OxLDL treatment compared to control buffer treatment. The cells exposed to Ang1 (400 ng/ml) are more adherent than the cells exposed to control buffer. Magnifications are 200x. Brown TUNEL-positives cells are apoptotic with fragmented DNA. Magnifications: 400x. (B) Quantification of apoptosis. Bars represent the mean±S.D. of five experiments. CB, control buffer; LD, LDL (50 µg/ml); 1A, 2A and 4A; 100 ng/ml, 200 ng/ml and 400 ng/ml of Ang1; T2, rTie2–Fc (2 µg/ml); WT, wortmanin (30 nM); LY, LY294002 (100 nM). * P<0.05 versus CB; # P<0.05 versus LDL; P<0.05 versus OxLDL only.

 
Ang1 inhibited the apoptotic events in OxLDL-treated PCAECs in a dose-dependent manner (Fig. 5B). Pretreatment of five-fold molar excess of soluble receptor, rTie2–Fc and phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitors, wortmanin or LY294002 almost completely inhibited Ang1's anti-apoptotic effect during OxLDL treatment (Fig. 5B). These results suggest that Ang1 exerts a strong cell survival effect through the Tie2 receptor and intracellular PI 3'-kinase.

We explanted porcine coronary arteries and treated them with OxLDL (100 µg/ml) for 12 h. This treatment clearly caused apoptosis and/or necrosis that increased TUNEL staining and decreased the staining of the endothelial marker, CD31 (Fig. 6A). Ang1 (200 ng/ml) noticeably reduced the number of TUNEL stained endothelial cells and the number of detached endothelial cells (Fig. 6A). Quantitative analysis revealed that administration of Ang1 increased endothelial cell survival approximately 58.4% in the coronary arterial rings following OxLDL treatment (Fig. 6B).

View larger version (158K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of Ang1 (200 ng/ml) on OxLDL (100 µg/ml)-induced cell death in endothelial cells of explanted porcine coronary arteries. Sampling was performed at 24 h after OxLDL treatment. (A) Upper panels, representative hematoxylin and eosin-stained cross section of an arterial segment (HE). Middle panels, representative TUNEL-assay in the adjacent section of the arterial segment (TU). Arrowheads indicate dark brown TUNEL-positive apoptotic endothelial cells. Lower panels, representative light microscopy of CD-31-positive endothelial cells in the other adjacent section of the arterial segment (CD). Arrowheads indicate the areas of detached endothelial apoptotic cells. Note that there are more TUNEL-positive and detached endothelial cells at 24 h after OxLDL. Ang1 reduced TUNEL-positive and detached endothelial cells. (B) Quantification of survival. The percentage of surviving cells per section was calculated as described in Methods. Bars represent the mean±S.D. from seven independent experiments. CB, control buffer; * P<0.05 versus CB; # P<0.05 versus OxLDL only.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 
Blood vessels form through two distinct processes, vasculogenesis and angiogenesis. Vasculogenesis involves the de novo differentiation of endothelial cells from mesodermal precursors, whereas angiogenesis generates new vessels that sprout from pre-existing ones [29]. In embryos, both processes are essential for normal development. In adults, angiogenesis is essential for wound healing and for the tissue restructuring of the endometrium and ovary during the female reproductive cycle [30,31]. Neovascularization in adults may occur during pathological processes such as the formation of collateral circulation, tumor growth, and metastasis [32,33]. For both angiogenesis and vasculogenesis, VEGF, Ang1, and Ang2 are essential endothelial cell specific ligands [34]. However, our recent study indicated that Ang1 is expressed in adult coronary artery smooth muscle cells in which no vasculogenesis or angiogenesis is occurring [21]. In addition, previous reports indicated that Tie2 is mainly expressed in the endothelial cells of normal adult vessels. Moreover, Tie2 is present in its phosphorylated (active) form [22]. Because Ang1 is a functional agonist to the Tie2 receptor, we speculated that they could have a biological significance in normal adult vessels. Furthermore, given the high incidence of coronary artery diseases, we chose the coronary artery for clarifying our speculation.

Our immunohistochemical analyses confirm that Ang1 is localized in coronary vascular smooth muscle cells, whereas Ang2 and Tie2 are selectively localized to coronary artery endothelial cells. Consistent with in vivo analyses, the primary cultured PCAVSMCs still mainly express Ang1 mRNA, whereas the primary cultured PCAECs still mainly express Ang2 and Tie2 mRNA. To date, the regulation of Ang1 mRNA expression is unknown, but we do have some information about the regulation of Ang2 mRNA expression. Hypoxia, VEGF, basic fibroblast growth factor and tumor necrosis factor- upregulate Ang2 expression in endothelial cells [25,35,36]. Furthermore, in cultured PCAECs, as we expected, Ang1 potently induced Tie2 phosphorylation, while Ang2 did not produce any changes in Tie2 phosphorylation. Therefore, we speculate that constitutive expression of Ang1 is involved in constitutive Tie2 activation as a paracrine factor in adult coronary artery endothelial cells in vivo. Thereby, Ang1 and Tie2 are essential components for maintaining the normal integrity of coronary endothelium.

In order to maintain normal coronary vessel morphology and function, coronary endothelial cells must survive even under onslaught by various biochemicals and metabolites in the blood. Of these, OxLDL is one of the most threatening metabolites to endothelial cells [4,5]. OxLDL-induced apoptosis in endothelial cells occurs through the activation of several mediators including caspases, Fas, and membrane sphingomyelinase [6–8]. The present results indicate that OxLDL induces significant apoptosis in primary cultured PCAECs and in endothelial cells of explanted coronary artery. Here, as we expected, Ang1 increased cell survival in a Tie2- and PI 3'kinase-dependent manner. We recently demonstrated that Ang1-induced endothelial cell survival is mediated through activation of Tie2, PI 3'-kinase, and Akt [21]. Thus, our present results suggest that Ang1-induced endothelial cell survival during OxLDL toxicity occurs through the same pathway. Akt has been shown to promote cell survival through its ability to phosphorylate Bad and procaspase-9 [37,38]. Furthermore, Papapetropoulos et al. [39] recently demonstrated that Ang1 can induce cell survival by an induction of the apoptosis inhibitor, survivin, through PI 3'-kinase/Akt activation. Therefore, in future studies, we will examine the exact downstream pathways of Ang1-induced PI 3'-kinase/Akt activation for endothelial cell survival during OxLDL-induced apoptosis.

In summary, our results indicate that in the adult coronary artery, Ang1 and Tie2 interact to boost endothelial cell survival in response to apoptotic injuries, including OxLDL. This defensive process may help to maintain the normal integrity of the coronary artery endothelium.

	5 Footnotes

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 
The full nucleotide sequences for the porcine angiopoietin-1 and porcine angiopoietin-2 genes, and the partial nucleotide sequence for the porcine Tie2 gene were deposited in the GenBank database under accession numbers AF233227 [GenBank] , AF233228 [GenBank] , and AF251494.

Time for primary review 22 days.

	Acknowledgments

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 
This work was supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology. We thank Peter C. Maisonpierre and George D. Yancopoulos for providing critical angiopoietin- and Tie-related reagents. We thank Jennifer Macke for help in preparing the manuscript.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Footnotes Acknowledgments References

 

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