This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 94/6/785    most recent 01.RES.0000121103.03275.ECv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ohashi, H. Articles by Honda, Y. Search for Related Content PubMed PubMed Citation Articles by Ohashi, H. Articles by Honda, Y. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Genetics Home Reference Hazardous Substances DB OXYGEN Related Collections ACE/Angiotension receptors Angiogenesis Apoptosis

(Circulation Research. 2004;94:785.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Phosphatidylinositol 3-Kinase/Akt Regulates Angiotensin II–Induced Inhibition of Apoptosis in Microvascular Endothelial Cells by Governing Survivin Expression and Suppression of Caspase-3 Activity

Hirokazu Ohashi, Hitoshi Takagi, Hideyasu Oh, Kiyoshi Suzuma, Izumi Suzuma, Noriko Miyamoto, Akiyoshi Uemura, Daisuke Watanabe, Tomoaki Murakami, Takeshi Sugaya, Akiyoshi Fukamizu, Yoshihito Honda

From the Department of Ophthalmology and Visual Sciences Graduate School of Medicine (H. Ohashi, H.T., H. Oh, K.S., I.S., N.M., A.U., D.W., T.M., Y.H.), Kyoto University, Kyoto, Japan; Tanabe Seiyaku Co, Ltd (T.S.), Osaka, Japan; and the Center for Tsukuba Advanced Research Alliance (A.F.), the University of Tsukuba, Ibaraki, Japan.

Correspondence to Hitoshi Takagi, Department of Ophthalmology and Visual Sciences Graduate School of Medicine, Kyoto University, 54 Shogoinkawaharacho Sakyo-ku, Kyoto, 606-8507, Japan. E-mail hitoshi{at}kuhp.kyoto-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin II (Ang II) plays essential roles in vascular homeostasis, neointimal formation, and postinfarct remodeling. Although Ang II has been shown to regulate apoptosis in cardiomyocytes and vascular smooth muscle cells, its role in vascular endothelial cells (ECs) remains elusive. To address this issue, we first performed TUNEL and caspase-3 activity assays with porcine microvascular ECs challenged by serum deprivation. Ang II significantly reduced the ratio of apoptotic cells and caspase-3 activity. The Ang II type 1 receptor (AT₁) was responsible for these effects. Among the signaling molecules downstream of AT₁, we revealed that PI3-kinase/Akt pathway plays a predominant role in the antiapoptotic effect of Ang II. Interestingly, the expression of survivin, a central molecule of cell survival, increased after Ang II stimulation. Overexpression of a dominant-negative form of Akt abolished both Ang II–induced antiapoptosis and survivin protein expression. In a murine model of hyperoxygen-induced retinal vascular regression, AT_1a knockout mice showed a significant increase in retinal avascular areas. Our data indicate that Ang II plays a critical antiapoptotic role in vascular ECs by a mechanism involving PI3-kinase/Akt activation, subsequent upregulation of survivin, and suppression of caspase-3 activity.

Key Words: angiotensin II • apoptosis • endothelium • survivin • caspase-3

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin II (Ang II), the most important effector peptide of the renin-angiotensin system, plays central roles in volume and salt homeostasis. Ang II is implicated in cardiovascular and renal pathology including cardiac left ventricular hypertrophy, neointimal formation, postinfarct vascular remodeling, and nephrosclerosis.^1,2 Apoptosis and cellular proliferation are important mechanisms causing these pathological conditions. In this respect, Ang II has been recognized as a growth-promoting and apoptosis-regulating factor contributing to vascular structural alteration.³

Ang II initiates its effects by interaction with at least two pharmacologically distinct subtypes of cell-surface receptors, AT₁ and AT₂. In mice, the AT₁ receptor is further subdivided into AT_1a and AT_1b. Major functions of Ang II in the cardiovascular system are mediated through AT₁, whereas AT₂ exerts antigrowth and antihypertrophic effects.⁴

Ang II activates multiple signaling pathways, including protein kinase C (PKC),⁵ and mitogen-activated protein kinase (MAPK).⁵ MAPKs are key regulatory proteins that control the cellular response to growth, apoptosis, and stress signals. More recently, stimulation of AT₁ has been shown to trigger the activation of phosphatidylinositol 3 (PI3)-kinase and Akt,⁶ which is a common feature in the signal transduction of the antiapoptotic effects of growth factors. Although both MAPK and PI3-kinase/Akt contribute to apoptosis, the precise role of Ang II in apoptosis has been a subject of continuing controversy. Ang II has been reported to induce apoptosis in fibroblasts,⁷ cardiomyocytes,⁸ and vascular smooth muscle cells (VSMCs),⁹ whereas it has been shown to prevent apoptosis of SMCs,¹⁰ neuronal cells,¹¹ muscle cells of aortic media,¹² and the subepicardium area of the heart.¹³ In addition, the role of Ang II receptor subtypes in the regulation of apoptosis has remained elusive.

In endothelial cells (ECs), Ang II has been reported to potentiate vascular endothelial growth factor (VEGF)–mediated angiogenic activity.¹⁴ Although apoptosis of ECs is important for the regulation of physiological and pathological angiogenesis,¹⁵ little is known about whether Ang II plays either an anti- or a proapoptotic role, especially in microvascular ECs such as retinal vascular ECs. For this article, we studied the potential roles of Ang II in apoptosis, and found that this molecule plays an antiapoptotic role in cultured microvascular ECs challenged by serum deprivation. We further dissected the underlying molecular mechanisms by examining the effects of Ang II on diverse signaling pathways related to apoptosis. The signaling molecules that were proven to be involved included AT₁, epidermal growth factor receptor (EGFR), PI3-kinase, and Akt. We further showed for the first time that both survivin and caspase-3, but not MAPK, participate in this antiapoptotic effect of Ang II downstream of Akt. Finally, we confirmed the antiapoptotic effects of Ang II in vivo with the use of a murine model of hyperoxygen-induced retinal vascular regression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction and Quantitative Determination of Apoptosis
To induce apoptosis, a serum deprivation method was used as described previously.¹⁶ The percentage of apoptotic cells is based on the sum of floating cells plus apoptotic adherent cells in a given cell population.

Murine Model of Hyperoxygen-Induced Retinal Vascular Regression
AT_1a-deficient homozygous (AT_1aKO; AT_1a^–/– C57BL/6J) mice were provided by the Discovery Research Laboratory, Tanabe Seiyaku Co (Osaka, Japan). One-week-old (postnatal day 7; P7) wild-type (WT) and AT_1aKO mice were placed in an airtight incubator and exposed to an atmosphere of 75±3% oxygen for 5 days. All procedures involving animals were conducted in accordance with both the guidelines for animal experiments of Kyoto University and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Blocks Apoptosis in Retinal ECs Through the AT₁ Receptor
To study the effect of Ang II on apoptosis of ECs, we first used serum-starved cells. Approximately 30% of porcine retinal endothelial cells (PRECs) underwent typical apoptosis after 24 hours of serum deprivation, which is consistent with a previous study using human umbilical venous endothelial cells¹⁶ (Figure 1B). Additionally, the cells shrank in size and the nuclei became condensed and fragmented (Figure 1A). Inhibition of apoptosis by Ang II was dose-dependent (Figures 1A and 1B), which was also confirmed by reduced DNA fragmentation in a DNA ladder assay (Figure 1C). To determine the receptor responsible for mediating the antiapoptotic effects of Ang II, we next used receptor-type specific inhibitors. Candesartan, an AT₁ antagonist, completely suppressed the antiapoptotic effect of Ang II, whereas PD123319, an AT₂ antagonist, had no effect (Figure 1D). These results indicate that Ang II exerts its antiapoptotic effect in ECs through the AT₁ receptor.

View larger version (28K): [in this window] [in a new window]   Figure 1. Ang II blocks serum deprivation–induced apoptosis in retinal ECs through the AT1 receptor. Cells were treated with either Ang II (1 to 100 nmol/L) or vehicle after changing to serum-free medium and the ratio of apoptotic cells was determined after 24 hours. A, Fluorescence images of TUNEL assays from cells treated with 100 nmol/L Ang II (middle) or without Ang II (left). Apoptotic cells are shown in green. High-magnification view (right). B, Dose-response analysis of the antiapoptotic effects of Ang II (n=6). C, DNA laddering assay. D, Role of AT1 and AT2 in Ang II–induced antiapoptosis. Cells were pretreated with 1 µmol/L of either candesartan or PD123319 and incubated for 24 hours in serum-free medium with or without Ang II (100 nmol/L). *P<0.0001, **P<0.05, ***P<0.01 vs serum-deprived control.

Ang II–Induced EC Survival Is Mediated by the PI3-Kinase/Akt Pathway
To further study the mechanisms underlying the antiapoptotic effect of Ang II in PRECs, we investigated the signaling molecules in pathways downstream of AT₁. MAPKs, PI3-kinase/Akt, and reactive oxygen species (ROS) are key regulatory pathways that control the cellular response to apoptosis.^5,6,15 LY294002, a PI3-kinase inhibitor, completely reversed this effect, whereas PD98059, a MAPK inhibitor, diphenylene iodonium (DPI), a NAD(P)H oxidase inhibitor, and N-acetyl-L-cysteine (NAC), an ROS inhibitor, did not exhibit a significant effect (Figure 2A). These results indicated that PI3-kinase plays a predominant role in the antiapoptotic effect of Ang II and prompted us to further examine the potential link between Ang II and Akt, a major downstream effector of PI3-kinase.

View larger version (29K): [in this window] [in a new window]   Figure 2. Ang II–induced EC survival is mediated by a PI3-kinase/Akt pathway, but not by MAPK- or ROS-mediated pathways. A, Role of PI3-kinase–, MAPK-, or ROS-mediated pathways in the Ang II–induced antiapoptotic effect in PRECs. Cells were pretreated for 30 minutes with either control buffer, LY294002 (100 µmol/L), PD98059 (10 µmol/L), DPI (10 µmol/L), or NAC (20mmol/L) before stimulation with Ang II (100 nmol/L) and incubated in serum-free medium for 24 hours (n=6). B and C, Cells were incubated for 24 hours in serum-free medium before addition of Ang II and Western blot analyses were performed. Phosphorylated-Akt (top), total Akt (middle). B, Time course studies of Akt phosphorylation by Ang II (100 nmol/L) stimulation (n=5). C, Dose-response analysis of Akt phosphorylation by Ang II stimulation (1 to 100 nmol/L) for 5 minutes (n=5). D, Role of Akt activation in Ang II–induced antiapoptotic effect in PRECs. Cells infected with recombinant adenoviruses were incubated in serum-free medium with or without Ang II (100 nmol/L) for 24 hours (n=6). #P<0.01, ##P<0.001 vs Ang II; *P<0.05, **P<0.01, ***P<0.001 vs control.

We found by Western blot analyses that Ang II stimulated phosphorylation of Akt in both a time- and dose-dependent manner. In our time course experiments, 100 nmol/L of Ang II caused maximal phosphorylation of Akt (3.75±0.69-fold; P<0.001) at 5 minutes (Figure 2B). Ang II also stimulated phosphorylation of Akt in a dose-dependent manner with a maximal fold increase of 3.89±0.69 (P<0.001) at a dosage of 100 nmol/L (Figure 2C). To study the functional relevance of Akt activation in Ang II–induced antiapoptosis, we infected PRECs with adenoviruses encoding either constitutively active (CA) or dominant-negative (DN) form of Akt. The ratio of apoptosis induced by serum deprivation was similar in PRECs infected with or without control vector (LacZ) (Figure 2D). Ang II significantly inhibited apoptosis of PRECs infected with control vector (20.4±2.8%; P<0.0001), whereas infection of PRECs with DN-Akt abrogated the antiapoptotic effect of Ang II (45.3±7.4%) (Figure 2D). Additionally, PRECs infected with CA-Akt showed a significant antiapoptotic effect compared with the control (10.6±2.0% versus 35.4±8.08%; P<0.0001). Collectively, these results suggest that Ang II exerts its antiapoptotic effect in PRECs through PI3-kinase and subsequent Akt activation, but not through a MAPK or ROS-mediated pathway.

Ang II Stimulates Phosphorylation of Akt Through an AT₁/PI3-Kinase but Not Through an AT₂/PKC Pathway in PRECs
We next investigated the molecular mechanisms leading to Akt activation in Ang II signaling. We revealed that the AT₁ receptor inhibitor candesartan completely blocked Ang II–induced phosphorylation of Akt, whereas the AT₂ inhibitor PD123319 had no effect (Figure 3A). This indicates that AT₁ is responsible for Ang II–induced Akt activation. Because both PI3-kinase and PKC were reported to mediate Akt activation,⁶ we next tested the effects of wortmannin and LY294002, two distinct PI3-kinase inhibitors, and GF109203X, a PKC inhibitor. Both PI3-kinase inhibitors completely abolished Akt phosphorylation in response to Ang II, whereas GF109203X failed to show a significant effect (Figure 3B).

View larger version (23K): [in this window] [in a new window]   Figure 3. Ang II stimulates phosphorylation of Akt through AT1- and PI3-kinase–, but not through AT2- or PKC-, mediated pathways. A, Effect of AT1 and AT2 antagonists on Akt phosphorylation via Ang II stimulation (n=5). B, Role of PI3-kinase and PKC in Ang II–induced phosphorylation of Akt. Cells were pretreated for 30 minutes with control buffer, candesartan (Cand, 1 µmol/L), PD123319 (1 µmol/L), wortmannin (wort, 100 nmol/L), LY294002 (LY, 100 µmol/L), or GF109203X (GFX, 1 µmol/L) before stimulation with Ang II (100 nmol/L) for 5 minutes (n=6). C, Role of Ang II in PI3-kinase activation. Cells were pretreated for 30 minutes with control buffer, candesartan (1 µmol/L), or PD123319 (1 µmol/L) before stimulation with Ang II (100 nmol/L) for 2 minutes and immunoprecipitation assays were performed. *P<0.05, **P<0.01 vs Ang II.

Immunoprecipitation experiments with phosphotyrosine antibodies further confirmed PI3-kinase–mediated Akt activation in Ang II signaling by showing that Ang II specifically phosphorylates the p85 subunit of PI3-kinase and that candesartan but not PD123319 blocked this phosphorylation (Figure 3C).

Ang II Stimulates Phosphorylation of Akt Through EGFR Transactivation and Not Through an ROS-Mediated Pathway
Because recent studies have shown that two additional signaling events, via EGFR transactivation and via ROS generation, could mediate Ang II/Akt activation,^5,17 we performed immunoprecipitation assays to study their potential roles in Ang II/Akt phosphorylation. Ang II stimulated phosphorylation of EGFR, and this effect was blocked by candesartan but not by PD123319 (Figure 4A). Furthermore, AG1478, an EGFR blocker, completely reversed Akt phosphorylation stimulated by Ang II (P<0.05 versus Ang II), and thus confirmed that EGFR transactivation is required for Akt activation in Ang II signaling (Figure 4B). We could not observe any significant effect on Akt activation in the presence of either DPI or NAC (Figure 4C). These results demonstrate that Ang II stimulates phosphorylation of Akt through the AT₁/EGFR pathway, but not through an ROS-mediated pathway in PRECs.

View larger version (36K): [in this window] [in a new window]   Figure 4. Ang II induces Akt phosphorylation via EGFR transactivation and not by an ROS-mediated pathway. A, Role of Ang II in EGFR transactivation. Cells were pretreated for 30 minutes with control buffer, candesartan (1 µmol/L), or PD123319 (1 µmol/L) before stimulation with Ang II (100 nmol/L) for 2 minutes and immunoprecipitation assays were performed to evaluate the phosphorylation status of EGFR. B, Role of EGFR transactivation in Ang II–induced phosphorylation of Akt. Cells were pretreated for 30 minutes with control buffer or AG1478 (250 nmol/L) before stimulation with Ang II (100 nmol/L) for 5 minutes (n=4). C, Role of ROS-mediated pathways in Ang II–induced phosphorylation of Akt. Cells were pretreated for 30 minutes with control buffer, DPI (10 µmol/L), or NAC (20 mmol/L) before stimulation with Ang II (100 nmol/L) for 5 minutes (n=6). *P<0.05 vs Ang II.

Ang II Upregulates Survivin Expression via PI3-Kinase/Akt Pathway
The signaling molecules involved downstream of the PI3-kinase/Akt pathway in the context of Ang II–triggered responses, have not yet been determined. Survivin is a molecule under the control of Akt¹⁸ and mediates survival in cells treated by cytokines such as VEGF¹⁹ and angiopoietin-1.²⁰ Because survivin had not been tested in Ang II signaling, we examined if there was a link between Ang II and survivin expression. By Western blot analyses, we observed that Ang II stimulated upregulation of survivin expression in both a dose- (Figure 5A) and time-dependent manner (Figure 5B). The maximum upregulation of survivin expression was induced at 24 hours after Ang II stimulation (7.00±0.80-fold; P<0.01) (Figure 5B).

View larger version (40K): [in this window] [in a new window]   Figure 5. Ang II stimulates upregulation of survivin mRNA and protein expression via PI3-kinase/Akt pathway in serum-deprived PRECs. A through C, Western blot analyses. A, Dose-response study: cells were stimulated with the indicated doses of Ang II for 24 hours in serum-free medium (n=5). B, Time course study: cells were stimulated with 1 µmol/L Ang II for the indicated time periods. C, Role of Akt activation in the increased survivin protein expression after Ang II stimulation. Cells infected with recombinant adenoviruses were incubated in serum-free medium with or without Ang II (1 µmol/L) for 24 hours (n=5). D, Northern blot analyses: cells were stimulated with 1 µmol/L Ang II for the indicated time periods (n=4). E, Role of PI3-kinase in the increased survivin mRNA expression after Ang II stimulation. Cells were pretreated for 60 minutes with control buffer or LY294002 (100 µmol/L) and stimulated with Ang II (1 µmol/L) for 6 hours (n=4). F, Half-life of survivin protein. Graph shows the decay of survivin protein in the presence or absence of 1 µmol/L Ang II. Square, Control cells; Circle, Cells with Ang II stimulation. Values in the graph indicate the percentage of remaining survivin protein compared with the initial amount in the specific conditions and are plotted in logarithmic scale. Representative data of 2 independent experiments are shown. **P<0.05, *P<0.01 vs serum-deprived control.

Next, we evaluated the role of Akt activation on survivin expression. The control vector had no effect on the upregulation of survivin expression after Ang II stimulation, whereas CA-Akt without Ang II stimulation was able to stimulate survivin expression significantly (6.88±1.68-fold; P<0.01). Infection of PRECs with DN-Akt abrogated Ang II–stimulated survivin upregulation (Figure 5C).

To study survivin expression at the mRNA level, we performed Northern blot analyses and found that survivin mRNA expression increased after Ang II stimulation in a time-dependent manner (Figure 5D). The maximal response (2.86±0.45 folds of control; P<0.01) was observed at 6 hours after stimulation with Ang II. We next investigated the effect of PI3-kinase on survivin mRNA. We found that the upregulation of survivin mRNA after Ang II stimulation was almost completely abolished by LY294002 (Figure 5E). These data revealed the upregulation of survivin mRNA on Ang II stimulation via AT₁/PI3-kinase pathway, consistent with our data of protein expression.

To further clarify whether the survivin protein upregulation after Ang II stimulation is due to de novo protein synthesis or prevention of degradation, we next studied the half-life of survivin using cycloheximide to inhibit protein synthesis. We found that the half-life of survivin protein in serum-free condition was approximately 40 minutes regardless of the presence of Ang II in PRECs (Figure 5F). Thus, we revealed that Ang II stimulates upregulation of survivin expression at both mRNA and protein levels and that the increase in protein expression is derived from de novo synthesis but not from prevention of degradation.

Ang II Stimulates the Inhibition of Serum Deprivation–Induced Caspase-3 Activation in PRECs
Because survivin has been reported to interact directly with caspase family members, especially caspase-3, in 293 cells,²¹ we further studied the effect of Ang II on caspase-3 activation in low serum conditions. As shown in Figure 6A, Ang II stimulated the inhibition of serum-deprived caspase-3 activation in PRECs in a dose-dependent manner. Ang II (100 nmol/L) stimulated the reduction of caspase-3 activity to approximately 60% of control levels (P<0.05). Candesartan and LY294002, but not PD123319 or PD98059, completely abolished the suppressive effect of Ang II on caspase-3 activation (Figure 6B). These data suggest that Ang II stimulates the reduction of caspase-3 activity and exerts its antiapoptotic effect through AT₁ and a PI3-kinase dependent mechanism.

View larger version (15K): [in this window] [in a new window]   Figure 6. Ang II stimulates the inhibition of serum deprivation–induced caspase-3 activation in PRECs. A, Dose-response study. Cells were stimulated with the indicated doses of Ang II for 24 hours (n=5). B, Role of downstream signaling molecules of Ang II in caspase-3 activity. Cells were pretreated for 30 minutes with control buffer, candesartan (Cand, 1 µmol/L), PD123319 (1 µmol/L), LY294002 (LY, 100 µmol/L), or PD98059 (10 µmol/L) before stimulation with Ang II (100 nmol/L) and incubated in serum-free medium for 24 hours (n=5). *P<0.05 vs serum deprivation; **P<0.05 vs Ang II.

Ang II Prevents Hyperoxygen-Induced Retinal Endothelial Apoptosis In Vivo
To study the role of Ang II in apoptosis in vivo, we utilized a murine model of hyperoxygen-induced retinal vascular regression. We found that hyperoxia causes retinal capillary regression via apoptosis of ECs (Figures 7A through 7C). We also analyzed the number of apoptotic retinal ECs in mice exposed to hyperoxia for 24 hours (P8). The retinas from AT_1aKO mice had significantly more apoptotic retinal ECs than that from WT mice (WT versus AT_1aKO mice; 6.78±2.07 versus 9.44±2.78 cells; P<0.01) (Figure 7D). These data suggested that Ang II acts as an antiapoptotic factor through AT₁ receptor also in retinal ECs in vivo.

View larger version (40K): [in this window] [in a new window]   Figure 7. Ang II acts as a survival factor in hyperoxygen-induced apoptosis of retinal ECs in vivo. A through C, Double-immunofluorescent staining to detect apoptotic ECs in the retina. A, ECs (red). B, Apoptotic cells (green). C, Merged image (A and B). Arrowheads indicate the apoptotic retinal ECs. D, Role of Ang II in hyperoxgen-induced apoptosis of retinal ECs. Number of apoptotic retinal ECs in paraffin sections from mice (P8) was counted (n=5). E through H, Real-time PCR analyses of retinal mRNA expression of VEGF (E), KDR (F), Flt-1 (G), and survivin (H) in the WT and AT1aKO mice subjected to hyperoxygen (n=5). White columns indicate WT mice; black columns, AT1aKO mice. I and J, Immunohistochemical analyses of survivin expression in the hyperoxic retina (P8). Retinal ECs (red) and survivin (green). P8 retinas from WT (I) and AT1aKO mice (J). Arrowheads indicate the vessels colabeled with the survivin antibody and arrows indicate the survivin-negative vessels. K, Comparison of the percentage of survivin-negative vessels in the hyperoxic retinas (P8) from WT and AT1aKO mice (n=5). P<0.01 vs WT mice, P<0.01 between both genotypes, P<0.001 vs WT mice. *P<0.01, ***P<0.05 vs P7 WT mice, **P<0.01, #P<0.05 vs P7 KO mice.

Because VEGF and its receptors, KDR and Flt-1, are the key regulators of EC survival, we studied their expression using real-time PCR analyses. We found that hyperoxygen significantly reduced the mRNA expression of these molecules in both genotypes compared with the level of P7 of corresponding genotypes (Figures 7E through 7G). As for the comparison of mRNA expression level between genotypes, there was no significant difference at all time points examined (Figures 7E through 7G). These results suggested that VEGF and its receptors have minor contribution to the differences of retinal EC survival between both genotypes.

Ang II Plays as a Retinal Endothelial Survival Factor via AT₁/Survivin Pathway in a Murine Model of Hyperoxygen-Induced Retinal Vascular Regression
Next, we focused on the changes of the retinal survivin expression. To study the changes in survivin mRNA expression in the hyperoxic retina, we first performed real-time PCR analyses with the retina from both genotypes of P7, P8, and P12. We found that survivin mRNA expression was significantly decreased in both genotypes of mice during hyperoxia compared with the level of corresponding genotypes of P7 (WT mice; 0.59±0.17- and 0.51±0.10-fold, AT_1aKO mice; 0.40±0.19- and 0.25±0.13-fold, respectively, at P8 and P12). Although the difference in expression level between genotypes was not significant at P7, we observed significantly reduced survivin mRNA expression in AT_1aKO mice both at both P8 and P12 (Figure 7H). To study in vivo survivin protein expression, we next performed immunohistochemical analyses in the hyperoxic retinas. By double-immunofluorescent staining, we observed that a subset of retinal vessels (Figures 7I and 7J, arrowheads) was colabeled with the survivin antibody. By comparing the percentage of vessels negative for survivin expression, we found that AT_1aKO mice had significantly higher percentage of survivin-negative vessels than that of WT mice (WT versus AT_1aKO mice; 34.8±3.6% versus 69.5±6.6%; P<0.001) (Figure 7K). These data suggested that Ang II signaling plays an important role in the in vivo expression of survivin.

Finally, we elucidate the vascular survival in a murine model of hyperoxygen-induced retinal vascular regression. In this study, WT and AT_1aKO mice showed similar extension and network formation of the retinal vessels (Figures 8A through 8D). There was no significant difference between the ratio of the retinal vascular bed to the total retinal areas of WT and AT_1aKO mice at P7 (Figure 8E). At P12, after hyperoxia, AT_1aKO mice showed significantly more capillary dropout and less vascular networks than WT mice (Figures 8F through 8I). AT_1aKO mice retained only 32.2±2.7% of the retinal vascular area, whereas WT mice retained 50.5±5.5% of this area (P<0.0001; Figure 8J). These results confirmed that Ang II plays an important role in the retinal vascular EC survival via its AT₁ receptor.

View larger version (53K): [in this window] [in a new window]   Figure 8. Ang II prevents endothelial apoptosis via AT1 in a murine model of retinal vascular regression. A and C, Retinal vasculature at P7. B and D, High-magnification images around the optic nerve of A and C, respectively. E, Ratio of vascularized areas to total retina at P7 (n=8). F and H, Retinal vasculature at P12, after hyperoxic exposure. G and I, High-magnification images of the vascular regression of F and H, respectively. J, Ratio of the retinal vascular bed to the total retinal areas at p12, after hyperoxic exposure (n=8). *P<0.0001 vs WT mice P12.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II is reported to be an inducer of apoptosis in several cell types, including pheochromocytoma cells,²² fibroblasts,²⁰ cardiomyocytes,⁸ VSMCs,²³ and renal proximal tubular cells.⁹ In contrast, other investigators have shown that Ang II prevented apoptosis in SMCs,¹⁰ in muscle cells of aortic media,¹² neuronal cells,¹¹ and in the subepicardium area of the heart.¹³ Thus, the role of Ang II in apoptosis has been a subject of some controversy. With regard to vascular ECs, however, little is known at all about the effect of Ang II in apoptosis.

Angiogenesis is controlled by endothelial apoptosis,¹⁵ and the disruption of endothelial cell-matrix contacts or interference with extracellular survival signals initiates caspase-dependent apoptosis in ECs.²⁴ A recent study showed that Ang II plays an angiogenic role via AT₁ in pathological angiogenesis, including hind-limb ischemia.²⁴ Moreover, we recently found that Ang II significantly contributes to VEGF-induced angiogenesis via upregulation of VEGF receptor 2.¹⁴ Although these findings suggest that Ang II might play antiapoptotic roles in ECs, the molecular mechanisms remain poorly understood. In this study, we intended to delineate the effects of Ang II on apoptosis in microvascular endothelial cells and the underlying signaling pathways.

We first revealed by TUNEL and DNA Ladder assays that Ang II protects PRECs against apoptosis induced by serum deprivation in a dose-dependent manner. This finding is the first demonstration of an antiapoptotic role for Ang II in ECs. The role of Ang II receptor subtypes in the regulation of apoptosis in different tissues also remains controversial. Ang II regulates apoptosis in cardiomyocytes⁸ and aortic SMCs¹⁰ via AT₁. Additionally, Ang II has also been reported to regulate apoptosis in VSMCs,²⁵ ovarian granulosa cells,⁶ and fibroblasts²² via AT₂. A previous study showed that AT₂ but not AT₁ is responsible for inducing apoptosis in macrovascular ECs,²⁶ but the precise role of AT₁ in apoptosis of ECs remained elusive. In this study, we clearly show that Ang II plays an antiapoptotic role in microvascular ECs and that AT₁ but not AT₂ is the receptor that mediates this mechanism. The apparent discrepancy between the study of Dimmeler et al²⁶ and our data may be due to cell type specificity, which has been shown to be a critical factor in determining how Ang II exerts its effect on apoptosis in fibroblasts⁷ and pheochromocytoma cells.²²

We next investigated apoptosis-related signaling pathways, including MAPK, ROS, and PI3-kinase, that were previously reported to be downstream of AT₁.^5,27 TUNEL assays revealed that PI3-kinase and its downstream effector, Akt, are essential components of the antiapoptotic mechanism of Ang II in PRECs but that neither MAPK nor ROS-mediated pathways are significantly involved. These results indicate that the PI3-kinase/Akt pathway has an essential role in antiapoptosis signaling by Ang II. Because the PI3-kinase/Akt pathway in ECs has been also shown to play a critical role in antiapoptosis triggered by other growth factors such as angiopoietin-1¹⁶ and VEGF,²⁸ our results may further support a general concept that this pathway is indispensable for protection against apoptosis.

We next studied the pathways leading to Akt phosphorylation in Ang II/AT₁ signaling. In VSMCs, previous reports have shown that PI3-kinase⁶ and ROS²⁹ are required for Ang II–induced Akt phosphorylation. In ECs, our study demonstrated that Akt phosphorylation is mediated through an AT₁/PI3-kinase pathway, but not through ROS-mediated pathways. These data suggest the presence of a cell type–independent mechanism for Akt activation in Ang II signaling.

Recent report has shown that AT₁-mediated activation of Akt¹⁷ is induced via downstream signals of transactivated EGFR in VSMCs. In ECs, our immunoprecipitation results demonstrate that Ang II induces phosphorylation of EGFR via AT₁ but not AT₂, and that Ang II–induced Akt phosphorylation was completely abolished by the EGFR blocker, AG1478. Thus, EGFR transactivation seems to play a critical role also in Ang II–induced Akt activation.

To investigate the downstream pathways after Ang II–induced Akt phosphorylation, we focused on the expression of survivin, which belongs to the family of genes known as inhibitors of apoptosis and has been implicated in both prevention of cell death and control of mitosis.³⁰ Although survivin plays important roles in suppression of cell death in response to diverse stimuli such as VEGF¹⁹ and angiopoietin-1,²⁰ its role in Ang II–induced signaling remains completely unknown in any cell types. We examined a potential link between Ang II and survivin, and revealed that both survivin mRNA and protein expression increased after Ang II stimulation. This is, to our knowledge, the first demonstration that Ang II can stimulate survivin mRNA and protein expression. As for the downstream signaling of Ang II, our results indicate that the PI3-kinase/Akt pathway plays a predominant role in this response in PRECs. On the other hand, the cell cycle also can regulate survivin expression.²⁰ Because Akt activation had reported to have significant contribution to the cell cycle progression³¹ and Ang II might play a role in the cell cycle regulation,³² the cell cycle can possibly contribute to the observed upregulation of survivin by Ang II. Further studies are required to study whether Ang II/AT₁/PI3-kinase/Akt pathway regulates survivin expression in a direct or the cell cycle–dependent manner in ECs.

Recent reports have shown that survivin interacts with and inhibits caspase-3.²¹ Additionally, it has also been shown that survivin has an indirect suppressive effect on caspase-3–related apoptosis.³³ Although caspase-3 is involved in events associated with apoptosis induced by diverse stimuli, little is known about its role in Ang II–induced signaling. Having demonstrated in this study that Ang II can induce survivin expression, we wanted to determine any potential effect of Ang II on caspase-3 activity. Our results using a caspase-3 activity assay revealed for the first time that Ang II can stimulate the inhibition of caspase-3 activation, which had been stimulated by serum deprivation. These findings together with our results showing survivin upregulation after Ang II stimulation highlight a novel mechanism involved in the signaling pathways for Ang II–induced antiapoptotic effects in ECs.

Because this in vitro data strongly indicated an antiapoptotic function of Ang II, we further investigated the potential role of Ang II in apoptosis in vivo by using a murine model of hyperoxygen-induced retinal vascular regression. The vascular regression in this model is reported to involve apoptosis of vascular ECs, but the molecular mechanisms underlying this phenomenon remain to be elucidated. Our results revealed that AT_1aKO mice had significantly more apoptosis of retinal ECs, capillary dropout and suppression of survivin expression compared with WT mice, which suggests that Ang II also plays a critical role in antiapoptosis in vivo and further validates our in vitro results.

In summary, our results first demonstrate that Ang II is a prominent antiapoptotic molecule in retinal vascular ECs and the underlying molecular mechanism for this effect involves AT₁, EGFR transactivation, PI3-kinase activation, and Akt phosphorylation. We further uncovered two novel signaling pathways responsible for Ang II–induced antiapoptosis, including upregulation of the broad spectrum apoptosis inhibitor survivin and inhibition of caspase-3 activation. Finally, we confirmed the antiapoptotic effects of Ang II in a murine model of hyperoxygen-induced retinal vascular regression. Recent studies have shown that inhibition of Ang II is effective for the treatment of diabetic retinopathy in both rodent³⁴ and human subjects.³⁵ Our results are indicative of molecular mechanisms that are consistent with these findings such that Ang II promotes the survival of microvascular cells and that suppression of Ang II signaling actually reduces retinal vascular viability.

	Acknowledgments

 
This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of the Japanese Government (70283596). The authors are grateful to Dr Kohjiro Ueki (Joslin Diabetes Center, Boston, Mass) for his generous gift of adenoviruses.

	Footnotes

 
Original received August 21, 2003; resubmission received December 2, 2003; revised resubmission received January 23, 2004; accepted January 29, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Falkenhahn M, Franke F, Bohle RM, Zhu YC, Stauss HM, Bachmann S, Danilov S, Unger T. Cellular distribution of angiotensin-converting enzyme after myocardial infarction. Hypertension. 1995; 25: 219–226.[Abstract/Free Full Text]
2. Rakugi H, Kim DK, Krieger JE, Wang DS, Dzau VJ, Pratt RE. Induction of angiotensin converting enzyme in the neointima after vascular injury: possible role in restenosis. J Clin Invest. 1994; 93: 339–346.[Medline] [Order article via Infotrieve]
3. Dzau VJ. Cell biology and genetics of angiotensin in cardiovascular disease. J Hypertens Suppl. 1994; 12: S3–S10.[Medline] [Order article via Infotrieve]
4. Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995; 95: 651–657.[Medline] [Order article via Infotrieve]
5. Griendling KK, Ushio-Fukai M. Reactive oxygen species as mediators of angiotensin II signaling. Regul Pept. 2000; 91: 21–27.[CrossRef][Medline] [Order article via Infotrieve]
6. Takahashi T, Taniguchi T, Konishi H, Kikkawa U, Ishikawa Y, Yokoyama M. Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells. Am J Physiol. 1999; 276: H1927–H1934.[Medline] [Order article via Infotrieve]
7. Li W, Ye Y, Fu B, Wang J, Yu L, Ichiki T, Inagami T, Ichikawa I, Chen X. Genetic deletion of AT2 receptor antagonizes angiotensin II–induced apoptosis in fibroblasts of the mouse embryo. Biochem Biophys Res Commun. 1998; 250: 72–76.[CrossRef][Medline] [Order article via Infotrieve]
8. Cigola E, Kajstura J, Li B, Meggs LG, Anversa P. Angiotensin II activates programmed myocyte cell death in vitro. Exp Cell Res. 1997; 231: 363–371.[CrossRef][Medline] [Order article via Infotrieve]
9. Bhaskaran M, Reddy K, Radhakrishnan N, Franki N, Ding G, Singhal PC. Angiotensin II induces apoptosis in renal proximal tubular cells. Am J Physiol Renal Physiol. 2003; 284: F955–F965.[Abstract/Free Full Text]
10. Pollman MJ, Yamada T, Horiuchi M, Gibbons GH. Vasoactive substances regulate vascular smooth muscle cell apoptosis: countervailing influences of nitric oxide and angiotensin II. Circ Res. 1996; 79: 748–756.[Abstract/Free Full Text]
11. Kakinuma Y, Hama H, Sugiyama F, Goto K, Murakami K, Fukamizu A. Anti-apoptotic action of angiotensin fragments to neuronal cells from angiotensinogen knock-out mice. Neurosci Lett. 1997; 232: 167–170.[CrossRef][Medline] [Order article via Infotrieve]
12. Nakamura M, Tanaka M, Abe S, Fujiwara H. Sudden pressure elevation can trigger acute muscle cell death of the heart and aorta. Atherosclerosis. 1999; 146: 25–32.[CrossRef][Medline] [Order article via Infotrieve]
13. Tea BS, Dam TV, Moreau P, Hamet P, deBlois D. Apoptosis during regression of cardiac hypertrophy in spontaneously hypertensive rats: temporal regulation and spatial heterogeneity. Hypertension. 1999; 34: 229–235.[Abstract/Free Full Text]
14. Otani A, Takagi H, Suzuma K, Honda Y. Angiotensin II potentiates vascular endothelial growth factor-induced angiogenic activity in retinal microcapillary endothelial cells. Circ Res. 1998; 82: 619–628.[Abstract/Free Full Text]
15. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res. 2000; 87: 434–439.[Abstract/Free Full Text]
16. Kim I, Kim HG, So JN, Kim JH, Kwak HJ, Koh GY. Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Circ Res. 2000; 86: 24–29.[Abstract/Free Full Text]
17. Auge N, Garcia V, Maupas-Schwalm F, Levade T, Salvayre R, Negre-Salvayre A. Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways. Arterioscler Thromb Vasc Biol. 2002; 22: 1990–1995.[Abstract/Free Full Text]
18. Zhu L, Fukuda S, Cordis G, Das DK, Maulik N. Anti-apoptotic protein survivin plays a significant role in tubular morphogenesis of human coronary arteriolar endothelial cells by hypoxic preconditioning. FEBS Lett. 2001; 508: 369–374.[CrossRef][Medline] [Order article via Infotrieve]
19. Tran J, Rak J, Sheehan C, Saibil SD, LaCasse E, Korneluk RG, Kerbel RS. Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. Biochem Biophys Res Commun. 1999; 264: 781–788.[CrossRef][Medline] [Order article via Infotrieve]
20. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998; 396: 580–584.[CrossRef][Medline] [Order article via Infotrieve]
21. Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T, Reed JC. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 1998; 58: 5315–5320.[Abstract/Free Full Text]
22. Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci U S A. 1996; 93: 156–160.[Abstract/Free Full Text]
23. Bascands JL, Girolami JP, Troly M, Escargueil-Blanc I, Nazzal D, Salvayre R, Blaes N. Angiotensin II induces phenotype-dependent apoptosis in vascular smooth muscle cells. Hypertension. 2001; 38: 1294–1299.[Abstract/Free Full Text]
24. Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA. Integrin _vß₃ antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 1994; 79: 1157–1164.[CrossRef][Medline] [Order article via Infotrieve]
25. Suzuki J, Iwai M, Nakagami H, Wu L, Chen R, Sugaya T, Hamada M, Hiwada K, Horiuchi M. Role of angiotensin II–regulated apoptosis through distinct AT₁ and AT₂ receptors in neointimal formation. Circulation. 2002; 106: 847–853.[Abstract/Free Full Text]
26. Dimmeler S, Rippmann V, Weiland U, Haendeler J, Zeiher AM. Angiotensin II induces apoptosis of human endothelial cells: protective effect of nitric oxide. Circ Res. 1997; 81: 970–976.[Abstract/Free Full Text]
27. Eguchi S, Inagami T. Signal transduction of angiotensin II type 1 receptor through receptor tyrosine kinase. Regul Pept. 2000; 91: 13–20.[CrossRef][Medline] [Order article via Infotrieve]
28. Fujio Y, Walsh K. Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner. J Biol Chem. 1999; 274: 16349–16354.[Abstract/Free Full Text]
29. Gorin Y, Kim NH, Feliers D, Bhandari B, Choudhury GG, Abboud HE. Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase. FASEB J. 2001; 15: 1909–1920.[Abstract/Free Full Text]
30. Altieri DC. The molecular basis and potential role of survivin in cancer diagnosis and therapy. Trends Mol Med. 2001; 7: 542–547.[CrossRef][Medline] [Order article via Infotrieve]
31. Stabile E, Zhou YF, Saji M, Castagna M, Shou M, Kinnaird TD, Baffour R, Ringel MD, Epstein SE, Fuchs S. Akt controls vascular smooth muscle cell proliferation in vitro and in vivo by delaying G1/S exit. Circ Res. 2003; 93: 1059–1065.[Abstract/Free Full Text]
32. Tian Y, Smith RD, Balla T, Catt KJ. Angiotensin II activates mitogen-activated protein kinase via protein kinase C and Ras/Raf-1 kinase in bovine adrenal glomerulosa cells. Endocrinology. 1998; 139: 1801–1809.[Abstract/Free Full Text]
33. Suzuki A, Ito T, Kawano H, Hayashida M, Hayasaki Y, Tsutomi Y, Akahane K, Nakano T, Miura M, Shiraki K. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death. Oncogene. 2000; 19: 1346–1353.[CrossRef][Medline] [Order article via Infotrieve]
34. Moravski CJ, Skinner SL, Stubbs AJ, Sarlos S, Kelly DJ, Cooper ME, Gilbert RE, Wilkinson-Berka JL. The renin-angiotensin system influences ocular endothelial cell proliferation in diabetes: transgenic and interventional studies. Am J Pathol. 2003; 162: 151–160.[Abstract/Free Full Text]
35. Chaturvedi N, Sjolie AK, Stephenson JM, Abrahamian H, Keipes M, Castellarin A, Rogulja-Pepeonik Z, Fuller JH. Effect of lisinopril on progression of retinopathy in normotensive people with type 1 diabetes. The EUCLID Study Group: EURODIAB Controlled Trial of Lisinopril in Insulin-Dependent Diabetes Mellitus. Lancet. 1998; 351: 28–31.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				M. M. de Resende and A. S. Greene Effect of ANG II on endothelial cell apoptosis and survival and its impact on skeletal muscle angiogenesis after electrical stimulation Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2814 - H2821. [Abstract] [Full Text] [PDF]

				M. M. de Resende, S. L. Amaral, C. Moreno, and A. S. Greene Congenic strains reveal the effect of the renin gene on skeletal muscle angiogenesis induced by electrical stimulation Physiol Genomics, March 10, 2008; 33(1): 33 - 40. [Abstract] [Full Text] [PDF]

				M A Redondo-Muller, M Stevanovic-Walker, S Barker, J R Puddefoot, and G P Vinson Anti-cancer actions of a recombinant antibody (R6313/G2) against the angiotensin II AT1 receptor Endocr. Relat. Cancer, March 1, 2008; 15(1): 277 - 288. [Abstract] [Full Text] [PDF]

				J. Gong, J. Lee, H. Akio, P. N. Schlegel, and R. Shen Attenuation of Apoptosis by Chromogranin A-Induced Akt and Survivin Pathways in Prostate Cancer Cells Endocrinology, September 1, 2007; 148(9): 4489 - 4499. [Abstract] [Full Text] [PDF]

				L. Pattacini, B. Casali, L. Boiardi, N. Pipitone, L. Albertazzi, and C. Salvarani Angiotensin II protects fibroblast-like synoviocytes from apoptosis via the AT1-NF-{kappa}B pathway Rheumatology, August 1, 2007; 46(8): 1252 - 1257. [Abstract] [Full Text] [PDF]

				D. Liu, H. Si, K. A. Reynolds, W. Zhen, Z. Jia, and J. S. Dillon Dehydroepiandrosterone Protects Vascular Endothelial Cells against Apoptosis through a G{alpha}i Protein-Dependent Activation of Phosphatidylinositol 3-Kinase/Akt and Regulation of Antiapoptotic Bcl-2 Expression Endocrinology, July 1, 2007; 148(7): 3068 - 3076. [Abstract] [Full Text] [PDF]

				H. A. Himburg, S. E. Dowd, and M. H. Friedman Frequency-dependent response of the vascular endothelium to pulsatile shear stress Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H645 - H653. [Abstract] [Full Text] [PDF]

				N. Yano, D. Suzuki, M. Endoh, T. C. Zhao, J. F. Padbury, and Y.-T. Tseng A Novel Phosphoinositide 3-Kinase-dependent Pathway for Angiotensin II/AT-1 Receptor-mediated Induction of Collagen Synthesis in MES-13 Mesangial Cells J. Biol. Chem., June 29, 2007; 282(26): 18819 - 18830. [Abstract] [Full Text] [PDF]

				Y. Zhang, T. S. Park, and J. M. Gidday Hypoxic preconditioning protects human brain endothelium from ischemic apoptosis by Akt-dependent survivin activation Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2573 - H2581. [Abstract] [Full Text] [PDF]

				Y. Mukai, C.-Y. Wang, Y. Rikitake, and J. K. Liao Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1937 - H1942. [Abstract] [Full Text] [PDF]

T. Murakami, K. Suzuma, H. Takagi, M. Kita, H. Ohashi, D. Watanabe, T. Ojima, M. Kurimoto, T. Kimura, A. Sakamoto, et al. Time-Lapse Imaging of Vitreoretinal Angiogenesis Originating from Both Quiescent and Mature Vessels in a Novel Ex Vivo Sy