Role of autophagy in angiogenesis in aortic endothelial cells

Abstract

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.

angiogenesis is a biological process of forming new blood vessels from preexisting vessels that has emerged as an important therapeutic target for treating ischemic heart disease and peripheral vascular disease (1, 2). Angiogenesis is regulated by many angiogenic factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and several integrins that are involved in the activation of complex signaling pathways (3, 23, 37). During ischemic conditions, decreased blood flow leads to diminished supply of nutrients especially glucose and oxygen. This, in turn, promotes angiogenesis by stimulating the production of angiogenic factors and certain signaling pathways as a protective mechanism (22, 24). Accumulating evidence has shown that ischemia stimulates angiogenesis in both acute and chronic models of myocardial ischemia (4, 31). In endothelial cells, glucose deprivation has been shown to increase angiogenesis as determined by increased tube formation and endothelial cell migration (39). Although most of tube formation studies have been performed under low nutrient conditions, the mechanism(s) of angiogenesis under nutrient deprivation has not been fully explored.

Autophagy is a dynamic process of subcellular degradation (10) that is critical for cell survival under nutrient-deprived conditions. When autophagy occurs, part of the cellular components is sequestered in double membrane-bound vesicles called autophagosomes and subsequently degraded upon fusion with lysosomes to provide essential elements for maintaining cell metabolism (11).

Interestingly, inhibition of nutrient deficiency-induced angiogenesis leads to autophagy (6, 15, 28). Recently, it was reported that angiogenesis inhibition increases autophagy even in the absence of nutritional stress or hypoxia (28, 35). In the myocardium, ischemia enhances autophagy (12, 25, 49), which protects the cell during ischemic injury (12, 25, 49). Nevertheless, it remains unknown whether autophagy has any influences on angiogenesis in endothelial cells.

3-Methyladenine (3-MA), a synthetic intermediate and a cell-permeable autophagic sequestration blocker, has been widely used in autophagy-related studies. 3-MA has been proposed to suppress autophagy by inhibiting the class III phosphoinositide 3-kinase (PI3K) to block the production of phosphatidylinositol 3-phosphate (PI3P) (32), which is essential for the initiation of autophagy (48). ATG5 has been characterized as a protein specifically required for autophagy (34). In this study, we used two different approaches to determine the role of autophagy in angiogenesis; one by using 3-MA to pharmacologically inhibit autophagy and the other by using small interfering RNAs (siRNAs) against AGT5 to block ATG5 expression and inhibit autophagy. We used this approach to verify whether results of experiments using genetic and pharmacological tools are aligned and internally consistent since pharmacology tools may not be as specific as they are first described.

Autophagosome formation and expression of one of the important autophagy-related proteins, the light chain of the microtubule-associated protein 1 (LC3), were used in our study to assess autophagy. During autophagy, phosphatidylethanolamine will conjugate to the cytosolic form of LC3 (LC3-I) to form LC3-II (43), and the amount of LC3-II is a commonly used indicator for autophagy (26). When cells are transfected with LC3 constructed with green fluorescent probe (GFP) plasmid (LC3-EGFP) (19), autophagosome formation can be visualized as green fluorescent punctate (26). In this study, we determined whether and how autophagy affects angiogenesis in aortic endothelial cells induced by nutrient deprivation.

MATERIALS AND METHODS

Materials.

3-MA was purchased from Sigma (St. Louis, MO). DMEM, Opti-MEM, HBSS, penicillin-streptomycin, negative siRNA control (NS), pcDNA 3.1 plasmid (pcDNA), Lipofectamine 2000, and Lipofectamine LTX and Plus reagent were from Invitrogen (Carlsbad, CA). ATG5-pcDNA 3.1 plasmid (ATG5, Plasmid 13095) (40) and LC3 constructed with GFP plasmid (LC3-EGFP, Plasmid 11546) (19) were from Addgene (Cambridge, MA). ATG5 siRNA and antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), VEGF, and PDGF were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against ATG5, LC3, integrin αV, phospho-AKT, AKT, phospho-extracellular-regulated kinase (ERK), and ERK were from Cell Signaling Technology (Boston, MA). Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) was from Enzo Life Sciences (Farmingdale, NY). FPA-124 (FPA), an Akt inhibitor, was from EMD Biosciences (La Jolla, CA).

Primary cell culture and treatment.

Bovine aortic endothelial cells (BAECs) were isolated from bovine aorta. Briefly, calf heart along with aorta was freshly obtained from a local slaughterhouse and transported to the lab on ice. Bovine thoracic aorta was cut out from the heart and washed in phosphate-buffered saline (PBS) containing 200 U/ml penicillin and 200 μg/ml streptomycin. Clean aorta segments were transferred to individual 10-cm Petri dishes. Then bovine aortic segments were cut open longitudinally, and the endothelia were removed by gently scraping with a sterile scalpel. The cells were then digested with 0.1% collagenase in HBSS for 15 min at 37°C, centrifuged, resuspended, and grown in RPMI-1640 containing 20% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). BAECs were maintained in complete medium (DMEM with 10% FBS) incubated at 37°C in a humidified 95% room air with 5% CO₂. BAECs were then expanded and used at passage 3–5 for all experiments. To induce autophagy, BAECs were deprived of nutrients by culture in DMEM without glucose and serum (nutrient-deprived medium) or Opti-MEM without serum. Nutrient-deprived medium was used in autophagy inhibition experiments with 3-MA or ATG5 siRNA. In autophagy induction experiments by ATG5 overexpression, the Opti-MEM without serum was used becauses it provides the basic nutrients for the cells and will not induce strong autophagy like nutrient-deprived medium, which overshadows the effect of ATG5 overexpression. In addition, we found that overexpressing ATG5 in nutrient-deprived medium but not Opti-MEM would increase cell death.

Transfection of siRNA and plasmids.

BAECs were cultured in 60-mm plates (4×10⁵). Fresh medium was added 18 h later preparing for transfection. BAECs were transiently transfected with 100 pmol siRNA using Lipofectamine 2000, and the cells were subsequently incubated in complete medium (DMEM with 10% FBS) for 48 h before starvation. Plasmids (LC3-EGFP, 2 μg; pcDNA and ATG5-pcDNA 3.1, 8 μg) were transfected into BAECs in 60-mm plates (5×10⁵) using Lipofectamine LTX and Plus reagent for 20 h according to the manufacturer's instructions.

Autophagy assay.

BAECs were lysed and processed for Western blot analysis as previously described (8) for determination of LC3-II/LC3-I ratio. The transfected BAECs with LC3-EGFP were reseeded into a Lab-Tek II four-well chamber slides (2×10⁴/well) and cultured for 12 h before treatment. The cells were then fixed and examined using a fluorescence microscope (Olympus IX50) for the GFP signals. About 50 pictures were randomly taken for each treatment, and cells with GFP punctate were counted.

Tube formation assay.

In vitro tube formation was assayed as previously described (41). Growth factor-reduced Matrigel (BD Biosciences) was pippetted into a 96-well plate (50 μl/well) and allowed to solidify (2 h at 37°C). Culture medium (100 μl) was added on top of the solidified Matrigel with or without 3-MA (5 mM), and then BAECs (2×10⁴/well) were added into each well with gentle mixing. Cells with 3-MA were incubated in nutrient-deprived medium while cells made to over express ATG5 were incubated in Opti-MEM for 4 h. Representative pictures were taken per well in bright field using an inverted microscope (Olympus IX50) at ×20 magnification. Total tube length was measured only for the tubular structures connecting two cell clusters in high-power field (HPF). Cell clusters with at least three tubular structures emanating out were considered to be a branching point. The number of branching points was used as another parameter to quantify tube formation.

Migration assay.

Transwell cluster plates (24-well, 8 μm pore size, Costar) were precoated with 0.1% gelatin in the outer membrane (41). The outer chamber was filled with nutrient-deprived medium or Opti-MEM containing VEGF (20 ng/ml) as chemoattractant. BAECs (2×10⁴) were placed into the insert of the transwell and placed in the incubator for 4 h.The inserts were removed, and the nonmigrating cells were removed by cotton-tipped swabs. The inserts were then stained and examined using an Olympus IX50 microscope. Three randomly selected fields were imaged per each insert, and migrating cells were counted in each HPF.

Measurement of reactive oxygen species production.

BAECs with or without transfection were seeded onto Lab-Tek II four-well chamber slides (5×10⁴/well) and maintained in complete medium for 12 h. After cultured in nutrient-deprived medium or Opti-MEM for 2 h, BAECs were then treated with dihydroethidium (DHE, 10 μM) in HBSS for 30 min to detect intracellular reactive oxygen species (ROS) production. Fluorescence was imaged under a Nikon Eclipse TE200 fluorescence microscope with excitation and emission at 510 and 590 nm, respectively. The relative light units of integrated fluorescence were measured from the images.

Western blot analysis.

Western blot analysis was performed as previously described (8). Cell lysate (30 μg protein) was loaded onto and separated by SDS/PAGE. The separated proteins were transferred to nitrocellulose membranes, which were then blocked with TBS-T buffer containing 5% nonfat milk, incubated with primary antibodies overnight, followed by incubation with secondary antibodies for 1 h at room temperature. Signals were visualized with Super Signal West Pico kit (Pierce Biotechnology.)

Statistical analysis.

Data are expressed as means ± SE. Significance of differences between means was determined by unpaired two-tailed t-tests or ANOVA with an appropriate post hoc test. A P value < 0.05 was considered to be significant.

RESULTS

Inhibition of autophagy decreased angiogenesis in BAECs.

After incubation in nutrient-deprived medium for 4 h, BAECs with GFP punctate were about 35–40% of the total GFP-positive cells (Fig. 1, A and C), whereas BAECs incubated in complete medium had only ∼10% cells with GFP punctate (data not shown). These data demonstrate that nutrient deprivation significantly increases autophagy in BAECs. Incubation with 3-MA (5 mM) decreased the number of GFP punctate cells in nutrient-deprived BAECs down to ∼13% (Figure 1, A–C). 3-MA also inhibited LC3-II conversion in nutrient-deprived BAECs (Fig. 1, D and E). 3-MA treatments significantly decreased total tube length and the number of branching points, respectively (Fig. 1, F and G). Similarly, 3-MA dramatically decreased the average number of migrated cells per HPF from 55 to 20 (Fig. 1H). To further determine whether inhibition of autophagy decreases angiogenesis in BAECs, we knocked down ATG5 in BAECs by using siRNA. Transfection of ATG5 siRNA reduced ATG5 expression by about 60∼70% (Fig. 2A) and significantly decreased the number of GFP punctate cells (inhibited autophagy) compared with cells transfected with negative control siRNA (Fig. 2B). Consistently, ATG5 siRNA decreased total tube length, number of branching points, and the average number of migrated cells (Fig. 2, C–E). These results show that inhibition of autophagy decreases BAECs tube formation and migration, signifying that autophagy promotes angiogenesis in BAECs.

Fig. 1.

Inhibition of autophagy with 3-methyladenine (3-MA) decreases angiogenesis in bovine aortic endothelial cells (BAECs). BAECs were transiently transfected with LC3 constructed with green fluorescent probe (GFP) plasmid (LC3-EGFP) for 20 h and then incubated in nutrient-deprived medium for 4 h with or without 3-MA (5 mM). GFP-positive cells and cells with GFP punctate were counted under fluorescence microscope. Representative pictures of cells without 3-MA (A) and with 3-MA (B) are shown. The percentage of cells with GFP punctate over total GFP-positive cells was counted among 150 cells in 3 repeats (C). Representative blot of LC3 I/II with or without 3-MA treatment (D), and LC3-II expression (E) was normalized by GAPDH. Total tube length (F) and number of branching points (G) were calculated from 4 repeats. BAECs were grown in transwell with or without 3-MA in nutrient-deprived medium, and the migrated cells were counted under brightfield microscope (H). The number of migrated cells per high-power field (HPF) was calculated from 3 independent experiments. Values are means ± SE. *P < 0.05 vs. cells without 3-MA treatment.

Fig. 2.

Inhibition of autophagy with ATG5 small interfering RNA (siRNA) decreases angiogenesis in BAECs. BAECs were transfected with negative siRNA (NS) or ATG5 siRNA for 48 h. A: cell lysates were immnublotted with antibody against ATG5 or GAPDH. B: BAECs were transfected with LC3-EGFP plasmids, and percentage of punctate GFP cells were calculated after cells were incubated in nutrient-deprived medium for 4 h. Total tube length (C), number of branching point (D) and the migrated cells (E) were calculated from BAEC on Matrigel or transwell after nutrient deprivation for 4 h. All the experiments were repeated 3 times. Values are means ± SE. *P < 0.05 vs. cells transfected with NS.

Induction of autophagy increased angiogenesis in BAECs.

Twenty hours after transfection, ATG5 expression was significantly increased about three- to fourfold (Fig. 3A), and the number of cells with GFP punctate increased by about 60% in the ATG5 overexpressing BAECs compared with BAECs transfected with vector alone (Fig. 3B). The overexpression of ATG5 also increased total tube length, the number of branching points, as well as migrated cells (Fig. 3, C–E). These results provide further evidence that autophagy promotes angiogenesis in BAECs under nutrient-deprived condition.

Fig. 3.

Induction of autophagy with ATG5 overexpression increases angiogenesis. A: BAECs transfected with either pcDNA or ATG5 for 20 h were lyzed for immunoblots to determine the expression level of ATG5. B: BAECs were cotransfected with pcDNA (or ATG5 plasmids) and LC3-EGFP plasmids in complete medium for 20 h and then incubated in Opti-MEM for 4 h. The GFP-positive cells and the cells with GFP punctate were counted under fluorescent microscope. After transfection with either pcDNA or ATG5, BAEC were reseeded on either matrigel or transwell in Opti-MEM for 4 h to measure the total tube length (C), the number of branching point (D), and the migrated cells (E) (n = 3). Values are means ± SE. *P < 0.05 vs. cells transfected with pcDNA.

Inhibition of autophagy suppresses VEGF-induced angiogenesis in BAECs.

As VEGF is a key angiogenic growth factor that stimulates tube formation and migration of endothelial cells (27), we determined whether inhibiting autophagy affects VEGF-induced angiogenesis. VEGF (20 ng/ml) significantly increased the total tube length and number of branching points by about two- to threefold in BAECs (Fig. 4, A and B). Treatment with 3-MA or with ATG5 siRNA decreased tube length and branching points by 75%, 66%, 62%, and 58%, respectively (Fig. 4). These data indicate that autophagy also plays critical roles in VEGF-induced angiogenesis.

Fig. 4.

Inhibition of autophagy decreases vascular endothelial growth factor (VEGF)-induced angiogenesis. BAECs were either treated with 3-MA or transfected with ATG5 siRNA and then grown in nutrient-deprived medium with VEGF (20 ng/ml) for 4 h. Cells [no 3-MA treatment or transfected with negative siRNA control (NS)] without VEGF were used as basal controls. Total tube length (A) and the number of branching points (B) for BAECs with or without 3-MA are shown. The total tube length (C) and the number of branching points(D) for BAECs transfected with or without ATG5 siRNA (n = 3) are shown. Values are means ± SE. *P < 0.05 vs. cells without VEGF (no 3-MA or transfected with NS). #P < 0.05 vs. cells with VEGF but no 3-MA treatment or transfected with NS.

Autophagy did not alter the expression of VEGF, PDGF, or integrin αV.

Many pro-angiogenic factors mediate angiogenesis in endothelial cells such as VEGF, PDGF, FGF, TGF-β, and integrin. To examine whether these pro-angiogenic factors mediate angiogenesis induced by autophagy, we examined VEGF, PDGF, and integrin αV expression levels in nutrient-deprived (4 h) BAECs. No changes in the expression of these pro-angiogenic factors were observed regardless of whether autophagy was induced by overexpression of ATG5 or inhibited by treating BAECs with 3-MA and ATG5 siRNA (Fig. 5, A–F).

Fig. 5.

The effect of autophagy on expressions of pro-angiogenic factors. The expressions of VEGF, platelet-derived growth factor (PDGF), and integrin αV from BAEC with 3-MA treatment (A) for 4 h in nutrient-deprived medium or ATG5 siRNA transfection (B) incubated in nutrient-deprived medium for 4 h, and ATG5 overexpression incubated in Opti-MEM (C) were examined by Western blot analysis. The signals of each protein were normalized by that of GAPDH. The representative blots were from three independent repeats. The expression of VEGF, PDGF, and integrin αV in A, B, and C were quantified by normalization of their densitometries with GAPDH and presented as means ± SE in D, E, and F, respectively. NS, no significant difference compared with control or vector groups. V, pcDNA vector. N = 3.

Autophagy induced ROS production in BAECs.

Next, we examined whether ROS, a stimulator of angiogenesis in endothelial cells (42, 44), is involved in autophagy-induced angiogenesis. Two hours of 3-MA treatment significantly reduced superoxide production (induced by nutrient deprivation) in BAECs as shown by a significant decrease in DHE fluorescent intensity (Fig. 6A). Similarly, treating BAECs with ATG5 siRNA reduced superoxide production after nutrient deprivation (Fig. 6B). To determine whether or not superoxide is increased by autophagy, we measured superoxide production in ATG5 overexpressing BAECs. Overexpression of ATG5 significantly increased DHE fluorescence in BAECs compared with transfection of the pcDNA vector alone (Fig. 6C).To assess whether ROS production contributes to the autophagy-induced angiogenesis, we used superoxide dismutase mimetic, which is a strong antioxidant in our ATG5 overexpresion study. MnTMPyP (25 μM) treatment for 4 h significantly decreased the total tube length and branching point numbers in BAECs with ATG5 but not pcDNA vector transfection (Fig. 6, D and E), indicating that ROS plays critical roles in autophagy-induced angiogenesis.

Fig. 6.

The effect of autophagy on reactive oxygen species (ROS) production in BAEC. The intracellular superoxide production from BAECs with 3-MA treatment (A) for 2 h in nutrient-deprived medium, or ATG5 siRNA transfection (B) incubated in nutrient-deprived medium for 2 h, or ATG5 overexpression (C) incubated in Opti-MEM was estimated by dihydroethidium (DHE) fluorescence. Top, integrated DHE fluorescence intensity; bottom, representative pictures (n = 3). NS, negative siRNA control. Total tube length (D) and the number of branching points (E) were determined after BAECs were treated with or without manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) (25 μM) for 4 h. Values are means ± SE. *P < 0.05 vs. cells without 3-MA or transfected with NS or pcDNA, and #P < 0.05 vs. BAECs overexpressing ATG5 without MnTMPyP.

Autophagy activated AKT phosphorylation but not ERK.

AKT and ERK are two major downstream protein kinases that have been shown to mediate angiogenesis. To examine whether nutrient deprivation-induced autophagy activates AKT phosphorylation, we incubated BAECs with DMEM containing 10% FBS or nutrient-deprived medium for 0.5, 2, and 4 h. Nutrient deprivation dramatically decreased AKT phosphorylation in BAEC at 30 min but increased AKT phosphorylation considerably at 2 and 4 h. As expected, autophagy indicator LC3-II formation increased with 30 min nutrient deprivation and lasted through the 4 h (Fig. 7A).To compare the effect of ATG5 on AKT activation under normal or nutrient-deprived conditions, BAECs transfected with pcDNA or ATG5 were incubated with 10% FBS DMEM or nutrient-deprived medium. ATG5 activated AKT more potently in the nutrient-deprived medium than nutrient-rich medium (Fig. 7B). To explore the role of AKT in autophagy-induced angiogenesis, we treated BAECs with specific AKT inhibitor FPA (50 μM)for 4 h after BAEC was transfected with either ATG5 or pcDNA. The inhibition of AKT significantly decreased the total tube length and branching point number in the ATG5-overexpressing BAECs (Fig. 7, C and D), which indicates that AKT activation is an important mechanism in autophagy-induced angiogenesis. We further observed that autophagy inhibitor 3-MA robustly inhibited AKT phosphorylation stimulated by nutrient deprivation at 30, 60, and 120 min in BAECs but had no effect on ERK phosphorylation (Fig. 8A). Similarly, ATG5 siRNA inhibited phosphorylation of AKT but not ERK in nutrient-deprived BAECs (Fig. 8B). In addition to data shown in Fig. 7B, interestingly, we found that ATG5 overexpression increased AKT phosphorylation but not ERK (Fig. 8, C and D). All these data show that autophagy activated AKT phosphorylation and this activation may be involved in autophagy-induced angiogenesis.

Fig. 7.

The role of AKT in autophagy-induced angiogenesis. A: BAECs were incubated either with DMEM containing 10% FBS or with nutrient-deprived medium for different time and analyzed by immunoblots for expression levels of AKT, phospho-AKT, LC3-I/II, and GAPDH. B: BAECs transfected with pcDNA or ATG5 were analyzed by immunoblots for phosphor-AKT, AKT, and GAPDH. Total tube length (C) and the number of branching points (D) were determined when BAECs were treated with or without AKT inhibitor FPA-124 (FPA, 50 μM) for 4 h. P-AKT, phosphor-AKT. Values are means ± SE. *P < 0.05 vs. cells transfected with pcDNA, and #P < 0.05 vs. BAECs overexpressing ATG5 without FPA treatment.

Fig. 8.

The effect of autophagy on AKT and extracellular regulated kinase (ERK) pathways. The activation of AKT and ERK from BAECs with 3-MA treatment ranging from 30 to 120 min in nutrient-deprived medium (A), with ATG5 siRNA incubated in nutrient deprived-medium for 30 min (B), and with ATG5 overexpression incubated in Opti-MEM (C) are shown. D: densitometry of C normalized with total AKT and GAPDH (n = 3). *P < 0.05 vs. cells transfected with pcDNA. NS, negative siRNA control.

DISCUSSION

Our findings indicate that autophagy plays an important role in nutrient deprivation-induced angiogenesis. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, even VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms in autophagy-mediated angiogenesis.

Autophagy has been considered an important survival mechanism and has been recognized as being critical for survival during the starvation period that occurs in the first few days immediately after birth (21). Autophagy increases and is protective under hypoxia and chronic ischemia conditions (18, 49). Interestingly, angiogenesis is also robustly induced by both hypoxia and ischemia (4, 31, 33). Our studies show that autophagy may be an important angiogenesis promoter, as both pharmacological and genetic inhibition of autophagy significantly impair tube formation and migration in BAECs (Figs. 1 and 2). Furthermore, inhibition of autophagy suppresses VEGF-induced angiogenesis (Fig. 4) and stimulation of autophagy increases angiogenesis (Fig. 3). Thus autophagy might help endothelial cells adapt to and be protected from ischemic and hypoxic conditions that occur after a myocardial infarction by increasing angiogenesis. In agreement with our study, it has been reported recently that inhibition of autophagy with 3-MA potentiates anti-angiogenic effects of a pro-apoptotic agent in human umbilical vein endothelial cells (30). Furthermore, lysosome-associated membrane proteins (LAMPs), proteins associated with autolysosome formation, were found in immature endothelium of proliferating infantile hemangioma, a tumor of the microvasculature characterized by aggressive angiogenesis during infancy. LAMPs were increased in involuting hemangioma, which might be associated with augmented autophagy required for tissue remodeling during tumor involution (36). Additionally, cathepsin D, a lysosomal hydrolase associated with autophagy, has been found to enhance angiogenesis of cancer cells probably by degrading extracellular matrix (20). However, excessive autophagy under some circumstances may result in autophagic cell death, also known as programmed cell death II. The autophagic cell death pathway converges with the apoptotic pathway (29). Our preliminary data showed that overexpression of ATG5 for more than 26 h induced significant autophagic cell death, which actually reduced angiogenesis (data not shown). Therefore, it is also possible that autophagic cell death and apoptosis accompany with or result in decreased angiogenesis (13, 14, 38). We speculate that that acute increase in autophagy helps to recover perfusion, whereas chronic increase in autophagy may turn to apoptosis that will lead to damaging effect.

Angiogenesis is a highly controlled process regulated by the balance between angiogenic promoters and angiogenic inhibitors (2). We focused on several important angiogenic promoters, VEGF, PDGF, and integrin αV in our study, but we didn't find any changes in their expression during either inhibition or induction of autophagy. It is possible that we did not catch the best timing of detecting their changes or autophagy might induce other angiogenic factors such as FGF and TGF or inhibit angiogenesis inhibitors. VEGF is an essential angiogenic promoter in angiogenesis, and we found that inhibition of autophagy significantly inhibited VEGF-induced angiogenesis (Fig. 4). We speculated that autophagy might affect the receptor or postreceptor signaling of VEGF.

ROS function as key signaling molecules to mediate angiogenesis (42, 44) and are essential for full activation of VEGF receptor 2 to signal endothelial cell proliferation and migration (7, 45, 46). As we found that inhibition of autophagy decreases ROS production, autophagy may influence the VEGF receptor activation and VEGF signaling. The antioxidant MnTMPyP decreased ATG5-induced tube formation, which suggests that ROS mediate autophagy-induced angiogenesis. Additionally, when we examined AKT and ERK pathways, two major downstream signaling targets for VEGF in angiogenesis (5, 17, 47), we found that autophagy activates AKT phosphorylation and inhibiting autophagy suppresses activation of AKT but not ERK. AKT is activated by all class I, II, and III PI3K. The class I and II PI3K are activated by growth factor receptor tyrosine kinases and G protein-coupled receptors (9). Nutrient deprivation of BAECs for 30 min decreased AKT phosphorylation (Fig. 7A), which might be triggered by the sudden withdrawal of growth factors. However, the enhanced activation of AKT in 2 h in nutrient-deprived medium accompanied by consistently higher amount of LC3-II formation indicate that autophagy activates AKT in BAECs (Fig. 7A). The inhibition of AKT blocked ATG5-induced tube formation (Fig. 7, C and D), indicating that the class III PI3K may be linked to the activation of AKT in angiogenesis. Further studies are needed to define how AKT and ROS are activated by autophagy, which AKT isoforms are responsible for the activation, and to identify the sources of ROS. Thus our current study implicates autophagy, ROS, and AKT in the regulation of angiogenesis. Our future studies will explore the mechanistic roles of AKT and ROS in the angiogenesis process by determining the signal transduction steps before and after AKT and whether AKT is the key to prevention of apoptosis during induction of autophagy as well as the activation of sources of ROS.

Finally, we want to point out the limitations of our study. The first limitation of our study is the pharmacological tool we used in our study 3-MA. Vesicular protein sorting 34 (Vps34), also known as class III PI3K, is well established to be the regulator of phagophore formation and autophagy. 3-MA is a widely used inhibitor of VPS34 in studies of autophagy (26). Recently, it was found that 3-MA may also inhibit class I and II PI3K when used at high concentrations (16). To limit this nonspecific effect, we used low concentration of 3-MA in our study. The second limitation of our study is that we only used BAECs as our model of endothelial cells. As different phenotypes of endothelial cells committed to angiogenesis may respond to stimuli, such as nutrition deprivation differently, further studies using other types of endothelial cells as well as in vivo models are warranted to fully explore the role of autophagy in angiogenesis. The third limitation is that we used a limited number of pharmacological inhibitors or siRNAs for AKT, ROS, and autophagy. Additional inhibitors and siRNAs will be used in future studies to account for their side effects or off-target effects. Nevertheless, our study observations are significant as we present evidence that autophagy may play critical roles in regulating angiogenesis by modulating superoxide production and AKT activation in BAECs. The new knowledge gained from our studies showing that autophagy promotes angiogenesis in endothelial cells may advance our understanding of mechanisms of ischemic heart disease and provide new targets for treating cardiovascular disease.

GRANTS

This work was supported by National Institutes of Health Grant HL-080468 (to Y. Shi) and American Heart Association Postdoctoral Fellowship Award 09POST2250335 (to J. Du).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: J.D. and Y.S. conception and design of research; J.D., R.-J.T., T.G., A.E., and S.K. performed experiments; J.D. and Y.S. analyzed data; J.D., R.-J.T., G.G.K., and Y.S. interpreted results of experiments; J.D. and Y.S. prepared figures; J.D. drafted manuscript; J.D., R.-J.T., S.K., G.G.K., and Y.S. edited and revised manuscript; J.D., R.-J.T., G.G.K., and Y.S. approved final version of manuscript.