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Am J Physiol Heart Circ Physiol 294: H1119-H1129, 2008. First published January 18, 2008; doi:10.1152/ajpheart.00713.2007
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Cardiovascular-Renal Mechanisms in Health and Disease

Lipid mediators of autophagy in stress-induced premature senescence of endothelial cells

Susann Patschan, Jun Chen, Alla Polotskaia, Natalja Mendelev, Jennifer Cheng, Daniel Patschan, and Michael S. Goligorsky

Departments of Medicine and Pharmacology, New York Medical College, Valhalla, New York

Submitted 19 June 2007 ; accepted in final form 16 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our group (Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS; Am J Physiol Renal Physiol 294: F100–F109, 2008) previously observed an accumulation of gangliosides coincident with development of cell senescence and demonstrated lysosomal permeabilization in human umbilical vein endothelial cells exposed to glycated collagen I (GC). Therefore, we investigated whether the lysosome-dependent, caspase-independent or type 2-programmed cell death (autophagy) is involved in development of premature senescence of endothelial cells. The cleaved microtubule-associated protein 1 light-chain 3 (LC3), a marker of autophagosome formation, was overexpressed within 24 h of GC treatment; however, by 4–5 days, it was nearly undetectable. Early induction of autophagosomes was associated with their fusion with lysosomes, a phenomenon that later became subverted. Autophagic cell death can be triggered by the products of damaged plasma membrane, sphingolipids, and ceramide. We observed a clustering of membrane rafts shortly after exposure to GC; later, after 24 h, we observed an internalization, accompanied by an increased acid sphingomyelinase activity and accumulation of ceramide. Pharmacological inhibition of autophagy prevented development of premature senescence but did lead to the enhanced rate of apoptosis in human umbilical vein endothelial cells exposed to GC. Pharmacological induction of autophagy resulted in reciprocal changes. These observations appear to represent a mechanistic molecular cascade whereby advanced glycation end products like GC induce sphingomyelinase activity, accumulation of ceramide, clustering, and later internalization of lipid rafts.

advanced glycation end product; lysosomes; sphingomyelinase activity; lipid rafts

DIABETES IS ASSOCIATED WITH an accelerated rate of cardiovascular complications (2) that is in part secondary to the premature senescence of vascular endothelium exposed to advanced glycation end products (AGEs) (5, 7). Chronological analyses of endothelial cell fate after exposure to the AGE-modified long-lived extracellular matrix protein glycated collagen I (GC) convincingly demonstrated an early induction of apoptotic cell death followed within 3–4 days by the development of premature senescence (27). These most recent studies demonstrated lysosomal permeabilization early after exposure to pathophysiologically relevant concentrations of GC and demonstrated the role of gangliosides in apoptotic cell death but rejected the hypothesis that accumulation of gangliosides, as a consequence of lysosomal dysfunction, is responsible for development of premature cell senescence. Because apoptotic mechanisms could not solely account for the cell death after exposure to GC, we reasoned that an additional mechanism may be represented by the recently recognized lysosome-dependent, caspase-independent or type 2 programmed cell death (autophagy). Autophagic cell death requires lysosomal integrity for efficient fusion and digestion of autophagosomes inside lysosomes. The general mechanism is the sequestration of cargo material into a large double-membrane vesicle, called an autophagosome, which then fuses with a lysosome or vacuole. Autophagy plays an essential role in development and differentiation and may be involved in lifespan extension and type II programmed cell death (28).1

Autophagy is involved in the most important cardiac pathologies (36) and plays a role in regulation of aging (24). Inhibition of macroautophagy has led to premature senescence (12) and has been shown to result in accumulation of lipofuscin in cultured human fibroblasts (33). Autophagic cell death can be triggered by products of the damaged plasma membrane, sphingolipids and ceramide (16, 34), and the age-related decline in macroautophagy has been associated with deterioration of cell membranes (13). Functional microdomains of cell membranes called membrane rafts (21) consist of glycosphingolipids together with cholesterol, the phosphosphingolipid sphingomyelin, and glycosylphosphatidyl-anchored proteins. We utilized a glycosylphosphatidylinositol (GPI)-anchored thermotolerant green fluorescent protein (ttGFP) as a probe to visualize membrane rafts and to analyze the changes in proximity of GPI-anchored ttGFP expressed in human umbilical vein endothelial cells (HUVECs) (26).

The lysosomal degradation of these plasma membrane glycosphingolipids, especially of the ganglio series, has been well studied. Gangliosides are composed of a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids. Our group (27) previously observed accumulation of gangliosides GM3, GD1b, and GT1b that was coincident with development of cell senescence. Glycosphingolipids of the gala series, but also sphingomyelin, are degraded into ceramide. The sphingolipid ceramide stimulates autophagy and cell death with autophagic features in cancer cells (23).

Previously, our group (27) established a time course of events following the exposure of endothelial cells to GC beginning with an early peak of apoptosis (16 h), antedated by the production of reactive oxygen species, lysosomal pH collapse, and lysosomal permeabilization and mitochondrial membrane depolarization, From day 3 onward, endothelial cells became senescent. In the late phase of incubation with GC, the ganglioside content of the cell increased (27).

Our hypothesis is that lysosomal permeabilization influences autophagy as a cell defense mechanism, which leads to a senescent phenotype of endothelial cells. In addition, lipid mediators such as ceramide and sphingosine-1-phosphate, known to be involved in autophagic cell death, are regulated by exposure of endothelial cells to GC (30).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. The following antibodies were used: anti-light-chain 3 (LC3; MBL, Nagoya, Japan), FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories), anti-ceramide (Alexis Biochemicals, San Diego, CA), Cy3-conjugated donkey anti-mouse IgM (Jackson Immunoresearch), anti-Lamp-1 (H4A3) (Developmental Studies Hybridoma Bank), and Texas red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch). pGEX plasmid containing ttGFP was used to generate GPI-ttGFP and wild-type ttGFP constructs, as previously detailed (10, 26). FuGene transfection reagent was from Roche Diagnostics. Type 1 collagen was from Vitrogen (Cohesion, Palo Alto, CA). Triton X-100, EDTA, and fibronectin were purchased from Sigma-Aldrich. GC was prepared as previously detailed (27). Native collagen (NC) and GC were used at a concentration of 100 µg/ml. Low-dose GC refers to a mixture of NC and GC of 1:3.

Cell culture. HUVECs and EGM-2 growth medium were purchased from Clonetics (Walkersville, MD). HUVECs were used between passages 3 and 6 and maintained at 37°C in a 95% air-5% CO2 humidified atmosphere.

Detection of autophagy. HUVECs were solubilized in Triton X-100-based lysis buffer consisting of 1% Triton X-100, 2 mM EDTA, 0.1 mM Na3VO4, 1 mM sodium fluoride, 1 mM PMSF, complete protease inhibitor cocktail (Roche), and PBS (pH 7.4). The protein concentration was determined with the Bradford protein assay (Bio-Rad). Equal amounts of protein (25 µg) were separated by SDS-PAGE (18%) and transferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore). After blocking procedure with 3% BSA, the membranes were incubated with primary antibody against LC3, the mammalian orthologue of Aut7/Apg8, (1:1,000 dilution) at 4°C overnight followed by horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution, 1 h) and developed with the enhanced chemiluminescence system (Amersham Biosciences). Experiments were repeated at least three times, and equal loading of protein was ensured by measuring β-actin expression.

Immunofluorescence. Cells (~104/well) were seeded on circular microscope coverslips (diameter 12 mm; Fisher) precoated with fibronectin (final concentration of 10 µg/ml) and UV light sterilized. Slides were placed into 24-well tissue culture plates (Falcon). HUVECs were incubated with NC or GC (100 µg/ml) from 3 h up to 5 days. The cells were washed at room temperature with PBS and fixed with 4% paraformaldehyde for 10 min. Permeabilization was performed with 0.2% Triton X-100 in BSA 1% for 10 min and followed by blocking with 1% BSA for 30 min. HUVECs were incubated with the Lamp-1 antibodies (concentration 1:100) overnight at 4°C and secondary antibody for 1 h (concentration 1:500). Cells were costained with LC3 antibody (concentration 1:100) for 1 h at 4°C and secondary antibody FITC-conjugated donkey anti-rabbit IgG for 1 h at 4°C. Finally, HUVECs were stained with Hoechst 33342 for 30 min, mounted with Slowfade (Molecular Probes), and imaged with a Nikon TE-2000U microscope equipped with a Spot Insight camera (Diagnostic Instruments). The green staining of the LC3 antibody was overlapped with the red Lamp-1 staining, and yellow pixels were counted with the Metamorph program. The percentage of red (Lamp-1) to yellow pixels was calculated. The experiment was done in triplicate, and 15 cells were counted per time point.

Imaging of lipid rafts. GPI-anchored ttGFP and wild-type ttGFP plasmids were constructed as previously described (10). Plasmid purification was accomplished with Endofree plasmid maxi kit (Qiagen). The purified DNA was resuspended in endotoxin-free Tris-EDTA buffer at a concentration of 2 µg/µl. The purity of plasmid preparation was verified by measurement of 260 nm-to-280 nm absorbance ratio and agarose gel electrophoresis. Cells were passaged 1–2 days before transfection and were transfected at 70–80% confluency using FuGENE 6 reagent. The optimal ratio of DNA to FuGENE was determined to be 1:3. HUVECs were grown on fibronectin-coated glass coverslips, transiently transfected with GPI-anchored ttGFP for 24 h (26), and subjected to different treatments as specified in RESULTS. The cells were washed with PBS at room temperature and fixed with 4% paraformaldehyde for 10 min at room temperature. Finally, all slides were stained with Hoechst 33342, mounted with Slowfade, and imaged with a Nikon TE-2000U microscope. Images were digitally analyzed using MetaMorph software to quantify the proportion of clusters of cell membrane-associated GPI-ttGFP as a percentage of cell perimeter.

Detection of ceramide. For detection of ceramide, endothelial cells were grown on fibronectin-coated glass coverslips and subjected to NC and GC (100 µg/ml) for 3, 6, 16, 24, 48, 72, 96, and 120 h. Cells were washed with 1x PBS, fixed with paraformaldehyde for 10 min, and washed twice with PBS. Samples were blocked with 1% BSA in PBS. Cells were stained overnight with antibodies to ceramide (1:100), washed twice in PBS, and stained for 1 h with Cy3-coupled anti-mouse IgM antibodies. Nonspecific fluorescence was excluded by performing a control without primary antibody. Finally, all slides were stained with Hoechst 33342, mounted with Slowfade, and imaged using a Nikon TE-2000U microscope equipped with a Spot Insight camera. To compare the intensities of fluorescence, images were captured by analogous exposure time and equally processed. Images of endothelial cells subjected to GC were visually compared with evaluated time points for quantification of ceramide levels by HPLC-tandem mass spectrometry (MS).

Measurement of ceramide levels by HPLC/MS. HUVECs were grown in standard tissue culture dishes (100 x 20 mm; BD) and subjected to GC or NC for 24, 72, and 120 h. Cells from four separate experiments were pooled by trypsinization, washed in ice-cold PBS, centrifuged at 500 g, and stored at –80°C until further analysis in the lipidomics core facility at the Medical University of South Carolina. The simultaneous quantitative analysis of bioactive sphingolipids was achieved by HPLC/MS, as previously detailed (3). Simultaneous electrospray ionization (ESI)/MS/MS analyses of sphingoid bases, sphingoid base 1 phosphates, and ceramides were performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring positive ionization mode. Biological samples were fortified with internal standards, extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 HPLC/MS system. Qualitative analysis of sphingolipids (SPLs) was performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of internal standard.

Assay for acid sphingomyelinase activity. The enzymatic hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine by sphingomyelinase was measured at pH 5.0 with the Amplex red reaction kit (39) according to manufacturer's instructions with minor modifications. Briefly, after stimulation with NC or GC, cells (5–10 x 105) were pelleted by centrifugation at 1,500 g for 10 min at 4°C and washed twice with ice-cold PBS. Catalase was added (50 µl, 1.0 U/ml) and incubated with HUVECs at room temperature for 10 min to remove endogenous H2O2. Cells were washed with PBS three times, and the pellet was resuspended and lysed in 0.6 ml of buffer containing 1% Triton X-100, 1x proteinase inhibitor, 1 mM EDTA, and 50 mM sodium acetate (pH 5.0) for 60 min on ice. The supernatant fraction was saved by centrifugation at 17,000 g for 10 min at 4°C to remove nuclei. The protein concentration in the supernatant fraction was measured with the Bio-Rad protein assay. Cell membrane-free supernatant fractions (adjusted to pH 5.0) were assayed for sphingomyelinase activity in a two-step reaction system. First, to generate phosphocholine and ceramide, 0.5 mM SM was added to the supernatant fraction and incubated for 60 min at 37°C. The reaction was then placed on ice, and the fluorogenic probe Amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), which is sensitive to H2O2, was added and further incubated at 37°C for 60 min to generate H2O2 (through alkaline phosphatase hydrolysis of phosphocholine and choline oxidation by choline oxidase to generate betaine and H2O2). H2O2 in the presence of horseradish peroxidase reacts with Amplex red to generate the fluorescent resorufin. Each reaction mixture contained 5 µM Amplex red reagent, 1 U/ml horseradish peroxidase, 0.1 U/ml choline oxidase, and 4 U/ml alkaline phosphatase, as detailed by Zhou et al. (40). Fluorescence intensity was measured at excitation and emission wavelengths of 528 and 620 nm, respectively.

Senescence and apoptosis. Senescence-associated β-galactosidase (SA-β-gal) expressed in HUVECs was analyzed according to the protocol described by Dimri et al. (11). In brief, subconfluent HUVECs were stained in the working buffer (pH 6) at 37°C overnight. Stained cells were viewed under an inverted microscope at x200. The percentage of SA-β-gal-positive cells was determined by counting the number of blue cells under bright-field illumination and the total number of cells in the same field under phase contrast. At least eight random fields were counted for each culture dish. In addition, quantification of apoptotic cells was performed using Hoechst and annexin V. The data presented were obtained by counting the number of apoptotic cells per 500 cells using fluorescence microscopy. In each experiment at least 15–20 randomly chosen fields were examined.

Statistical analysis. All experiments were repeated at least three times. Values are given as means ± SE. ANOVA was used for multiple comparisons. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Autophagy is enhanced early and subverted in the later phase of exposure to GC. LC3, the first protein identified on the autophagosomal membrane, undergoes posttranslational modifications (37). The product of LC3 conversion, LC3-II, tightly associates with the autophagosomal membrane and migrates faster than LC3-I on SDS-PAGE. Consequently, immunoblotting of LC3 may detect two bands: LC3-I with an apparent mobility of 18 kDa and LC3-II (16 kDa). Because the amount of LC3-II correlates with the number of autophagosomes, immunoblot analysis of LC3 is an easy method to predict autophagic activity of mammalian cells (19). We utilized this property of LC3 to monitor the dynamics of the autophagic process in HUVECs subjected to GC. Exposure of HUVECs to GC resulted in an increase in LC3-II at 24 h (Fig. 1). By day 3, the LC3-II expression declined to the level seen in NC-treated cells and exhibited a further decline by 5 days in the GC-treated HUVECs.


Figure 1
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  		Fig. 1. Dynamics of autophagosomal formation in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to glycated collagen (GC) or native collagen I (NC) for the indicated times, and total cell lysates were harvested. Autophagosomal formation was detected with Western blot analysis with light-chain 3 (LC3) antibody (n = 3). Top band: LC3 I. Bottom band: LC3 II, a typical marker of autophagosomes. Equal protein loading was confirmed with β-actin antibody. Autophagosomal formation was robust during the early time points (24 h) of stimulation with GC (n = 3). Later time points (days 3–5) showed a decrease in formation of autophagosomes. C, control (PBS treatment); hd, high dose.

 
Immunocytochemistry showed the intracellular localization of LC3 in HUVECs. Untreated control cells or cells treated with NC showed a diffuse cytosolic distribution of LC3, consistent with the distribution of LC3-I. Within 24–72 h of treatment with GC, LC3 showed a delicate punctated pattern of distribution and was also localized to the plasma membrane (Fig. 2). Longer exposure to GC led to a decline in LC3 immunostaining.


Figure 2
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  		Fig. 2. Cellular distribution of autophagosomal marker LC3. HUVECs were grown on fibronectin-coated glass coverslips and incubated with 100 µg/ml NC (B, E, H) or GC (C, F, I) for the indicated periods of time. Control cells (A, D, G) were incubated with vehicle (PBS). After fixation and permeabilization, cells were stained with an LC3 antibody and Hoechst 33342 nuclear stain. A and B: after 24 h, control (A) and NC-treated (B) HUVECs show a homogeneous cytoplasmic staining typical of absent or low-level autophagosomal formation. In contrast, cells treated with GC (C) show early (24 h) and to a lesser extent after 72 h (F) a punctated pattern and membrane location of LC3. This punctuated pattern is typical for autophagosomal formation. The late time points (120-h incubation) again show a homogenous LC3 pattern, representing a decline of autophagy (I). Images c, f, and i show magnified areas of the corresponding cells. Calibration bar = 10 µm (I).

 
When autophagy is induced, autophagosomes fuse with lysosomes to form autolysosomes, the site of ultimate degradation of their cargo. Therefore, the colocalization of the lysosomal (Lamp-1) and autophagosomal (LC3) markers was examined to gain insight into this final step of autophagy. The distribution of LC3 and Lamp-1 showed a largely nonoverlapping pattern in control cells. HUVECs treated for 24 h with GC showed overlapping structures, indicative of the formation of autolysosomes (Fig. 3, A and B). By 72 h, however, these markers were dissociated, again exhibiting a nonoverlapping pattern, suggestive of a failed fusion of autophagosomes with lysosomes. By 5 days of GC treatment, giant confluent lysosomal structures marked by Lamp-1 and nonoverlapping LC3 pattern were consistently observed. This latter pattern was similar to the distribution of LC3 in cells with subverted last step of phagocytosis, i.e., fusion and lysosomal degradation (1).


Figure 3
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  		Fig. 3. Cellular distribution of autophagosomes and lysosomes. HUVECs were cultured on fibronectin-coated glass coverslips and incubated with NC or GC (100 µg/ml). After fixation and permeabilization, cells were triple stained with antibodies against LC3 (green) and Lamp-1 (red) and with Hoechst 33342 stain (blue). Top A: merged images. GC-treated endothelial cells show after 24 h overlapping fluorescence (yellow), indicative of the late step of autophagy (fusion of lysosomes and autophagosomes), and the formation of autolysosome (C, c). At 72 h in GC-treated group, the overlap is reduced (F, f) and after 120 h "giant" lysosomal structures become conspicuous, and the staining pattern representing autophagosomes disappears (I, i). Images c, f, and i represent magnified areas of the corresponding cell. Calibration bar = 10 µm (I). Bottom B: statistical evaluation of the merged images, showing the percentage of overlap between LC3 and Lamp-1 as an indicator of autolysosome formation. ld, Low dose. *P < 0.05.

 
Collectively, these data show that endothelial cells exhibited a biphasic response to GC: initial induction followed by the suppression of autophagy. Hence, we investigated the contribution of lysosomal- and lipid-mediated mechanisms to the observed dynamic changes in autophagy.

Ceramide in endothelial cells. Among the diverse mechanisms of lysosomal permeabilization, the prominent role is played by the detergent action of sphingosine (20), a metabolic product of ceramide. Ceramide has been shown to induce autophagy (30). Hannun and Obeid (17) provided strong evidence of participation of these products in cell death.

From these and other observations, we first analyzed sphingomyelinase activity. Measurements of the activity of the acid sphingomyelinase, a lysosomal enzyme responsible for the hydrolysis of sphingomyelin to ceramide (29), showed an increase after 24-h exposure of HUVECs to GC compared with that shown with NC exposure (Fig. 4). The measurement of neutral sphingomyelinase showed no detectable changes (data not shown).


Figure 4
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  		Fig. 4. Acid sphingomyelinase activity in HUVECs. After stimulation with NC + low-dose GC [1:3 mixture of GC and NC] or GC (100 µg/ml each) for 24 h, the enzymatic hydrolysis of sphingomyelin to ceramide and phosphocholine by sphingomyelinase was measured at pH 5.0 with the Amplex red reaction kit. Fluorescence intensity was measured at excitation and emission wavelengths of 528 and 620 nm, respectively. Values were normalized to NC-treated group; n = 3. *P < 0.05.

 
To localize any possible intracellular accumulation of the product of the increased sphingomyelinase activity (ceramide), HUVECs treated with NC or GC (from 3 h to 5 days) were stained an anti-ceramide antibody. Images obtained at the analogous exposure times indicated an increased expression of ceramide from 3 days up to 5 days of GC treatment (Fig. 5). This observation was further investigated with HPLC/MS.


Figure 5
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  		Fig. 5. Cellular distribution of ceramide. HUVECs were grown on fibronectin-coated glass coverslips and incubated with NC or GC (100 µg/ml) for different time points (3, 6, 16, 24, 48, 72, 96, and 120 h). After fixation, cells were stained with an antibody against ceramide. Images obtained at the analogous exposure time demonstrate a more robust expression of ceramide at 3–5 days of GC treatment.

 
The data showed that different concentrations of GC resulted in an increase in the cellular ceramide at all times, which was particularly pronounced for C18 and C20 ceramides (~2-fold) and to a lesser extent for C14 and C16 ceramides (~1.6-fold) (Fig. 6A). Sphingosine-1-phosphate was not increased in endothelial cells exposed to low-dose GC for 24–120 h. Higher concentrations of GC resulted in a 1.5-fold increase after 5 days compared with that shown in cells treated with NC (Fig. 6B).


Figure 6
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  		Fig. 6. Mass spectroscopy (MS) analysis of ceramides and sphingosines. Time courses of the concentration of C14, C16, C18, C18:1, C20, and C24, and C24:1 ceramide (A; Cer) and sphingosine-1-phosphate (Sph-1P) and sphingosine (Sph) (B) in the presence of NC, low-dose GC, and high-dose GC, measured by HPLC/MS, are shown. Cells from 4 separate experiments were pooled, and measurements were performed. Values were normalized to the levels of phospholipids extracted from tissue homogenate by the Bligh and Dryer extraction method (3) (Pi) in the same sample and expressed as pmol/nmol Pi.

 
GC affects lipid-rich domains in endothelial cells. The observed increase in the activity of acid sphingomyelinase, an enzyme responsible for hydrolysis of sphingomyelin to ceramide (29), prompted us to examine also the expression of lipid-rich domains, also known as membrane rafts, which are constitutively enriched in sphingomyelin and cholesterol. We made use of GPI-anchored ttGFP to examine the plasma membrane distribution and expression of lipid rafts in HUVECs. Previously, we confirmed the usefulness of GPI-anchored ttGFP beacon for localizing and monitoring lipid rafts in endothelial cells (26). HUVECs transfected with GPI-anchored ttGFP were treated with GC or NC (100 µmol/l each) for the indicated periods of time before fluorescence microscopy. In untreated cells, membrane rafts (measured as GPI-anchored ttGFP fluorescence) occupied on average 1.4 ± 0.018% of the cell surface. Treatment of HUVECs with NC for 1 h did not significantly change the surface area occupied by GPI-ttGFP clusters at the perimeter of the cell (1.4 ± 0.22%) (Fig. 7). In contrast, treatment of HUVECs with GC led within 1 h to a significant increase in the surface area occupied by lipid rafts (GC: 2.53 ± 0.28%, P < 0.01; 1:3 diluted GC: 2.6 ± 0.33%, P < 0.01). Longer exposure, for 3 h, also showed an increase in GPI clustering at the plasma membrane (P < 0.05, NC vs. GC). After 24 h, the differences disappeared or were inversed. After 24 h, "low-dose" GC treatment resulted in a decrease in the surface area occupied by lipid rafts compared with that shown with NC treatment (control: 1.88 ± 0.15%, NC: 2.52 ± 0.188%, low-dose GC: 1.29 ± 0.3%, GC for 24 h: 1.54 ± 0.2%; P < 0.05, NC vs. low-dose GC and GC). This decline in the surface area occupied by ttGFP-GPI could indicate the loss of membrane rafts, consistent with the concomitantly increased acid sphingomyelinase activity at 24-h exposure to GC.


Figure 7
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  		Fig. 7. Lipid raft modification by GC. Top A: glycosylphosphatidylinositol (GPI)-anchored thermotolerant green fluorescent protein (ttGFP) clusters in transfected HUVEC. A–C: endothelial cells incubated with NC for indicated times and costained with Hoechst 33342 show a nearly equal distribution of lipid rafts. a–c: Magnified areas of corresponding images. D–F: HUVECs incubated with a low-dose GC (1:3 GC). Note an increase in GPI-anchored ttGFP clusters at 1 and 3 h of treatment, followed by the later decrease. d–f: Magnified areas of corresponding images. G–I: incubation of HUVEC with GC for indicated times demonstrated the increase of lipid rafts in short-term and their later disappearance from the plasma membrane. g–i: Magnified areas of corresponding images. Calibration bar = 10 µm (G). Bottom B: area occupied by GPI-ttGFP clusters on the perimetric surface of transfected HUVECs. Data show a significant increase in the area occupied by GPI-ttGFP after application of GC at 1 and 3 h of incubation. This difference diminishes and inverts after 24 h. Values are means ± SE; experiments were performed 3 times, and 25 cells were analyzed for each treatment in each experiment. *P < 0.05.

 
Effects of inhibition and stimulation of autophagy on apoptosis and senescence of HUVECs. To gain insight into the role of autophagy in the development of cell senescence, we manipulated the process pharmacologically. A widely used inhibitor of autophagy at the initial sequestration step, 3-methyladenine (3-MA) (31), and an inducer of autophagy, tamoxifen (6), were applied to HUVECs cultured in the absence or presence of NC or GC. Under basal conditions, application of either 3-MA or tamoxifen had no detectable effects on the proportion of senescent cells (Fig. 8). When HUVECs were exposed to NC, tamoxifen-induced cell senescence increased about fourfold, whereas 3-MA affected senescence only marginally. In contrast, exposure of HUVECs to GC alone resulted in a greater than fivefold increase in the proportion of senescent cells, and the presence of 3-MA led to a significant attenuation of development of premature senescence. Addition of tamoxifen to GC-exposed HUVECs did not increase further the already high proportion of prematurely senescent HUVEC.


Figure 8
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  		Fig. 8. Effect of the inhibitor of autophagy, 3-methyladenine, and an inducer of autophagy, tamoxifen, on the rate of senescence and apoptosis in endothelial cells. HUVECs were cultured with NC or GC and treated with 3-methyladenine (10 mM) or tamoxifen (2 µM). A: senescence-associated β-galactosidase (SA-β-gal) staining was performed, and the proportion of stained cells was enumerated. B: proportion of annexin V-stained HUVEC. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The data presented herein demonstrate that endothelial cells subjected to the long-lived glycated extracellular matrix protein exhibit a biphasic autophagic response: an initial period of induction of autophagy followed by the inhibition of autophagosomal-lysosomal fusion (subverted autophagy). Although the initial response coincides with the enhanced apoptosis, the latter occurs concomitantly with development of cell senescence. Moreover, inhibition of autophagy led to attenuation of senescence, but it increased the rate of apoptosis. Furthermore, HUVECs exposed to GC showed elevated levels of ceramide, whereas the sphingosine-1-phosphate level was found to be elevated only at later times, when cells developed premature senescence. The finding of elevated levels of ceramide is consistent with the observed increase in the activity of acid sphingomyelinase, which is responsible for the hydrolysis of sphingomyelin to ceramide (29). A mechanism of lysosomal permeabilization involving ceramide and its product sphingosine has been proposed, which is based on the ability of the exogenous ceramide to induce reversible cell cycle arrest and appearance of SA-β-gal (25). Elevated activity of acid sphingomyelinase appeared to be responsible for the reduction in the surface area occupied by lipid rafts in GC-treated HUVECs. Hence, all of these findings appear to represent a mechanistic molecular cascade whereby GC induces acid sphingomyelinase activity and enhances degradation of sphingomyelin, accumulation of ceramide, and reduction in the surface area occupied by lipid rafts.

Recent studies have placed sphingolipids, including ceramide, sphingosine, and sphingosine-1-phosphate, at the center stage of biological processes specifically related to signal transduction and determination of cell fate, in which their levels change in a highly regulated temporal and spatial manner (14). Possessing opposite actions on cell death and survival, ceramide and sphingosine-1-phosphate represent the key players of the concept of "spingolipid rheostat (sphingostat)" as a determinant of cell fate (9). Our findings are consistent with this concept: proapoptotic action of elevated ceramide when it is unopposed by sphingosine-1-phosphate, marking the early response to GC. Furthermore, our data suggest that the anti-apoptotic action of sphingosine-1-phosphate, in conjunction with the elevated level of ceramide, could result in cell senescence, marking the later response to GC.

Indeed, the dynamics of HUVEC responses to GC is of note. Initially, exposure of endothelial cells to GC leads to an enhanced rate of autophagy. Autophagy has emerged as an essential function for cell homeostasis, cell defense, and adaptation to adverse environments (24). A protein known to be involved in autophagy is the processed form of microtubule-associated protein 1 LC3 (36). Although autophagy promotes a cell survival response, morphological features of autophagy have also been observed in dying cells, where caspases are suppressed or not sufficiently activated (32). Recently, it was described that autophagy-related gene 5 product, required for the formation of autophagosomes in addition to promoting autophagy, enhances the susceptibility to apoptotic stimuli (38). Thus a switch from apoptosis to autophagic cell death can occur, and, on the other hand, autophagy could promote apoptosis. In addition, recent findings point out that accumulation of autophagic vacuoles can precede apoptotic cell death, thus arguing against a clear-cut distinction between apoptotic and autophagic cell death (15).

More protracted exposure to GC resulted in a decreased rate of autophagy coincident with elevation of SA-β-gal and development of cell senescence. An age-related increase in oxidation of lipids or proteins within the lysosomal membrane has been linked to the increased fragility and reduced fusion of lysosomes with autophagic vacuoles and/or reduced levels of Lamp-2a in the lysosomal membrane (8). In addition, oxygen-free radical damage may inhibit macroautophagy and in turn enhance oxygen radical-induced damage. Inhibition of autophagy in starving cells activates apoptosis (4). In some cell types, where crucial apoptosis regulators are either lacking or inhibited, autophagic cell death compensates for apoptosis (32). This may predispose to the acquisition of a senescent phenotype.

The action of ceramide and its product sphingosine, lysosomal membrane detergent, on lysosomal permeability may have similarly important implications for the functioning of autophagic machinery. Considering the obligatory role of autophagosome-lysosome fusion and the subsequent degradation by lysosomal enzymes, the lysosomal leakiness may represent an obstacle to efficient culmination of autophagy. This particular scenario, perhaps, is unveiled at the later stage of exposure to GC. Such a tentative conclusion is supported by the finding of reduced levels of LC3 and nonfusion of lysosomes and phagosomes, consistent with subversion of autophagy. A similar process occurs during infection of human cells by RNA viruses (18). Could this subversion be conducive to the development of premature senescence? To address this question, we inhibited or stimulated autophagy while exposing HUVECs to GC. Only by inhibiting autophagy was it possible to reduce the number of senescent cells. However, this was accompanied by the reciprocal increase in the proportion of apoptotic HUVECs.

Integration of present findings with our previous observations on the mechanisms and time course of premature senescence and apoptosis of HUVEC subjected to long-lived glycated protein is depicted in Fig. 9. Endothelial cells succumb to apoptosis, with peak rates at 16 h, or premature senescence, peaking at 3–5 days (27). The earliest detectable event on exposure to GC was an increase in reactive oxygen species, followed by lysosomal permeabilization with collapse of lysosomal pH. In our experimental system of premature senescence, lysosomal changes predated mitochondrial damage, similar to observations by Brunk and colleagues (35) made in replicative senescence. The loss of acidic pH optimum of lysosomes resulted in the accumulation of uncleaved products, such as gangliosides (22). The levels of several gangliosides [GM3 (C16/C18), GD1b, and GT1b] were increased in HUVECs subjected to GC. Although senescence occurred in synchrony with the accumulation of nondegraded gangliosides, the latter did not appear to be causatively involved in its development.


Figure 9
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  		Fig. 9. Schematic integration of cellular events associated with apoptosis, autophagy, and premature senescence. Incubation of endothelial cells with AGE-modified collagen I culminates in 1 of 3 outcomes: an early peak of apoptosis (16 h), antedated by the production of reactive oxygen species (ROS), lysosomal pH collapse and lysomal permeabilization, and mitochondrial membrane depolarization. From day 3 onward, endothelial cells become senescent. In this late phase of incubation with GC, an increase in ganglioside content of the cell is observed (27). LC3, a marker of autophagy, is overexpressed within 24 h of GC treatment, declined by 3 days, and was nearly undetectable on day 5. Formation of autophagosomes was induced early but later became subverted because of the failure of fusion with lysosomes. Clustering of membrane rafts occurred shortly after exposure to GC, followed later (after 24 h) by their internalization accompanied by an increased acid sphingomyelinase (ASM) activity and accumulation of ceramide. d, Days.

 
There is an on-going debate on the relationship between autophagy and apoptotic cell death, which, however, did not take into the account yet another form of cellular response to stressors: premature senescence. Under experimental conditions described here, the existence of the seesaw relationship between premature senescence and apoptosis becomes apparent. The data also suggest a novel function for autophagy: that of a cellular switch between premature senescence and apoptosis.


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 MATERIALS AND METHODS
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These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54602 and DK-45462 (M. S. Goligorsky) and an American Heart Association grant (to J. Chen). The HPLC/MS measurements of ceramide and sphingosine-1-phosphate were conducted in a facility constructed with support from Grant C06 RR-018823 from the Extramural Research Facilities Program of the National Center for Research Resources.


   ACKNOWLEDGMENTS
 
J. Cheng was a PhD student.

We thank Dr. Dino A. DeAngelis for providing the ttGFP-GPI construct. The Lamp-1 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA 52242).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Patschan or M. S. Goligorsky, Depts. of Medicine and Pharmacology, New York Medical College, Valhalla, NY 10595 (e-mail: S.Patschan{at}gmail.com or Michael_goligorsky{at}nymc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This paper was presented at the 9th Cardiovascular-Kidney Interactions in Health and Disease Meeting at Amelia Island Plantation, Florida, on May 26–29, 2006. Back


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