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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1104-1111.)
© 1996 American Heart Association, Inc.

Articles

Human Endothelial Cells Exposed to Oxidized LDL Express hsp70 Only When Proliferating

Weimin Zhu; Paola Roma; Angela Pirillo; Fabio Pellegatta; Alberico Luigi Catapano

the Institute of Pharmacological Sciences, University of Milano (W.Z., P.R., A.P., A.L.C.), the Centro per lo Studio delle Vasculopatie Periferiche, Ospedale Bassini (A.L.C.), and the Cardiovascular Physiopathology Laboratory, Istituto Scientifico H. S. Raffaele (F.P.), Milano, Italy.

Correspondence to Prof Alberico L. Catapano, Institute of Pharmacological Sciences, Via Balzaretti, 9, 20133 Milano, Italy.

	Abstract

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Oxidized LDL (OxLDL), a causal factor in atherosclerosis, is cytotoxic and triggers the expression of various heat shock proteins (hsps), among which is hsp70, in cultured animal and human cells. hsps constitutively act as molecular chaperones and in situations of stress protect other cellular proteins from potential denaturation caused by cytotoxic stimuli. The sensitivity of endothelial cells to OxLDL toxicity and accordingly the level of hsp70 expression depend on cell density. While confluent cells were relatively resistant to OxLDL toxicity and were not induced to express hsp70 when challenged with the lipoprotein (up to 800 µg/mL), sparse cells exhibited a concentration- and time-dependent expression of inducible hsp70, which increased up to fivefold to sixfold in unchallenged cells. Neither the activity of receptors recognizing OxLDL nor potentially protective cell products affected the stress response. Rather, we demonstrated that cell proliferation, which is high for sparse cultures and wound-healing monolayers, is responsible for these observations. We also demonstrated that the lipid moiety of OxLDL essentially accounts for the hsp-inducing effect of the lipoprotein. OxLDL has been detected in atherosclerotic lesions, which also show an increase of immunoreactive hsp72/73. We speculate that, in vivo, rapidly growing cells, such as those of lesion-prone areas, are more sensitive to the toxicity of OxLDL than are quiescent cells and that an increased expression of hsp70 may allow proliferating cells an increased chance of survival.

Key Words: cytotoxicity • proliferation • stress response

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Oxidized LDL possesses several proatherogenic properties and is considered a key factor in the etiology of atherosclerosis.^{1 2} OxLDL specifically interacts with several receptors,^{3 4 5} leading to the engorgement of cells with lipids; inhibits endothelium-dependent vascular relaxation;⁶ is cytotoxic to proliferating cells;^{7 8} stimulates chemoattractant secretion;⁹ and induces the expression of adhesion molecules that mediate the interaction of leukocytes with the endothelium.^{10 11} It was recently reported that OxLDL also triggers in vitro the expression of hsps, namely hsp23 and hsp32 (or heme oxygenase) in mouse peritoneal macrophages¹² and the inducible form of hsp70 in human endothelial¹³ and smooth muscle cells.¹⁴ Also called stress proteins,^{15 16} hsps are a family of polypeptides serving the role of molecular "chaperones."^{17 18} In physiological conditions, this role is accomplished by the constitutive (or cognate) proteins, which assist in the correct folding¹⁸ and specific subcellular localization of newly synthesized proteins.¹⁹ In the presence of toxic stimuli, the cellular pool of hsps expands through de novo synthesis of the inducible forms, which protect other polypeptides from irreversible denaturation. The cytoprotective role of stress proteins is relevant to several pathological conditions,²⁰ including inflammation²¹ and ischemia/reperfusion,^{22 23} both of which may be linked to atherosclerosis.

Oxidatively modified LDL exists in vivo and has been detected in blood vessel walls at sites of atherosclerotic lesions^{1 24} ; here focal changes in the expression of hsp70, with increased presence of immunoreactive hsp72/73 in the most involved areas, have also been described.^{25 26}

We previously observed^{13 14} that the degree of confluence affects the sensitivity of endothelial and smooth muscle cells to OxLDL cytotoxicity and accordingly the expression of hsp70. With the present studies, we aimed at understanding which factors modulate hsp70 expression by analyzing the effect of different experimental conditions in sparse endothelial cells and by directly comparing the response of cocultured sparse and confluent cells to a challenge with OxLDL.

	Methods

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Cells
HUVECs were isolated according to established procedures²⁷ and cultured under standard conditions in M-199, with the addition of heparin (15 U/mL) and with or without ECGF (20 µg/mL, Boehringer Mannheim). HUVECs were routinely used at the third or fourth passage. Because of limited availability of these cells, experiments that required a conspicuous amount of cells (for example, uptake and degradation studies) were performed with the EAhy-926 line,²⁸ which similarly to HUVECs is induced to express hsp70 in response to OxLDL.¹³ The EAhy-926 line was cultured under standard conditions in MEM+10% FCS and 1% HAT. We also tested the response of the parallel experiments performed with both sparse cultures and postconfluent cultures, in which cells acquire a typical "cobblestone" morphology. Culture dishes, or glass coverslips for immunocytochemistry, were usually coated with gelatin (1%); alternatively, to test the effect of different extracellular matrix components as culture substrates on the expression of hsp70, cells were grown on coverslips coated with fibronectin (10 µg/mL) or polylysine (10 µg/mL), on uncoated coverslips, or on uncoated slide-shaped plastic wells (Lab-Tek Chamber Slide).

Lipoproteins
LDL (1.019 to 1.063 g/mL) was isolated from human plasma by preparative ultracentrifugation²⁹ and dialyzed in 0.15 mol/L NaCl, 0.01% EDTA. Just before oxidation, EDTA was removed by gel filtration in PBS on a Sephadex G-25 column (PD-10, Pharmacia). Protein was measured by the method of Lowry et al.³⁰ LDL (200 µg lipoprotein protein per milliliter) was oxidized in sterile conditions for 24 hours at 37°C in PBS containing CuSO₄ (20 µmol/L), concentrated by ultrafiltration under N₂ pressure in an Amicon ultrafiltration apparatus fitted with a YM 100 membrane (American Corporation), desalted in PBS on Sephadex G-25 columns (PD-10), and sterile filtered. LDL modification was assessed by nondenaturing gel electrophoresis in 0.8% agarose (Agarose A, Pharmacia) in 0.1 mol/L Tris, pH 8.6, at 200 V.³¹ According to the literature, electrophoretic mobility increased after oxidation and was slightly lower than that of acetylated LDL.

To test the influence of the interaction between receptors and protein ligands on hsp70 expression, aliquots of medium containing LDL or OxLDL were boiled for 15 minutes in sterile conditions. Lipids were extracted from LDL and OxLDL with chloroform/methanol 2:1 (vol/vol).³² Extracts were evaporated to dryness under an N₂ stream, and lipids were redissolved in absolute ethanol. The solution was filtered through a 0.45-µm pore filter to decrease turbidity, and aliquots were added to the incubation medium. The volume of ethanol never exceeded 1% of total medium volume. Control medium containing 1% ethanol did not affect cell viability or hsp70 expression.

Cell Viability
Cell viability was assessed by the release of [³H]adenine into the culture medium.³³ Cells were labeled for 1 hour with [³H]adenine (1 µCi/mL medium), washed twice, and incubated for 24 hours at 37°C in medium containing LDL or OxLDL (200 µg/mL). The medium was collected and the cells were dissolved in 1 N NaOH. Aliquots of the medium and of solubilized cells were counted and percent [³H]adenine release was expressed as the ratio of medium radioactivity to medium plus cell radioactivity.

[³H]Thymidine Labeling
Cells were washed with PBS and incubated for 1 hour in MEM containing [³H]thymidine (2 µCi/mL). Cells were then washed three times in PBS, incubated with 5% trichloroacetic acid (10 minutes), and washed twice in 5% trichloroacetic acid; 1 mL of 1 N NaOH was added to each well. Aliquots were used to measure protein and radioactivity content.

Uptake and Degradation of Iodinated Lipoproteins
Lipoproteins were labeled with [¹²⁵I]NaI according to Bilheimer et al,³⁴ desalted by gel filtration on Sephadex G-25 eluted with PBS, and sterile filtered. Specific activity was 200 to 400 cpm/ng lipoprotein protein. EAhy-926 cells were incubated for 6 hours at 37°C in MEM containing essential fatty acid–free BSA (2 mg/mL) and ¹²⁵I-OxLDL or in the same medium containing, in addition to ¹²⁵I-OxLDL, an excess (30-fold) of unlabeled lipoprotein. Specific uptake was calculated as the difference between uptake by cells incubated without and with unlabeled OxLDL.³⁵ Specific degradation was calculated as the difference between degradation of ¹²⁵I-OxLDL incubated in the presence and absence of cells.³⁵

Uptake was also measured in cells incubated with ¹²⁵I-OxLDL in the presence of fucoidin (20 µg/mL), which binds to scavenger receptors with high affinity.

Immunocytochemistry
Sparse or confluent cultures were incubated with OxLDL (400 µg/mL) for different time periods (up to 24 hours) or with different concentrations (200 to 800 µg/mL) of the modified lipoprotein. Cells were fixed in 3% paraformaldehyde in 150 mmol/L NaCl, 10 mmol/L Tris, and 2% sucrose, pH 7.5 (3% paraformaldehyde solution) (15 minutes at room temperature) and processed for immunostaining as described previously.¹³ Antibodies used were (1) mouse monoclonal antibody specific for hsp72, the inducible form of hsp70 (C92F3A-5, Stressgen, 1:200), followed by biotinylated anti-mouse IgG (Amersham, 1:250) and fluorescein-conjugated streptavidin (Amersham, 1:200); (2) mouse monoclonal antibody against BrdU (BUI/75-ICR 1, Sera-Lab, 1:50), followed by rhodamine-conjugated anti-mouse IgG (Amersham, 1:200); (3) mouse monoclonal antibody against OxLDL, obtained in our laboratory, followed by biotinylated anti-mouse IgG (1:100) and fluorescein-conjugated streptavidin (1:200). Stain specificity was assessed by omitting either the first or second antibody. For double labeling, the following steps were performed after staining for hsp70: a wash in PBST, fixation in 0.5% paraformaldehyde solution (30 minutes at 4°C and 30 minutes at room temperature), a wash in PBST, incubation in 2 N HCl+0.5% Tween 20 (10 minutes at 37°C), a wash in 0.1 mol/L Na₂B₄O₇ (pH 8.5), and a wash with PBST. Staining for BrdU was then performed. Coverslips were mounted on microscopy slides with glycerol/PBS 1:1 (vol/vol), examined with a Zeiss Axioscop fluorescence microscope (filter sets: BP 450-490, FT 510, LP 520 for fluorescein; BP 546, FT 580, LP 590 for rhodamine), and photographed using 400 ASA Diachrome film.

The expression of hsp70 was also assessed in cells incubated with OxLDL (400 µg/mL) in the presence of fucoidin (20 µg/mL) or of chloroquine (50 µmol/L), as well as in cells incubated with total lipids extracted from LDL or OxLDL.

To evaluate, within the same culture plate, the response of sparse and confluent cells to a challenge with OxLDL, a wounding experiment was performed. HUVECs were cultured to a postconfluent state, as judged on the basis of their cobblestone morphology; the monolayer was then wounded with a polytetrafluoroethylene cell lifter and the cells were allowed to recover for 24 hours in M-199 containing FCS (20%). After recovery, BrdU (10 µmol/L final concentration) was added to the culture medium for a further incubation of 6 hours. The medium was then replaced by M-199 containing OxLDL (400 to 600 µg/mL) for a period of 12 hours. At the end of the incubation, cells were processed for immunostaining of hsp70 and BrdU.³⁶

Immunoblotting
Sparse or confluent cultures were incubated with OxLDL for different time periods (up to 24 hours) or with different concentrations (200 to 800 µg/mL) of the modified lipoprotein.

Cells were lysed in Tris-glycine buffer (0.25 mol/L Tris, 0.173 mol/L glycine, pH 8.5) containing SDS (3%), and protein was measured by the method of Lowry et al.³⁰ Equal amounts of protein from the different samples were subjected to SDS-PAGE on a 10% polyacrylamide gel,³⁷ after the addition of ß-mercaptoethanol (2%), glycerol, and bromophenol blue. Electrophoresed proteins were transferred onto a nitrocellulose membrane using a Trans Blot Cell (Bio-Rad).³⁸ The membrane was incubated with a 1:1500 dilution of anti-hsp70 antibody (see above), then with a 1:2000 dilution of peroxidase-conjugated goat anti-mouse IgG (Amersham). Immunocomplexes were detected by an enhanced chemiluminescence method (ECL, Amersham), followed by autoradiography. Immunocomplexes were quantified by the Image program (ISF 1.47). Data were expressed as area units.

Metabolic Labeling
Sparse cells were incubated in M-199 containing OxLDL (600 µg/mL and 800 µg/mL) for 6 hours or heat shocked at 45°C for 15 minutes and allowed to recover for 2 hours in fresh M-199 containing 20% FCS. During the last hour of incubation a [³⁵S]protein-labeling mixture (EXPRE³⁵S Protein Mix, Du Pont-NEN, >1000 Ci/mmol, containing >77% L[-³⁵S]methionine and 18% L-[³⁵S]cysteine) was added to the medium (50 µCi/mL). Cells were washed three times in PBS and lysed in sample buffer (3% SDS, 10% ß-mercaptoethanol, 2% glycerol, and bromophenol blue in Tris-glycine buffer, pH 8.5). The content of radioactivity was evaluated in a beta counter and equal numbers of counts were loaded onto a 10% polyacrylamide gel for SDS-PAGE. Gels were dried and processed for autoradiography.

	Results

Top Abstract Introduction Methods Results Discussion References

 
In previous experiments, we observed that incubation in the presence of OxLDL is a stress capable of inducing the expression of hsp70 in sparse endothelial cells but not in confluent cells.¹³ We also observed that this induction takes place together with the cytotoxic effect of OxLDL, which is evident with sparse cells, while confluent monolayers are relatively resistant.¹³ Cytotoxicity experiments performed with the preparations of OxLDL used in the present studies gave results in agreement with those previously reported¹³ (data not shown). To clarify the issue of the relation between cell density and hsp70 expression, we performed time-course and concentration-course experiments. As indicated by immunoblotting results, OxLDL induced the expression of hsp70 in sparse endothelial cells in a concentration- and time-dependent manner (Fig 1A and 1B). Similar results were obtained by immunocytochemistry (data not shown). Immunostaining for hsp70 was negative in cells treated with cycloheximide (not shown), indicating that protein synthesis is necessary for the stress response. In fact, metabolic labeling with [³⁵S]amino acids indicated that a challenge with OxLDL increased the synthesis of hsp70 (Fig 2).

View larger version (22K): [in this window] [in a new window]   Figure 1. A, Sparse () and confluent () cultures of HUVECs were incubated for 12 hours at 37°C in M-199 containing the indicated concentrations of OxLDL. At the end of the incubation, cellular proteins were subjected to SDS-PAGE in 10% gels. Immunoblotting with anti-hsp70 antibody was then performed. Bands relative to hsp70 were analyzed by densitometry, and the intensity of hsp70 staining was plotted as area units (AU). B, Sparse () and confluent () cultures of HUVECs were incubated at 37°C in M-199 containing OxLDL (400 µg/mL) for the indicated times. Cells were then processed as above.

View larger version (115K): [in this window] [in a new window]   Figure 2. Sparse cultures of HUVECs were incubated at 37°C in M-199 containing OxLDL (600 µg/mL and 800 µg/mL) for 6 hours or heat-shocked at 45°C for 15 minutes and allowed to recover for 2 hours in fresh M-199 containing 20% FCS. During the last hour of incubation a [35S]protein-labeling mixture was added to the medium (50 µCi/mL). Cells were washed three times with PBS and lysed in sample buffer. Equal numbers of counts were loaded onto a 10% polyacrylamide gel for SDS-PAGE. Gels were dried and processed for autoradiography. Lane 1, control; 2, heat shock; 3, OxLDL (600 µg/mL); and 4, OxLDL (800 µg/mL).

Different scavenger receptors mediate the specific binding and internalization of OxLDL. We therefore speculated that if the response elicited by OxLDL required the internalization of the lipoprotein, different degrees of expression of scavenger receptors in sparse and confluent cells might explain our findings; however, specific uptake and degradation of ¹²⁵I-OxLDL by sparse EAhy-926 cells were similar to those measured with confluent cells per milligram of cell protein (Fig 3A and 3B). These data agreed with the results of immunostaining with anti-OxLDL, which showed similar amounts of internalized OxLDL in sparse and confluent HUVECs (not shown). The role of receptor-mediated internalization and degradation of OxLDL in the stress response activated by this lipoprotein (Fig 4; compare panels a and b for the effects of OxLDL and LDL) was assessed by immunofluorescence. Fucoidin did not hamper the expression of hsp70 triggered by these lipoproteins (panel c), although it competed with OxLDL for uptake via scavenger receptors. In fact, in the presence of fucoidin (20 µg/mL), uptake of ¹²⁵I-OxLDL decreased from 322±22 to 144±10 ng/mg cell protein in sparse EAhy-926 cells and from 342±36 to 98±8 ng/mg cell protein in confluent cells (mean±SD of triplicate experiments). Fucoidin alone did not trigger hsp70 expression (panel d). Similarly, blocking lysosomal degradation of OxLDL with chloroquine did not prevent hsp70 expression (panel e), while chloroquine alone did not induce its expression (panel f). Heat-denatured OxLDL, which lost its ability to interact with receptors, still induced hsp70 expression (panel g), while heat-denatured LDL was ineffective (panel h). Lipids extracted from OxLDL strongly induced hsp70 expression (panel i), while lipids extracted from native LDL did not (panel j). All together, these data support the conclusion that the activity of scavenger receptors and lipoprotein internalization and degradation are not determinants of the induction of hsp70 expression.

View larger version (17K): [in this window] [in a new window]   Figure 3. Sparse () and confluent () cultures of EAhy-926 cells were incubated for 6 hours at 37°C in MEM containing essential fatty acid–free BSA (2 mg/mL) and the indicated concentrations of 125I-OxLDL, with or without a 30-fold excess of unlabeled OxLDL. Specific uptake (A) and degradation (B) were evaluated (see "Methods").

View larger version (72K): [in this window] [in a new window]   Figure 4. Sparse cultures of HUVECs were incubated for 12 hours at 37°C in M-199 containing a, OxLDL (400 µg/mL); b, LDL (400 µg/mL); c, OxLDL (400 µg/mL) plus fucoidin (20 µg/mL); d, fucoidin alone (20 µg/mL); e, OxLDL (400 µg/mL) plus chloroquine (50 µmol/L); f, chloroquine alone (50 µmol/L); g, heat-denatured OxLDL (400 µg/mL); h, heat-denatured LDL; i, lipids extracted from 1 mg OxLDL; and j, lipids extracted from 1 mg LDL. At the end of the incubation, cells were processed for immunostaining of hsp70. Magnification x400.

Since the degree of confluence does not affect the expression of OxLDL receptors and their activity is not critical for the stress response, we wondered what factor peculiar to confluent cells might render them relatively resistant to OxLDL. We therefore tested the possibility that endothelial cells secrete factors protecting against OxLDL toxicity: in such case, only a large number of juxtaposed cells could potentially yield an effective concentration of cytoprotective agents. Sparse HUVECs were incubated with OxLDL (200 µg/mL) in conditioned media collected from sparse and confluent cultures. Because hsp70 expression was induced in both conditions in HUVECs (Fig 5, panels a and b) as well as in the EAhy-926 line (panels c and d), we concluded that the relative resistance of confluent cells to OxLDL does not depend on reaching a threshold concentration of a putative cytoprotective factor.

View larger version (93K): [in this window] [in a new window]   Figure 5. Panels a through d, Sparse cultures of HUVECs (a and b) and EAhy-926 cells (c and d) were incubated for 12 hours at 37°C in conditioned medium obtained from sparse (a and c) and confluent (b and d) cultures containing OxLDL (400 µg/mL). At the end of the incubation, cells were processed for immunostaining of hsp70. Magnification x400. Panels e through h, Sparse cultures of HUVECs grown on coverslips coated with polylysine (e), fibronectin (f), or gelatin (g), or on plastic slides (h) were incubated for 12 hours at 37°C in M-199 containing OxLDL (400 µg/mL). Cells were then processed for immunostaining of hsp70. Magnification x400.

Besides soluble factors, cells produce insoluble extracellular matrix components, which can influence their response to stress.³⁹ Cells sparsely grown on different culture substrates, such as polylysine, fibronectin, or gelatin, or on uncoated culture dishes were equally sensitive to OxLDL (Fig 5, panels e through h); therefore, neither the type nor amount of extracellular matrix, presumably more abundant in confluent cultures, can explain the relative resistance to OxLDL.

One major difference between sparse and confluent cells may be that the former are proliferating (ie, proceeding through the cell cycle) or migrating on the culture surface, while the latter are in a relatively quiescent state. Indeed, in our culture conditions, the incorporation of [³H]thymidine by sparse cells was usually more than six times that measured in confluent cells (not shown). We therefore designed an experiment that allowed us to compare, within the same culture plate, the response of proliferating/migrating and quiescent cells to a challenge with OxLDL. After wounding a confluent monolayer, we allowed enough time for the cells closest to the lesion to start healing it by proliferating and/or migrating. Cells were then incubated with a cytotoxic concentration of OxLDL known to markedly inhibit proliferation and induce hsp70 expression. Fig 6 depicts the immunostaining results. Cells located on the edge of the wound expressed hsp70, while those not involved in the repair process displayed a faint background staining (panel a). When BrdU, a thymidine analogue, was used to label cycling cells during wound repair, the majority of cells were positive for both hsp70 (panel b) and BrdU (panel c); this indicated that cells on the edge of the injured area expressed hsp70 (panels b and d) on challenge with OxLDL and were actively cycling (panels c and d). In fact, since recovery took place in serum-containing medium, healing of the wound monolayer was most likely due to cell proliferation. Immunostaining with anti–proliferating cell nuclear antigen (a marker of cell growth) showed a strong nuclear staining in cells on the edge of the injury, indicating that the majority of them were actively proliferating (not shown). Immunostaining of a wounded monolayer that was allowed to recover and then incubated in complete medium without OxLDL was negative, indicating that injury and recovery per se were not able to induce the expression of hsp70 in cells on the edge of the lesion.

View larger version (81K): [in this window] [in a new window]   Figure 6. HUVECs were cultured to a postconfluent state (judged by their cobblestone morphology). The monolayer was wounded by means of a polytetrafluoroethylene spatula, and cells were allowed to recover for 24 hours in M-199+20% FCS. After recovery, BrdU (10 µmol/L final concentration) was added to the culture medium for a further incubation of 6 hours. The medium was then replaced with M-199 containing OxLDL (600 µg/mL) for an incubation of 12 hours. Cells were then processed for immunostaining of hsp70 (a, b, and d) or BrdU (c and d). Magnification x100 (a, b, c) and x400 (d).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
OxLDL is thought to have a causal role in atherosclerosis, since it affects several cellular functions in a proatherogenic way.^{1 2} Because of the high concentration of antioxidants in plasma, oxidation of LDL can take place only in the subendothelial space^{1 2} ; therefore, cells of the vessel wall are the most likely target of OxLDL toxicity. The effect of sublethal cytotoxic stimuli is often counteracted by the synthesis of hsps, which protect other cellular proteins from irreversible denaturation.^{15 16} We recently reported that OxLDL triggers the expression of the inducible form of hsp70 in cultured endothelial and smooth muscle cells.^{13 14} The present study has shown that, in endothelial cells, the expression of the inducible form of hsp70, which is concomitant with the cytotoxic effect of OxLDL,¹³ depends on the time of exposure to the lipoprotein and on its concentration in the culture medium. As previously observed,^{13 14} cell density critically influences the stress response; in fact, induction of hsp70 occurred only in sparse cells. Results obtained with both the EAhy-926 cell line (not shown) and human umbilical vein smooth muscle cells¹⁴ suggest that the observed response is a general feature of cell response to OxLDL. Our data indicate that the activity of the scavenger receptors does not influence the expression of hsp70, suggesting that internalization and degradation of the lipoprotein are not necessary for it. Furthermore, neither soluble cell products nor constituents of the extracellular matrix are determinant for inducing the stress response, although the latter are known to modulate protein synthesis.³⁹ Rather, lipid components of OxLDL are responsible for the observed effect. Since lipids extracted from OxLDL contain several oxysterols,^{40 41} many of which are cytotoxic,^{42 43} we evaluated the role of these cholesterol derivatives in triggering the stress response. Yet none of the oxysterols affected hsp70 expression (data not shown), and further work is in progress to identify the lipid(s) responsible for hsp70 synthesis. We are presently focusing on more hydrophilic components of OxLDL, resulting from oxidation of fatty acids, as potentially responsible for the activation of the stress response. Interestingly, it has been reported that 4-hydroxynonenal, also a product of fatty acid oxidation, induces heat shock factor.⁴⁴

The expression of hsp70 mostly depended on whether cells exposed to OxLDL were in a postconfluent or in a nonconfluent state; that is, whether they were quiescent or proceeding through the cell cycle. This was clear when a monolayer of endothelial cells displaying the cobblestone morphology was mechanically wounded and then allowed to recover. Only the cells at the edge of the wound, which were proliferating and most probably responsible for healing the lesion, expressed hsp70 on incubation with OxLDL. Omission of ECGF from incubation medium did not affect the expression of hsp70 in injured cells exposed to OxLDL, suggesting that hsp70 induction by OxLDL is not dependent on the presence of growth factors (not shown). The noncomplete colocalization of induced hsp70 and of BrdU (see "Results") may indicate the presence of migrating cells or of a small number of cells that, although proliferating, did not incorporate BrdU. In fact, we cannot rule out the possibility that the time allowed for BrdU incorporation was not sufficient to label every cycling cell. Since not only proliferating but also migrating cells may be involved in wound repair, we cannot exclude the possibility that the latter are also more sensitive than stationary cells of a contact-inhibited monolayer to the toxicity of OxLDL. However, the vast majority of the cells expressing hsp70 were also labeled with BrdU, suggesting a close relation between cell cycle and hsp70 expression driven by OxLDL. Cell-cycle dependence of hsp70 expression has been observed in heat-shocked Chinese hamster ovary cells.⁴⁵ It appears that the induction of hsp72 is essential for the accomplishment of the early S phase and is also necessary in G1.⁴⁵ Furthermore, OxLDL toxicity to fibroblasts appears to be selective for the S phase of the cell cycle.⁸

Regulation of endothelial cell proliferation in vivo is tightly controlled. Under normal conditions, these cells are quiescent and form an antithrombogenic monolayer. Different types of injury to the endothelial monolayer can result in endothelial activation or denudation and in adhesion of platelets to the subendothelium. Both the activated endothelium and the subendothelium may provide the source of growth factors for cell proliferation and rapid healing of the injured site.⁴⁶ Lesion-prone areas of the vasculature are characterized by enhanced endothelial regeneration, probably reflecting repeated injury to the intima.⁴⁷ These areas also show an increased permeability, which allows the diffusion of plasma lipoproteins into the subendothelial space and their subsequent oxidation. Therefore, in vivo, the risk of being challenged by OxLDL is higher for cells healing an injured vessel wall than for cells of unaffected areas. On the basis of the results obtained in vitro we speculate that, besides being disruptive for the structure and functionality of the arterial tissue, the cytotoxicity of OxLDL might also trigger the synthesis of hsps in the cells that are most sensitive to their toxicity. The stress response (namely, the expression of the inducible form of hsp70) may represent a defense mechanism against cytotoxic proatherogenic stimuli. Cytoprotection afforded by stress proteins may allow these healing cells a higher chance of surviving further cytotoxic challenges by factors other than the presence of oxidized lipoproteins. In step with the cytoprotective role of hsp70 are the observations that overexpression of hsp70 in transfected cells and in transgenic animals are associated with improved postischemic performance.^{22 48} Also, overexpression of hsp70 has been shown to increase the threshold for the release of stress mediators (IL-1, IL-6) induced by UV light and oxidants.⁴⁹ It is of interest, however, that the induction of hsp70 expression in hypertensive animals presents a form of tolerance; ie, chronic hypertension does not induce hsp70 expression, while an acute increase of blood pressure does.⁵⁰ If this is a general mechanism, it can be speculated that at early stages of atherosclerosis, hsp expression allows cells to cope with oxidative damage, while repeated oxidative insults will result in a lower ability to cope with them and eventually lead to cell death. Sustained hsp70 expression may then result in protection from chronic damage, and work is in progress to address this issue in cells overexpressing inducible hsp70.

In summary, we have demonstrated that OxLDL can trigger the expression of hsp70 (inducible form) only in nonconfluent endothelial cells and that cell proliferation is responsible for this expression. Whether this effect has in vivo relevance remains to be addressed; we speculate that cells rapidly growing are more sensitive to cytotoxicity of OxLDL and that expression of hsp70 could allow a better chance of survival. Confirmation of this observation might have therapeutic applications in atherosclerosis.

	Selected Abbreviations and Acronyms

 

BrdU = 5-bromo-2'-deoxyuridine ECGF = endothelial cell growth factor FCS = fetal calf serum hsp(s) = heat shock protein(s) HUVEC(s) = human umbilical vein endothelial cell(s) M-199 = Medium 199 MEM = minimum essential medium OxLDL = oxidized low-density lipoprotein PAGE = polyacrylamide gel electrophoresis PBST = PBS+0.1% Tween 20

	Acknowledgments

 
This work was supported in part by a grant from CNR, Progetto Finalizzato Invecchiamento (publication No. 963668), and a research grant of the European Community (PL931790). The authors are grateful to Dr A. Corsini and Dr E. Cattaneo for helpful discussion and to Maddalena Marazzini for typing the manuscript.

Received November 27, 1995; revision received February 20, 1996;

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