Ascorbic Acid Blocks the Growth Inhibitory Effect of Tumor Necrosis Factor-α on Endothelial Cells

Abstract

Impaired endothelial cell proliferation has been proposed to be an early, critical defect contributing to the development of atherosclerosis. Recent studies show that high plasma tumor necrosis factor (TNF)-α levels and low serum ascorbic acid (AA) levels correlate with atherosclerosis severity. Additionally, AA has been reported to have potential beneficial effects in preventing atherosclerosis. Based on these studies, we investigated the role of AA (≤ 1mM) on TNF-α-mediated vascular endothelial cell growth inhibition in vitro. In accordance with previous reports, we found that TNF-α alone inhibited endothelial cell proliferation. Further studies revealed that AA alone enhanced endothelial cell proliferation and that AA blocked endothelial cell growth inhibition induced by TNF-α. By contrast, we observed no effect of AA on endothelial cell activation or nuclear entry of nuclear factor-κB in response to TNF-α. The protective effect of AA on endothelial cell proliferation was not simply the result of its antioxidant activity but did correlate with collagen IV expression by endothelial cells. AA pre-treatment of proliferating endothelial cells promoted retinoblastoma protein (Rb) phosphorylation and decreased p53 levels when compared to untreated cells. Furthermore, the addition of AA to TNF-α-treated proliferating endothelial cells blocked both the inhibition of retinoblastoma protein phosphorylation and enhanced p53 expression induced by TNF-α. Consistent with these results, we found that AA protects endothelial cells against TNF-α-induced apoptosis. These studies highlight the potential therapeutic role of AA in promoting endothelial cell proliferation during inflammatory conditions, such as atherosclerosis and cardiovascular disease.

Endothelial cells (ECs) found lining the blood vessels are quiescent (0.1% replications per day) under normal physiological conditions (reviewed in Ref. 1). When necessary, these cells proliferate as determined by a balance of numerous pro- and antiangiogenic factors. An example where rapid and regulated angiogenesis is proposed in preventing disease is the ability of the endothelium to repair itself promptly following vessel wall denudation, which may occur during angioplasty. In the absence of appropriate endothelial cell proliferation (re-endothelialization), smooth muscle cell proliferation and neointimal thickening ensues leading to the development and/or progression of atherosclerosis.

In addition to angiogenic factors, the endothelium is an important target for cytokines released during inflammation and tumor growth. The inflammatory and immunomodulatory effects of tumor necrosis factor (TNF)-α on ECs include the following: enhanced adhesion molecule expression, secretion of chemokines and cytokines, and increased coagulant activity (reviewed in Ref. 1). TNF-α can either promote (2, 3) or inhibit (4–12) EC growth and/or angiogenesis depending on the biological model used. Some of these effects of TNF-α on ECs are mediated through its ability to generate reactive oxygen species (ROS). Accordingly, numerous studies implicate the role of TNF-α in the early stages of atherosclerosis where endothelial proliferation is impaired following vascular tissue damage. Moreover, recent clinical investigations correlate elevated plasma TNF-α levels with the incidence of atherosclerosis (13) and with common carotid intima-media thickness, an early sign of atherosclerosis (14).

Numerous studies demonstrate that ascorbic acid (AA), a required vitamin for humans, reduces oxidative stress and improves EC function (15). Furthermore, AA administration has been shown to protect against atherosclerosis in experimental animals (16) and is believed to have beneficial effects in protecting against atherosclerosis in humans (17). In addition, the ROS formed by the inflammatory response within atherosclerotic lesions may in turn reduce AA antioxidant levels (18), further promoting atherosclerosis. Thus, based on the observations that: (i) elevated plasma TNF-α levels correlate with atherosclerosis (13–14); (ii) AA deficiency is associated with the severity of atherosclerosis (16, 19); (iii) AA supplementation protects against atherosclerosis (17); and (iv) impaired EC proliferation (20) and enhanced EC apoptosis (21–23) are postulated to play an important role in the onset of atherosclerosis, we explored the effect of AA on TNF-α-mediated EC growth inhibition and apoptosis in vitro. For our in vitro experiments, we chose concentrations of AA that reflect the levels found in the cells (approximately 1 mM) of human subjects taking approximately 100 mg AA per day (24). This amount of AA is less than the amount typically found in the 4–5 daily servings of fruits and vegetables recommended by the USDA and the NCI. Our results suggest that the growth and survival promoting activities of AA on ECs may prevent EC damage by the host inflammatory response (TNF), which contributes to the evolution of pathological conditions, such as atherosclerosis.

Materials and Methods

Cells and Cell Culture.

Primary cultures of adult human dermal microvascular endothelial cells were obtained from Clonetics (San Diego, CA). ECs were maintained in complete growth media (EGM2-MV; Clonetics). Experiments were initiated using confluent cells (passages 4 to 8). Cycle synchronization by serum deprivation induces apoptosis in ECs (25), therefore confluent EC monolayers were G₀/G₁ synchronized by contact inhibition (in the absence of AA), as described previously (11). G₀/G₁ synchronized ECs were then harvested by trypsinization and following centrifugation, the cells were dispersed in moderate growth media (75% endothelial basal media (EBM) + 5% FCS/25% EGM2-MV complete growth media-with no AA added) and plated at 40–60% confluency for each experiment (i.e., growth promoting conditions). Moderate growth media was chosen for proliferation experiments because endothelial basal medium alone does not contain any growth factors and growth-arrested ECs require some growth factors to enter in the S phase. AA, TNF-α, or other drugs were added as described in the figure legends. For cytotoxicity studies, neutral red (0.015% w/v final) was added to the proliferating cultures after 28 hr. The cells were then incubated for 12–16 hr at 37°C in 5% CO₂ and analyzed for viability as previously described (26).

Antibodies.

Human retinoblastoma protein (Rb), vascular cell adhesion molecule (VCAM), ELAM (E-selectin), and intercellular adhesion molecule (ICAM) antibodies were purchased from BD Pharmingen (San Diego, CA). Human p53 antibody was obtained from NeoMarkers (Fremont, CA). NF-κB and IκBα antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Collagen IV antibody was purchased from Rockland Immunochemical (Gilbertsville, PA).

Analysis of Endothelial Cell Proliferation.

EC proliferation was assessed by measuring the incorporation of [³H]-thymidine into DNA, as described previously (27). Briefly, G₀/G₁ synchronized ECs were plated in 96-well plates (2–4 × 10³ cells/well in moderate media) in the presence or absence of AA, TNF-α, or drugs together with 0.5 μCi /well [³H]-thymidine (DuPont NEN, Boston, MA). Following 42–48 hr of incubation, the cells were harvested onto Unifilter-96 GF/B filter plates (Packard, Downers Grove, IL) and [³H]-thymidine incorporation was measured by β-scintillation counting (Packard). Each experiment was repeated at least 3 times. The data are reported as the average cpm± SD (n ≥ 8 wells/condition) and the Student’s t test was performed to determine significance.

Assessment of Cell-Associated Oxidative Stress.

Cell-associated lipid peroxidation, a biomarker of oxidative stress, was determined using the OXI-TEK thiobarbituric acid-reactive substances (TBARS) kit (ZeptoMetrix Corp., Buffalo, NY) according to the manufacturer’s protocol. Briefly, G₀/G₁ synchronized ECs were plated in 6-well plates (2–3 × 10⁵ cells/well) in moderate media containing TNF-α, AA, or drugs for 5 hr and then harvested. Cell pellets were washed, lysed with kit reagents, and then incubated with TBA reagent in glass tubes (covered with glass marbles) at 95°C for 1 hr. The samples were then centrifuged at 3000 rpm for 15 min and the absorbance was read at 532 nm against a malondialdehyde (MDA) standard curve. The data are reported as nmol/ml MDA equivalents ± SD (n ≥ 6 wells/condition).

VEGF receptor (VEGFR) Expression Analysis by Flow Cytometry.

G₀/G₁ synchronized ECs were harvested and plated on six-well plates (3 × 10⁵ cells/well) in moderate media containing TNF-α, AA, or drugs. After an overnight incubation, the cells were washed with phosphate-buffered saline and then analyzed for VEGF receptor expression using the biotinylated VEGF receptor kit (R & D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Samples were analyzed by flow cytometry using the FACSCalibur (Becton-Dickinson, San Diego, CA) and the data were analyzed using CellQuest software (Becton-Dickinson).

ICAM, VCAM, and ELAM Expression by Endothelial Cells.

EC surface expression of ICAM, VCAM, and ELAM (E-selectin) was determined using a cell-based enzyme-linked immunoadsorbent assay (ELISA) according to previously published methods (28). Briefly, G₀/G₁ synchronized ECs were grown in 96-well plates in EGM2-MV media until ≥90% confluent and then incubated in basal media containing 5% FCS for 2 days before. ECs were incubated AA, TNF-α, and/or drugs for 18–20 hr prior to analysis of ICAM, VCAM, and ELAM by cell-based ELISA (n = 4 per condition). Data are shown as relative OD ± SD (after subtraction of background using isotype control antibody).

VEGF and Chemokine Expression by Endothelial Cells.

G₀/G₁ synchronized ECs were plated in 24-well plates (4–6 × 10⁴ cells/well) in moderate media and incubated with TNF-α and/or AA. The culture supernatants were harvested at various time points, centrifuged, and stored at −80°C until assay for VEGF or IL-8 by ELISA (R&D Systems).

Western Blotting.

G₀/G₁ synchronized ECs were plated in 6-well plates in moderate media (2–3 × 10⁵ cells/plate) and incubated with TNF-α and/or AA for the indicated times. For collagen IV: Harvested cells were lysed in M-PER mammalian protein extraction reagent (Pierce, Rockford, IL) containing collagenase-1A, collagenase IV and NaCl by repeated pipetting and incubated on a shaker for 50 min at room temperature. The extract, after a brief centrifugation, was then concentrated using Centricon-100 units (Millipore, Bedford, MA). For IκBα and NF-κB analyses, cytoplasmic and nuclear extracts were prepared using NE-PER (Pierce). For p53 and P-Rb analyses, total cell lysates were prepared according to details provided by the antibody manufacturers. Sample protein concentrations (1:50) were determined by BCA assay (Pierce). Equal amounts of cell lysates (30 μg/lane) were loaded onto SDS/PAGE gels (BioRad, Hercules, CA), and subsequently electrophoresed. Separated proteins were then transferred to PVDF Immobilon-P membranes (Millipore, MA), and then probed with primary antiserum followed by incubation with HRP-conjugated secondary antibody. Specific proteins were revealed using the ECL detection system (Amersham Pharmacia, Piscataway, NJ). Immunoblots were scanned for specific protein band intensities using NIH Image 1.62f software.

Detection of Apoptosis.

To induce apoptosis, ECs were treated with TNF-α (200 ng/ml) in endothelial basal medium 5% FCS (± 1 mM AA) overnight. To detect apoptosis, the TACS Annexin V-FITC apoptotic detection kit (R&D Systems) was used according to manufacturer’s guidelines. The samples were assessed by flow cytometry (FACSCalibur, Becton Dickinson; 10,000 cells were analyzed for each condition). The data were analyzed using CellQuest software (Becton Dickinson). This experiment was repeated three times and the data are shown as the mean percentage of apoptosis ±SD for each condition.

Results

AA Promotes EC Proliferation.

Numerous reports that date back as far as the 1970s show that oxidative stress inhibits EC proliferation and that collagen matrices support EC growth in vitro. Based on these observations, we examined the effect of AA, an important factor regulating collagen production, on EC proliferation. In the presence of moderate growth media, AA potently enhanced EC proliferation in a dose-dependent manner (0.5–2.5 mM), as assessed by ³[H]-thymidine incorporation over 48 h (Fig. 1A). This increase in EC proliferation was similar to that induced by the addition of VEGF (2–20 ng/ml) to moderate growth media (data not shown). By contrast, the addition of AA at concentrations greater than 2.5 mM was growth inhibitory to ECs (Fig. 1A ). This growth inhibition at high doses of AA (>2.5 mM) is most likely because of its cytotoxic activity, as we observed a significant reduction in viability when assessed by neutral red staining (data not shown). Additional studies showed that AA (0.1–1 mM), when added to ECs grown in complete growth media (containing growth factors) only slightly enhanced EC proliferation (data not shown). Similarly, AA did not promote EC proliferation in the absence of growth factors (i.e., in media where ECs fail to proliferate) (data not shown).

AA Partially Abrogates the Growth Inhibitory Effects of TNF-α on ECs.

Numerous studies report that TNF-α potently inhibits EC proliferation in vitro. Accordingly, we found that the addition of TNF-α blocked EC proliferation in a dose-dependent manner (2–200 ng/ml), with up to 75% inhibition observed at 200 ng/ml TNF-α (Fig. 1B ). Next, we examined whether AA could block the growth inhibitory effects of TNF-α on EC proliferation (Fig. 1B ). The addition of AA (1 mM) protected ECs against the anti-proliferative effects of TNF-α (2–200 ng/ml). This effect was dose-dependent with maximum protection at 20 ng/ml TNF-α in the presence of 1 mM AA. Consistent with our observations with AA alone, we found that in the presence of TNF-α, AA at concentrations ≥2.5 mM inhibited EC proliferation when compared to cells treated with TNF-α alone (data not shown). This growth inhibition correlated with the increased cytotoxicity of AA (≥2.5 mM) in the presence of TNF-α, as assessed by neutral red staining (data not shown).

AA Has No Effect on TNF-α−Induced EC Activation.

In addition to its effect on proliferation, TNF-α exerts potent inflammatory and immunomodulatory responses on ECs resulting in EC activation. EC activation is characterized by enhanced cytokine/chemokine production, as well as increased expression of cell surface adhesion molecules. Based on previous reports showing that several antioxidants, including N-acetyl L-cysteine (NAC), vitamin E, and α-lipoic acid suppress the expression of adhesion molecules and/or IL-8 production by ECs in response to TNF-α (29–31), and our observation that AA reversed the growth inhibitory effects of TNF-α on ECs, we next examined whether AA could inhibit TNF-α-mediated EC activation. We found that AA (1 mM) did not inhibit EC production of IL-8 in response to TNF-α (Fig. 2A), nor did AA (1 mM) block TNF-α-induced expression of EC adhesion molecules (Fig. 2B ). Thus, it appears that AA blocks the growth inhibition of ECs after TNF-α treatment, but not EC activation in response to TNF-α.

AA Does Not Block the Nuclear Entry of NF-κB by TNF-α in Proliferating ECs.

Perhaps the most prominent response elicited by TNF-α that is required for EC gene transcription (which mediates the expression of adhesion molecules, cytokines, and chemokines) is the activation of NF-κB. TNF-α enhances the phosphorylation of IκBα (32), which permits the release NF-κB from its inactive state so that it can translocate to the nucleus and activate the transcription of NF-κB responsive genes (33). Based on previous reports suggesting the role of anti-oxidants (AA, NAC, and α-lipoic acid) on the inhibition of TNF-α-mediated NF-κB activation in ECs (29–31), we evaluated the localization and expression of NF-κB and IκBα in proliferating ECs treated with TNF-α ± AA. Consistent with our finding that AA did not protect ECs from TNF-α-induced EC activation, AA did not block the TNF-α-mediated reduction in cytoplasmic IκBα levels in ECs (Fig. 2C), nor did it block NF-κB translocation to the nucleus in response to TNF-α (Fig. 2C ), suggesting that AA did not block TNF-α-mediated NF-κB activation.

AA Suppresses the Growth Inhibitory Effects of Oxidative Stress on ECs.

To determine whether the antioxidant activity of AA promotes EC proliferation during oxidative stress, we evaluated the effects of AA on the inhibition of EC proliferation in the presence of H₂O₂. H₂O₂ (0–100 μM) inhibits proliferation in a dose-dependent manner (Fig. 3A ). This anti-proliferative effect is partially abrogated (approximately 60%) by 1mM AA with the maximum protective effect at 50 μM H₂O₂, suggesting that the antioxidant activity of AA may play a role in protecting ECs against the antiproliferative effects of oxidative stress.

More Potent Antioxidants Are Less Effective in Suppressing TNF-α-Induced Growth Inhibition When Compared with AA.

We next examined whether the antioxidant activity of AA suppressed TNF-α-mediated lipid peroxidation in ECs using TBARS, a sensitive technique that measures lipid peroxidation in terms of MDA production. Table I shows that incubation of ECs with TNF-α (20 ng/ml) induces lipid peroxidation, as measured by TBARS (141% of control) and that AA slightly inhibits basal peroxidation (93.3%) when compared to control cells (100%). Combined treatment of ECs with 1 mM AA and TNF-α (20 ng/ml) reversed the peroxidation induced by TNF-α to basal levels (AA+TNF-α: 104% versus TNF-α alone: 141%). Furthermore, other potent antioxidants such as vitamin E and the well-known radical scavenger, NAC, were tested for their ability to prevent basal cellular lipid peroxidation and TNF-α-mediated EC lipid peroxidation. Vitamin E potently blocked basal cellular lipid peroxidation, as measured by TBARS, to 23% of control, whereas NAC inhibited basal cellular lipid oxidation to only 95% of control (similar to AA). Furthermore, in response to TNF-α stimulation (141%), we found that vitamin E and NAC reversed lipid peroxidation to levels significantly below (39.5% and 79.6%, respectively) that observed in control cells (100%) and below that observed when ECs were treated with TNF-α in the presence of AA (104%).

Using these more potent anti-oxidants, we found that glutathione, NAC, and vitamin E protected against TNF-α-mediated cell growth inhibition (Fig. 3B ). However, even at their most potent doses they did not attenuate the effect of TNF-α on EC proliferation as well as AA (Fig. 3B ), nor did they promote basal EC proliferation in moderate growth media in the absence of TNF-α as effectively as AA (data not shown).

AA Does Not Promote EC Proliferation via VEGF or the VEGF Receptor.

Our observations suggest that AA blocks TNF-α-mediated growth inhibition of ECs by a mechanism other than suppressing TNF-α-induced lipid peroxidation. Vascular endothelial cell growth factor (VEGF) is a potent mitogen that promotes EC growth. We found no effect of AA on VEGF production (mRNA/protein) by ECs in the presence or absence of TNF-α (data not shown). VEGF (approximately 250 pg/ml) was found in the moderate growth media alone used for our assays. We found that the VEGF levels in the culture supernatants continue to decline over time (24 hr) independent of treatment. VEGF exerts its mitogenic activity via interaction with specific cell surface receptors, VEGFR1 (K _d = 10–20 pM) and VEGFR2 (K _d = 75–125 pM; Refs. 34, 35). Several conflicting studies report the link (both positive and negative) between TNF-α and EC-associated VEGFR1 and VEGFR2 surface expression/phosphorylation (9, 36, 37). Therefore, we examined the effects of TNF-α ± AA on VEGFR expression. Using biotinylated VEGFR1 and 2 ligands and flow cytometry methods, we found no significant decrease in VEGFR expression (as determined by the mean fluorescence intensity and the number of VEGFR1 and 2 positive cells) induced by TNF-α when compared to untreated cells (Table II). Likewise, we failed to observe an increase in VEGFR expression by ECs after their treatment with TNF-α (20 ng/ml) + AA (1 mM) when compared with TNF-α-treated ECs (Table II ). Together, these data suggest that the AA does not block TNF-α-mediated EC growth inhibition by altering VEGF production or VEGFR expression.

AA, in the Presence and Absence of TNF-α, Promotes Collagen IV Production.

Cultured vascular ECs produce collagen IV, a prominent protein of the extracellular matrix (ECM) within the vascular wall (38). AA promotes collagen synthesis by the endothelium (39) and published protocols indicate the use of collagen IV + laminin (40), as well as general collagen-coated plates for culturing ECs in vitro (41, 42). Given the observations that TNF-α potently inhibits EC proliferation (Fig. 1B) and prevents ECM accumulation by stimulating the degradation of connective tissue components (43), we evaluated the effect of AA, a co-factor for the synthesis of collagen, on collagen IV production in response to antagonizing the effect of TNF-α on ECs. We found that AA induces the production of high molecular weight collagen IV by ECs (by approximately 4-fold; Fig. 4A). In the presence of TNF-α, AA induced collagen IV expression by ECs to that observed with AA alone (Fig. 4A ). Moreover these observations were confirmed using the collagen synthesis inhibitor, cis-hydroxy proline (100 μg/ml), which attenuated the synthesis of collagen IV induced by AA (Fig. 4A ). Accordingly, Figure 4B illustrates that the proproliferative effect of AA on EC growth is blocked by cis-hydroxy proline (CHP), suggesting that collagen production is an important factor mediating the growth promoting activities of AA.

AA Induces Rb Phosphorylation and Inhibits p53 Expression.

Recent studies by Lopez-Marure and co-workers identified Rb and p53 as two cell cycle mediators involved in TNF-α-induced EC growth inhibition (11). Therefore, we examined the levels of phosphorylated Rb (pRb) and p53 proteins by Western blot analysis in the lysates of cells treated with TNF-α ± AA. As shown in Figure 5, the expression of p53 by ECs is markedly increased by TNF-α in a dose-dependent manner, whereas pRb expression is decreased in response to TNF-α. However, in the presence of AA + TNF-α, p53 expression is reduced and pRb expression is significantly upregulated when compared to TNF-α-treated ECs. Additionally, AA treatment alone enhanced pRb levels in ECs when compared to control cells. These data suggest the critical role of AA in modulating the cellular expression of p53 and phosphorylated Rb in response to TNF-α.

AA Protects ECs from TNF-α-Mediated Apoptosis.

TNF-α, at high concentrations (≥100–200 ng/ml), induces EC apoptosis (44–46). Therefore, we evaluated the effects of AA on TNF-α-induced EC apoptosis. Using flow cytometry after annexin V staining, we show that 15.5% of the ECs were apoptotic in response to TNF-α treatment as compared to 1.4% of the ECs in the untreated control group versus only 3.9% of the ECs treated with TNF-α + AA (Table III). Thus, in addition to its growth promoting activities, AA can promote EC survival in the presence of high TNF-α concentrations.

Discussion

Endothelial cell (EC) proliferation is crucial for the progression of certain diseases, including cancer and arthritis. By contrast, appropriate EC proliferation in response to vessel injury has been postulated to be important for the inhibition of atherosclerosis. Numerous studies highlight the potential role of inflammatory mediators (e.g., TNF-α, IL-6, and C-reactive protein) present at the sites of atherosclerotic plaques, in atherosclerosis. These inflammatory factors are considered critical for mediating the initiation, progression, and clinical outcome of atherosclerotic disease through their effects on EC migration, proliferation, and apoptosis, as well as their growth promoting activities for smooth muscle cells (reviewed in Ref. 47). TNF-α induces EC apoptosis and inhibits EC proliferation depending on the biological system used. Recent studies suggest that plasma TNF-α levels (13) and AA depletion correlate with atherosclerosis severity in humans (19) and experimental animals (16), whereas AA supplementation protects against atherosclerosis (16). Based on these observations and the well-documented role of AA in maintaining healthy vessels, we examined the effects of AA on TNF-α-mediated growth inhibition using proliferating ECs (after G₀/G₁ synchronization).

Our data show that AA alone is proproliferative to ECs (Fig. 1A) and that AA partially abrogates TNF-α-mediated EC growth arrest in vitro (Fig. 1B ). Despite the protective effects of AA on TNF-α-induced growth inhibition of ECs, AA did not block the upregulation of adhesion molecule expression or chemokine production by ECs in response to TNF-α (Figs. 2A and B ). Accordingly, we found that AA (1 mM) did not attenuate TNF-α-mediated NF-κB activation in ECs (Fig. 2C ). Our results are consistent with previous reports demonstrating that basal NF-κB expression is necessary for EC proliferation (48) and that AA (1 mM) does not attenuate the nuclear entry of NF-κB in TNF-α-treated human aortic ECs (31) or in LPS/IFNγ-treated rat ECs (49). By contrast, Bowie and co-workers report the blockade of TNF-α-induced NF-κB activation by AA (10–40 mM) in ECs (30). This apparent difference may be, in part, the result of different concentrations of AA, different types of ECs, as well as their proliferative state. We used 1mM AA and proliferating microvascular ECs (after G₀/G₁ synchronization), whereas Bowie and co-workers used 10–40 mM AA and confluent (nonproliferating) monolayers of human vascular endothelial cells (HUVECs) or a transformed endothelial cell line (ECV304). It is important to note that oral dosing with AA (200 mg/day) maintains a plasma concentration between 10–160 μM (50). However, intracellular AA levels can range between 1.4–3.4 mM (51). In our studies, we found that concentrations of AA alone >2.5 mM and concentrations of AA ≥2.5 mM in the presence of TNF (20 ng/ml) were quite toxic to ECs (as assessed by neutral red viability staining). High-dose AA toxicity is most likely the result of iron-catalyzed oxidation (52) and numerous reports of AA (>2 mM) toxicity using fibroblasts (53), ECs (54), and other cells types, have been described. Thus, in our study we show that the treatment of proliferating ECs with lower concentrations of AA (1 mM) can block TNF-α-mediated growth inhibition, but not TNF-α-induced EC activation or NF-κB nuclear entry.

Next, we investigated the potential mechanism by which AA abrogated the growth inhibitory effects of TNF-α on ECs. TNF-α treatment produces reactive oxygen species, which have been shown to inhibit the EC proliferation (55). Considering that AA is a potent antioxidant, we investigated whether AA could promote EC growth in the presence of other mediators of oxidative stress. We observed that AA abrogates the lipid peroxidation induced by TNF-α (Table I) and the growth inhibitory effects of H₂O₂ on ECs (Fig. 3A ). Interestingly, we found that other more potent anti-oxidants, such as vitamin E and NAC, did not protect against TNF-α growth inhibition as well as AA (Fig. 3B ). These data suggest that AA attenuates the growth inhibitory effect of TN-Fα on ECs by a mechanism other than via its anti-oxidant activity.

VEGF is considered one of the most potent angiogenic factors that induces EC proliferation and angiogenesis (56), and protects ECs from apoptosis (57). Therefore, we investigated whether the protective effects of AA on TNF-α-induced EC growth inhibition occurred via the VEGF/VEGFR pathway. We observed no effect of AA (±TNF-α) or TNF-α alone on VEGF production by proliferating ECs and no effect of AA (±TNF-α) on VEGFR expression. By contrast, when we stimulated confluent, quiescent (non-proliferating) ECs with TNF-α, we observed a slight reduction in VEGFR expression (data not shown). However, we found no increase in VEGR expression by ECs when TNF-α was combined with AA when compared to cells treated with TNF-α alone. Several published studies report the link between TNF-α treatment and altered VEGFR expression/activation. However, the results of these previous studies are conflicting. In two cases, the growth inhibitory effect of TNF-α (1–20 ng/ml) on confluent HUVECs correlates with reduced VEGFR1 and R2 expression (36) and suppressed VEGFR2 phosphorylation (9). By contrast, another study reports that TNF-α (20 ng/ml) induces VEGFR2 expression by confluent HUVECs (37). Again, the major difference between our study and these previous reports is the proliferative state of the cells (proliferating cells were used in our study versus confluent EC monolayers were used in the previous studies).

In addition to protecting cells against oxidative stress, AA promotes the production of ECM proteins, including collagen, elastin, and laminin. Collagen production is required for blood vessel formation (58–59). Collagen IV, which is proposed to be generated by proliferating ECs (60), is only found within the basal lamina of the blood vessel (61) and collagen IV production is profoundly reduced in AA-deficient guinea pigs (39, 62). Interestingly, collagen Type IV, but not collagen Types I and III, increases EC adhesion in the presence of serum (63). Endothelial cell adhesion is absolutely essential for cell survival and proliferation (64) and thus, may be considered a critical step in the prevention of an atherogenic lesion following vascular injury. Interestingly, atherosclerotic lesions are characterized by an increase in collagen Types I, III, and V (but not collagen IV) (65). Thus, we investigated the effect of TNF-α ± AA on collagen IV expression by proliferating ECs. As expected, we observed enhanced collagen IV expression (4-fold) by proliferating ECs treated with TNF-α ± AA when compared with cells treated with TNF-α alone (Fig. 4A). Although TNF-α promotes collagen IV mRNA stability (66–68), we found no increase in collagen IV expression by ECs in response to TNF-α (Fig. 4A). However, TNF-α has been shown to upregulate MMP-9 expression by ECs, which degrades basement membrane proteins, specifically collagens IV, V, and IX (69).

Our data, together with these reports, suggest that the protective effect of AA on TNF-α-induced growth inhibition may be mediated, in part, through its effect on ECM production. The best-studied family of receptors that mediate endothelial cell-matrix interactions is the integrins. Integrins, by serving both tethering and cell signaling functions, facilitate cell movement, maintain tissue stability, and activate pathways involved in cell replication and cell death (70). This hypothesis is supported by several studies showing enhanced proliferative responses by different cell types, including smooth muscle cells and epithelial cells, when plated on ECM in the presence of growth factors (71, 72). By contrast, growth inhibition by anchorage-deficiency (or detachment from extracellular matrix) is associated with increased p53 expression (73–75).

The cell cycle control mediators, p53 and the unphosphorylated form of Rb prevent the transition of G₁ cells to S phase (DNA synthesis). Unlike the constitutive cellular expression of Rb (which is regulated by phosphorylation), p53 expression is induced only under certain circumstances. Previous in vitro studies revealed that TNF-α causes an accumulation of ECs in the G₁ phase of the cell cycle by inhibiting the phosphorylation of the retinoblastoma (Rb) gene product (10) and by inducing p53 expression (11). Further studies demonstrated that TNF-α can promote EC apoptosis (76–78). Interestingly, Kawauchi and co-workers recently reported that TNF-α-induced EC apoptosis by enhancing p53 transcription, which can be blocked by overexpression of ATF3, a transcriptional repressor (45). Therefore, we examined the effects of AA on p53 and phospho-Rb expression by ECs treated with TNF-α. We found that AA alone inhibited p53 expression and promoted pRb expression by proliferating ECs and blocked the induction of TNF-mediated p53 expression and Rb hypophosphorylation (Fig. 5).

Concordant with the inhibitory effects of AA on TNF-α-induced p53 expression, we found that AA protected ECs from TNF-α-induced apoptosis (Table III ). In response to high levels of TNF, ECs produce pro-survival factors, so apoptosis is significantly higher in the presence of cyclohexamide or Actinomycin D (77). Therefore, the effect of TNF-α on EC apoptosis is variable depending on whether mRNA or protein expression is blocked (by cyclohexamide or Actinomycin D) (77, 79). We chose to look at a more ‘physiological’ state, i.e. in the absence of these inhibitors. Our results are consistent with a previous report showing that TNF-α alone (in the absence of cyclohexamide or Actinomycin D) induced apoptosis in approximately 14% of ECs within 8 hr (80). EC apoptosis has been implicated in preventing re-endothelialization following vascular injury and thus, promoting atherosclerosis. In accordance with our data, recent reports demonstrate the increased expression of proapoptotic proteins, such as Fas and Bax and decreased expression of anti-apoptotic factors by ECs overlying vascular lesions (21). Together, these observations suggest that AA protects ECs against TNF-α-mediated growth inhibition and TNF-α-induced apoptosis by suppressing p53 expression and promoting Rb phosphorylation.

There is enormous variability in the literature regarding the results of endothelial cell studies. For the experiments outlined in this paper, we employed proliferating human dermal microvascular endothelial cells. However, other studies have employed several different kinds of endothelial cells grown under various conditions. The variability of the effects of AA (and TNF) on endothelial cells is probably due to many factors, including the source (specific organ; vein versus artery) of ECs, the species used for the isolation of ECs (the requirement for AA is different for humans versus rodents versus bovine), the growth conditions of the cells (the presence of specific growth factors, extracellular matrix molecules, etc.), and finally, the proliferative state (growth arrested, i.e., normal EC state, versus proliferating ECs, which is similar to what occurs during pathological conditions).

Cell proliferation regulatory pathways, including genes that control the G₁/S phase checkpoint (such as p53 and Rb), have been associated with plaque progression during atherosclerosis. However, most of the studies have examined the expression of these genes by vascular smooth muscle cells whose uncontrolled cell proliferation leads to initial thickening in the absence of re-endothelialization. Both EC proliferation and apoptosis are two critical events postulated to contribute to the pathogenesis of atherosclerosis, which is characterized by enhanced TNF-α expression and associated with low serum AA levels. The results from this study suggest a molecular mechanism(s) by which AA significantly blocks the effects of TNF-α on EC proliferation and apoptosis, namely by inhibiting TNF-α-induced p53 expression and Rb hypophosphorylation, as well as by promoting collagen IV production, and further support the postulated role of AA as an antiatherogenic agent.

Table I.

Effect of Antioxidants on Lipid Peroxidation

Treatment	TBARs (percent control)
Endothelial cells were treated with media alone (untreated), TNF-α alone, H₂O₂ alone, indicated antioxidants alone, or indicated antioxidants in the presence of TNF-α (20 ng/ml) for 5 hr as described in the Materials and Methods section. Cell pellets were isolated and cell-associated lipid peroxidation was determined by measuring TBARS against an MDA standard curve.
Data are shown as nmol/ml MDA equivalents ± SD (n ≥ 6 wells per condition). The data are representative of two similar experiments.
Untreated	100 ± 3
Ascorbic acid (1 mM)	93.3 ± 4
Vitamin E (0.1 mM)	23 ± 4
NAC (1 mM)	96.1 ± 10.5
H₂O₂ (0.1 mM)	137 ± 10
TNF-α (20 ng/ml)	141 ± 16
TNF-α + Ascorbic acid	104 ± 12
TNF-α + Vitamin E	39.5 ± 12
TNF-α + NAC	79.6 ± 10

Table II.

VEGFR Expression by ECs

Treatment	M1 (% positive)	M2 (% positive)
ECs were incubated in media alone (untreated), media containing TNF-α (20 ng/ml), or TNF-α (20 ng/ml) + AA (1 mM). Cells were then analyzed for VEGFR expression using labeled VEGF ligand and flow cytometry methods as described in the Methods and Materials section. Data are shown as the mean fluorescence intensity for low affinity VEGFR (M1) and high affinity VEGFR (M2) and the number of positively labeled cells (% positive). The data are representative of two similar experiments.
Untreated	183 (72)	313 (27)
TNF-α	185 (77.5)	350 (27)
TNF-α + AA	149 (77)	302 (20)

Table III.

Effect of AA on TNF-α-Induced EC Apoptosis

Treatment	Apoptosis (%) + SD
ECs were treated with media alone (untreated), AA (1 mM), TNF-α alone (200 ng/ml), or with AA plus TNF-α, as indicated in the Methods and Materials section. Cells were harvested and assayed for apoptosis. Data are shown as the percent of apoptotic cells ± SD.
**p < 0.01, as determined by the Student’s t test (vs untreated cells).
Untreated	1.35 ± 0.69
Ascorbic acid (1 mM)	1.6 ± 0.54
TNF-α (200 ng/ml)	15.5 ± 3.1**
TNF-α + AA	3.9 ± 2.6

Figure 1.

AA promotes EC proliferation and blocks the growth inhibitory effects of TNF-α. G₀/G₁-synchronized ECs were plated in 96-well plates (2–4 × 10³ cells/well) and incubated with (A) AA alone (0–10 mM) or (B) in the absence or presence of TNF-α (0–200 ng/ml) ± AA (1 mM) in moderate growth media containing 0.5 μCi/well ³[H]-thymidine. Cellular proliferation over 48 hr was assessed by ³[H]-thymidine incorporation and β-scintillation counting (n = 8 per condition). Data are shown as counts per minute (cpm). *P < 0.05 and **P < 0.001, as determined by the Student’s t test. Data is representative of five similar experiments.

Figure 2.

AA does not block TNF-α-mediated adhesion chemokine production, adhesion molecule expression, or NF-κB activation by ECs. A and B, G₀/G₁-synchronized EC monolayers (in 96-well plates) were treated with media alone, TNF-α (20 ng/ml), AA (1 mM), or TNF-α (20 ng/ml) + AA (1 mM) for 20 hr. (A) Supernatants were collects and assayed by enzyme-linked immunosorbent assay for interleukin-8 (n = 4 per condition). (B) ECs (n = 4 per condition) then were assayed for ELAM, ICAM, and VCAM expression using a previously described cell-based ELISA method (27). (C) G₀/G₁-synchronized ECs (6-well plates) were treated with media alone, TNF-α (20 ng/ml) alone, AA (1 mM) alone, or TNF-α (20 ng/ml) + AA (1 mM). After 3 hr, cytoplasmic and nuclear extracts were prepared and analyzed for IκBα and NF-κB expression by western blotting methods. Data are representative of three similar experiments.

Figure 3.

AA suppresses the EC growth inhibition induced by H₂O₂ (oxidative stress), but the growth promoting activity of AA is not simply because of its antioxidant activity. G₀/G₁-synchronized ECs were plated in 96-well plates (2–4 × 10³ cells/well) incubated with moderate media containing (A) AA (1 mM) in the presence of H₂O₂ (0–100 μM) or (B) media alone or TNF-α ± AA, glutathione (Glut), Vitamin E (Vit E), or N-acetylcysteine (NAC) at indicated concentrations in the presence of 0.5μCi/well ³[H]-thymidine. Cell proliferation over 48 hr was assessed by ³[H]-thymidine incorporation followed by β-scintillation counting (n = 8 per condition). Data are shown as counts per minute (cpm). *P < 0.01; **P < 0.001; ***P < 0.000001, as determined by the Student’s t test. Data are representative of four similar experiments.

Figure 4.

AA promotes collagen IV production and collagen production correlates with the growth promoting activity of AA. (A) Proliferating ECs were incubated with media alone or treated with AA (1 mM) ± TNF-α (20 ng/ml) or AA (1 mM) ± cis-hydroxyproline (CHP, 100 μg/ml) for 48 h. Native ECM protein was isolated (see Materials and Methods), fractionated by PAGE, and blotted to PVDF membrane. Collagen IV was detected by immunoblotting with rabbit anti-anti-collagen IV antibodies (Rockland). This antibody recognizes high molecular weight collagen IV (Hi MW Col IV) and the 180-kDa form of collagen IV. The bands were scanned and assessed for intensity; upper values refer to the intensity of the Hi MW Col IV band and the lower values refer to the intensity of the 180 kD Col IV band. (B) EC proliferation was performed as described in Figure 1, in the presence or absence AA (1 mM) ± CHP (100 μg/ml). *P < 0.001, as determined by the Student’s t test. Data are representative of three similar experiments.

Figure 5.

AA blocks TNF-α-mediated p53 expression and Rb hypophosphorylation. (A) Total cell lysates were prepared from proliferating ECs treated for 18 hr with TNF-α (0–20 ng/ml) in the presence or absence of AA (1 mM). Protein extracts were electrophoresed and immunoblotted for p53 (upper panel) and pRb/Rb (lower panel). The intensity of the bands are indicated below the blots.

Footnotes

This work was supported by a grant from The Picower Foundation (CNM) and NS-LIJ Research Institute funds.

1

To whom requests for reprints should be addressed at North Shore-Long Island Jewish Research Institute, 350 Community Drive, Manhasset, NY 11030. E-mail:

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