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Cardiovascular Research 2004 61(4):693-704; doi:10.1016/j.cardiores.2003.12.008
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Copyright © 2004, European Society of Cardiology

p21Waf1/Cip1/Sdi1 mediates shear stress-dependent antiapoptotic function

Stefania Mattiussia,b,1, Paolo Turrinia,1,2, Lucia Testolina, Fabio Martellia, Germana Zaccagninic, Antonella Mangonic, Laura M Barlucchia, Annalisa Antoninia, Barbara Illia, Corrado Ciriellia, Julio Padrona,1, Chiara Nicolòb, Roberto Testib, Francesco Osculatid, Paolo Bigliolic, Maurizio C Capogrossia and Carlo Gaetano*,a

aLaboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, IRCCS, Via dei Monti di Creta, 104, 00167 Rome, Italy
bUniversità degli Studi di Roma "Tor Vergata", Italy
cIstituto Cardiologico Fondazione "I. Monzino", IRCCS, Milan, Italy
dDipartimento di Scienze Biomediche e Morfologiche, Sezione di Istologia ed Anatomia, Facoltà di Medicina e Chirurgia, Università di Verona, Verona, Italy

* Corresponding author. Tel.: +39-6-66464794; fax: +39-6-66462430. gaetano{at}idi.it

1 Current address: Istituto Ricerche di Biologia Molecolare (IRBM) "P. Angeletti", Pomezia, Roma, Italy.

Received 16 September 2003; revised 28 November 2003; accepted 5 December 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The antiapoptotic effect of p21Waf1/Cip1/Sdi1 (p21) was examined in human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress (SS) or to the nitric oxide donor sodium nitroprusside (SNP) and in a mouse model of hindlimb ischemia. Methods: In vitro: Cells were cultured without serum and in the presence of cobalt chloride to simulate hypoxia for 12 h (T0). Shear stress was applied to endothelial cells for additional 12 h. In vivo: Hindlimb ischemia was realized in mice by femoral artery ligation. SNP was acutely administered by subcutaneous injection or by Alzet osmotic pumps for a longer treatment. Results: At T0, HUVEC were either exposed to SS (15 dyn/cm2/s–1), treated with SNP or kept in static condition (ST) for 1–12 h; after additional 12 h in ST, 30–35% of cells still alive at T0 had died. In this condition, both SS and SNP treatments markedly increased p21 levels and reduced apoptosis in HUVEC. Recombinant adenoviruses carrying p21 (AdCMV.p21) or antisense p21 (AdCMV.ASp21) cDNA revealed that AdCMV.p21-infected HUVEC were protected from death while AdCMV.ASp21 reduced SS- and SNP-dependent protection from apoptosis. In mice, apoptosis was detected in endothelial cells of ischemic hindlimbs as early as 8 h after femoral artery ligation. Treatment with SNP enhanced p21 expression and protected ischemic tissue from damage. Remarkably, direct in vivo injection of AdCMV.p21 significantly reduced the number of apoptotic nuclei in the presence of ischemia. Conclusions: The present study establishes that, under our experimental conditions, (a) p21 plays an important role in SS and nitric oxide antiapoptotic effect in vitro, and (b) p21 gene transfer prevents apoptosis in vitro and in vivo, following acute interruption of blood flow.

KEYWORDS Shear stress; p21; Apoptosis; Flow; Hypoxia; Ischemia

This article is referred to in the Editorial by F. Aoudjit and J. Sévigny (pages 648–650) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The mechanism enabling laminar shear stress (SS) to have an antiapoptotic function on endothelial cells has been only partially characterized and includes the activation of the serine/threonine kinase Akt signaling, the upregulation of Cu/Zn superoxide dismutase and cyclooxygenase-2 activities as well as an enhanced nitric oxide (NO) production (see Ref. [1] for review). Recently, it has been shown that laminar SS increases the intracellular level of the CDK inhibitor p21Waf1/Cip1/Sdi1 (p21) and, via this mechanism, it induces dephosphorylation of the retinoblastoma gene product inhibiting endothelial cell proliferation. Although some evidences suggested that p21 upregulation may be associated with increased apoptosis [2], in addition to its cell cycle effects, p21 expression has been recently correlated with an increased cell survival [3–5]. Recent observations pointed out that in ECs exposed to SS p21 gene expression is upregulated [6]. Prior studies had shown that SS prevents ECs death induced by a variety of stimuli including growth factors deprivation [7], TNF-{alpha} [7,8], oxidative stress [9], oxidized LDL [8], as well as ECs death occurring in the absence of flow, when ECs are cultured in the presence of serum and growth factors [10]. In fact, SS induces an integrin-dependent [11] activation of the PI3K-AKT pathway [12] which, modulating NO production, inhibits caspase-3 activity and cell death [13]. The function of p21 in relationship to the ability of SS to prevent apoptosis is, however, still unknown. To address this question we investigated, in vitro, the role of p21 in a cell culture system characterized by the presence or absence of SS, growth factor deprivation (GFD) and reduced oxygen level, realized by adding CoCl2 to the culture media and, in vivo, during the process of vascular regression upon acute hindlimb ischemia. The results of the present study indicate that p21 expression in vascular cells is important for the inhibition of apoptosis in vitro and in vivo.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture and treatments
Human umbilical vein endothelial cells (HUVEC) were grown at 37 °C in 5% CO2 atmosphere in endothelial growth medium (EGM-2; Clonetics) supplemented with antibiotics. Apoptosis was induced in 50–70% confluent cultures by treatment with 100 µM cobalt chloride (CoCl2, Sigma-Aldrich) in EBM-2 serum-free medium (Clonetics); after 12 h of this treatment (T0), the cells were either kept in static culture conditions (ST) or exposed to laminar fluid shear stress (SS) of 15 dyn/cm2/s–1 for additional 1, 2, 4, 6 and 12 h. For each experiments, 1 x 106 cells were plated in 150-mm plastic dishes in the presence of complete EGM-2 medium 24 h before exposure to serum deprivation and CoCl2. In all experiments, HUVEC were used between passages 4 and 6.

2.2 Animals
Pathogen-free CD1 or Nudenu/nu male mice, 2–3 months of age and weighing 25–35 g (Harlan, Nossan, Italy), were anesthetized with 2% tribromoethyl alcohol, 2% tert-amyl alcohol (Avertin, 25 µl/g body weight) diluted in saline solution and injected intraperitoneally. Animal care procedures were performed in conformity with the Intramural Committee and Institutional guidelines in accordance with national and international laws and policies.

2.3 Hindlimb ischemia
Ischemia was induced in the left hindlimb. The left femoral artery was ligated at its proximal origin as a branch of the external iliac artery. Animals were sacrificed 8, 24, 48 h and 5–7 days after ischemia with an overdose of Avertin.

2.4 Elisa determination of free nucleosomes
Mono- and oligo-nucleosomes in cell lysates of HUVEC cultured in the absence of growth factors with or without CoCl2 were determined by Cell Death Detection ELISA (Roche). About 105 cells for each condition were lysed and processed according to manufacturer's instructions.

2.5 TUNEL assay
Apoptosis mediated by DNAse I and II was identified by TdT assay. Briefly, cells or muscle sections were incubated with 5U of TdT, 2.5% mM CoCl2, 0.2 M potassium cacodylate, 25 mM Tris–HCl, 0.25% BSA and 0.5 nM biotinylated 2'-deoxyuridine-5'-triphosphate (biotin-16-dUTP). Then, sections were incubated with ABC–rodhamine or ABC–horseradish peroxidase (HRP) complex and revealed with DAB.

2.6 Viral infections
Stably transfected HUVEC expressing E6 human papilloma protein were generated by retroviral infection with pBABE-puro retroviral vector (a kind gift from J.P. Morgenstern and H. Land; Imperial Cancer Research Fund, London). pBABE-puro E6 and the mutant {Delta}E6, unable of targeting p53, were generated from GST-HPV type 16 E6 [14]. HUVEC were infected as described [15] and selected in puromycin-containing medium (0.5 µg/ml). For transient gene expression, ECs were infected with replication-deficient, recombinant, Ad vectors carrying cDNAs expressing human sense (AdCMV.p21) and antisense (AdCMV.AS p21) p21 RNA [16], and nuclear-localized β-galactosidase (AdCMV.nlsβgal), as control. For in vivo analysis, normoperfused Nudenu/nu mice were infected with 1 x 108 pfu/muscle of AdCMV.nlsβgal or AdCMV.p21, suspended in a total volume of 30 µl of saline solution. Ad viruses were injected in the adductor muscle 48 h before ischemia. The HUVEC were infected, as previously described [17], with 250 pfu/cell and cultured for additional 48 h before use.

2.7 Antibodies
For Western blot analysis, the following antibodies were used: anti-p21 peptide rabbit polyclonal (C-19; Santa Cruz); anti-caspase-3 rabbit polyclonal (H-277; Santa Cruz); anti-p53 monoclonal (Ab-1; Oncogene Science); anti-pRB monoclonal (RB G3-245; BD-Pharmingen); anti-PARP rabbit polyclonal (H-250; Santa Cruz).

2.8 Shear stress apparatus
A cone-and-plate apparatus, maintained at 37 °C in humidified air with 5% CO2, was used as described previously [18].

2.9 Administration of sodium nitroprusside
In vitro, cells were treated with sodium nitroprusside (SNP; 50 µM) during a time course of 2, 4 and 6 h after T0 before harvesting for Western blot analysis or trypan blue exclusion assay to determine the number of viable cells. In vivo, immediately after the induction of ischemia, animals were either injected with SNP (1 µg/µl) in a total volume of 50 µl/injection or an equal amount of saline. Each animal was injected subcutaneously in the left ischemic hindlimb every 2 h for a total of four injections. Alternatively, in other animals, SNP was administrated with Alzet micropumps in the ischemic limb near the site of artery ligation. In these animals SNP was delivered at 10 µg/kg/min–1 either for 2 or 7 days.

2.10 Protein extraction and Western blotting
HUVEC were lysed with ice-cold RIPA buffer of the following composition: 10 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA (pH 7.4). The buffer contained the following protease inhibitors: 2 mM PMSF, 2 mM benzamidine, 10 mM aprotinin, 0.1 M leupeptin and 5 mM pepstatin (all from Sigma-Aldrich). The proteins were separated on a SDS-PAGE gel, electrotransfered onto a nitrocellulose membrane (Bio-Rad) and analyzed by Western blotting.

2.11 Immunohistochemistry
To evaluate p21 immunoreactivity, sections of the adductor muscle were incubated with rabbit polyclonal anti-p21 (Santa Cruz) and with anti-rabbit IgG. After incubation with ABC complex (Vectastain), the reaction was revealed with diaminobenzidine (DAB) according to manufacturer's instructions.

2.12 Capillary density
After paraffin embedding, 3 µm sections from each adductor muscle were cut. Muscle fibers were oriented in the transverse direction and stained with hematoxylin/eosin. The analysis of capillary network was performed using an ocular reticle at 1000 x magnification. In each section, 50–70 random fields were examined. The number of capillary profiles in each sampled area (10,000 µm2) was counted. We indicated the capillary density as ratio of capillary number/area in mm2. All samples were counted blind by two independent operators.

2.13 Statistical analysis
Results are expressed as mean±S.E. and were analyzed with a one-way ANOVA and post hoc Student's t-test for assessment of statistical significance. A value of P≤0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 SS and NO prevent apoptosis and upregulate p21 in the presence of hypoxia and serum starvation
CoCl2 was added to the culture media to accelerate the onset of apoptosis in human endothelial cells (Fig. 1A). Our experiments, in fact, indicate that, in medium deprived of growth factors (GFD), a significant amount of cell death occurred between 12 and 24 h of this treatment, and that CoCl2 added to the culture medium enhanced this process of about 250% (Fig. 1A). Based on these observations, the 12-h time-point of cells cultured with CoCl2 was chosen as the experimental T0 in which ECs were either kept in static culture (ST), exposed to laminar SS or treated with the NO donor sodium nitroprusside (SNP) for additional 1–12 h. Cell viability was assessed by trypan blue exclusion and TUNEL analysis (Fig. 1B). After the first 12 h of culture in ST in the presence of CoCl2 (this time-point has been defined as the experimental T0), only 10–15% of the cells were not viable. As expected, cells kept in ST continued to die and 12 h after T0 an additional 30% of the cells alive at T0 had died. In contrast, SS prevented apoptosis, and <5% HUVEC exposed to SS at T0 had died by the end of the experiment (Fig. 1B). In this context, p21 levels were evaluated and no significant changes were detected during the first 6 h after T0 in cells kept in ST while a reduction of about 50% appeared at 12 h, possibly due to the prolonged caspase-3 activation (not shown) and/or to the higher number of dead cells present at this time-point. In contrast, in cells exposed to SS, there was a significant increase in p21, which achieved its peak 4 h after T0 and it was still above control 12 h after T0 (see inset in Fig. 1B). Since SS is a potent inducer of NO [8], which is known to upregulate p21 [19], we next evaluated whether NO could protect cells from apoptosis and upregulate p21 in the presence of GFD and CoCl2. Fig. 1C shows that ECs exposed to SNP become resistant to the pro-apoptotic environment. Remarkably, as shown in the inset of Fig. 1C (right panel), SNP treatment induced about 3-fold increase in p21 levels after 6 h of treatment.


Figure 1
Figure 1
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  		Fig. 1 Effect of SS and NO on HUVEC apoptosis and p21 expression. (A) Apoptosis was induced by serum starvation and growth factor deprivation (GFD) for 12–24 h both in the presence and in the absence of CoCl2. The bar graph depicts the result of three independent experiments in which nucleosomal fragmentation was determined by ELISA. Control cells in normal serum (black bar); serum starvation (white bars); serum starvation and CoCl2 supplement (striped bars). The 12-h time-point was chosen as the experimental T0 at which cells were exposed to SS for additional 12 h. (B) The graph shows a progressive increase in the number of trypan blue-positive cells cultured in ST (black bars). Each point represents three separate experiments performed in quadruplicate. TUNEL assay was performed on HUVEC cultured in ST or SS condition for 12 h after T0. Representative pictures show the presence of TUNEL-positive cells (lower pictures) in ST and SS. The total number of cells was determined by HOECHST staining (upper pictures). The right panel shows the evaluation of TUNEL-positive nuclei 12 h after T0 in ST and SS cultures. At T0 10±4% of cells were TUNEL-positive. Cells present in 10 different fields were evaluated. For both experiments, points statistically different from T0 are indicated by * (P value <0.01). The inset shows the result of Western blot analysis of p21 levels, samples were normalized by tubulin (T) expression. Densitometric analysis shows that p21 expression increases about 3-fold after SS treatment. (C) HUVEC, cultured in serum-free medium and in the presence of CoCl2 for 4–6 h after T0 (black bar), were treated with 50 µM sodium nitroprusside (SNP) (striped bar). The effect of NO on apoptosis was then evaluated by trypan blue exclusion. The number of viable cells at T0 was 10±5%. These experiments were repeated three times. *P<0.01. The left panel in the inset shows that E6 papilloma virus protein (E6) was efficient at targeting human p53 protein compared to cells infected with the empty BABE vector. SS treatment induced p21 expression in E6+/p53– endothelial cells. The right inset panel shows a representative picture of the effect of NO on p21 protein level. HUVEC were treated with SNP (0.5 µM) for 2–6 h after T0. These experiments were repeated three times with similar results.

 
3.2 SS induces p21 through a p53-independent mechanism
p21 gene expression is, at least in part, controlled by p53; however, p53-independent mechanisms of regulation have also been described [20,21]. To explore the possible involvement of p53 in the SS-dependent induction of p21, HUVEC were infected with BABE retroviral vectors carrying wild-type papilloma E6 (wtE6) or the p53 incompetent delta-E6 ({Delta}E6) mutant as control. In these cells, as a consequence of wtE6 interaction with p53, the latter was rapidly inactivated by ubiquitin degradation and became undetectable (see inset in Fig. 1C, left panel). As a result, the suppression of p53 pathway generated a marked reduction in p21 protein. Surprisingly, SS treatment was still capable of enhancing p21 protein independently of p53 expression.

3.3 Targeting p21 impairs antiapoptotic properties of SS and NO
To determine whether p21 increase was functionally relevant to ECs survival in the presence of SS or NO, HUVEC were infected with adenoviral vectors carrying the human p21 cDNA in sense (AdCMV.p21) or antisense (AdCMV.ASp21) orientation. Both uninfected and AdCMV.LacZ-infected cells were used as control. Thirty-six hours after infection, cells were exposed to CoCl2 in GFD medium until T0 before starting ST and SS treatments for additional 12 h. Fig. 2A shows that, in cells kept in ST, adenovirus-mediated p21 sense expression reduced the incidence of cell death by approximately 60%. It is noteworthy that the antiapoptotic effect of SS was still present in these cells. The key role of p21 in cell death protection was further underlined by the observation that, in cells infected with AdCMV.ASp21, antisense expression significantly enhanced apoptosis in endothelial cell cultured in ST and abolished the protective effects of SS (Fig. 2A). Remarkably, at T0, the 17-kDa fragment, derived from active caspase-3, was not detectable in AdCMV.p21-infected cells while it was present in AdCMV.LacZ and AdCMV.ASp21 cell populations, thus indicating the presence of active apoptosis in these cells (see inset in Fig. 2A).


Figure 2
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  		Fig. 2 Effect of p21 targeting on SS and NO antiapoptotic effects. (A) The graph depicts the average number of trypan blue cells expressed as percent of live cells present at T0. Cells (1 x 106) were plated for each experimental time-point. Mock- (black bars), AdCMV.p21- (striped bars), AdCMV.LacZ- (dotted bars) and AdCMV.ASp21- (open bars) infected cells were cultured in GFD medium supplemented with CoCl2 for 12 h (T0) and then exposed to SS or kept under ST for additional 12 h. At T0 10±7% of cells were trypan blue-positive. Each point represents the mean value±S.E. of three separate experiments. Statistically significant differences between SS and ST are indicated by *P<0.01 or **P<0.001. The inset depicts p21 and caspase-3 active 17-kDa fragment in AdCMV.LacZ-, AdCMV.p21- and AdCMV.ASp21-infected cells after 48 h of culture in ST condition. This experiment was repeated three times with similar results. (B) The graph (left) shows that, in the presence of the eNOS inhibitor L-NMA (gray dotted bar), the number of dead HUVEC increased 18% above control (white dotted bar) at 6 h after T0. Cells infected with AdCMV.p21 were significantly protected from cell death 15% below control level (black striped bar). L-NMA treatment in the presence of AdCMV.p21 failed to increase the number of dead cells above control level (gray striped bar). The graph (right) shows that, in the presence of the NO donor SNP (black bar), HUVEC were protected from apoptosis. The number of dead cells significantly increased 15% above control level after infection with AdCMV.ASp21 (white bar) and in this condition (gray bar) SNP failed to rescue apoptosis. This experiment was repeated three times with similar results. *P<0.01; **P<0.001.

 
Further, we investigated the functional role of p21 in the presence of NO in a series of experiments in which ECs, infected with AdCMV.p21 or AdCMV.ASp21 adenovirus and cultured in GFD and CoCl2, were treated with SNP or the eNOS inhibitor L-NMA for 6 h after T0. AdCMV.LacZ-infected cells were used as control. Fig. 2B, left panel, shows that in cells treated with L-NMA (gray dotted bar) apoptosis significantly increased about 18% above control level (white dotted bar) while p21 expression drastically reduced (5%) apoptosis (black striped bar). However, in the presence of L-NMA p21 antiapoptotic effects were markedly reduced (20%) (gray striped bar) but failed to increase above control level. Fig. 2B, right panel, shows that increasing NO levels protected (3%) cells from apoptosis (black bar). In this context, targeting endogenous p21 expression by antisense adenovirus increased apoptosis of about 15% above control (white bar) and abolished the protective effect of NO (gray bar).

3.4 Femoral artery ligation induces tissue degeneration and capillary density regression in the adductor muscle
In light of the in vitro experiments reported in the present work, we reasoned that culturing ECs in the presence of GFD and CoCl2 partially reproduced some of the in vivo consequences of the abrupt interruption of blood flow such as the reduction in oxygen tension and nutrients levels ultimately leading to the ischemic tissue damage. Therefore, in additional in vivo experiments, the role of NO and p21, in mouse adductor muscles and blood vessels, in which SS was altered by femoral artery ligation (FAL), was examined. After FAL, the histology of adductor muscle showed that tissue degeneration and infiltration occurred 2 days after ischemia as shown in Fig. 3A. However, at all time-points tested, the number of arterioles of small, medium and large diameter did not change (not shown) while capillary density (mm/mm2) was strongly but transiently diminished in ischemic animals (Fig. 3B) [22]. Morphometric analysis revealed, in fact, that the increase in capillary density detectable at day 7 was preceded by a transient decrease in capillaries. This response achieved its peak at day 2 and at this time-point capillary density was about 50% of what was found in the normoperfused adductor muscle (Fig. 3B).


Figure 3
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  		Fig. 3 Effect of FAL on endothelial cell apoptosis and capillary density in ischemic adductor muscles. (A) The figure shows representative pictures of eosin-stained sections obtained from normoperfused and 2 and 7 days post-ischemic adductor muscles. (B) Morphometric analysis was performed after hematoxylin/eosin staining of tissue sections. The graph shows a 50% reduction in capillary density at day 2 after ischemia while a marked increase was evident between days 5 and 7. Results represent the mean±S.E. of six animals per group.

 
3.5 NO protects from apoptosis and induces p21 expression in vivo
Since the in vitro experiments shown in the present study indicates that p21 could play a role in the antiapoptotic pathway regulated by SS and NO, it was evaluated whether NO, a potent inducer of p21 [21], modulated the expression of this protein in ischemic tissues. Sodium nitroprusside (SNP), a NO donor, was administered to mice locally via intramuscle injection for the first 8 h immediately after FAL (Fig. 4A), or systemically, from 2 to 7 days, by Alzet micropumps (10 µg/kg/min–1) (Fig. 4B). Remarkably, the presence of NO prevented inflammatory tissue damage (not shown) and drastically reduced the number of TUNEL-positive nuclei observed as early as 8 h after FAL in capillary ECs of SNP-treated ischemic animals (Fig. 4A). In addition, quantitative analysis demonstrated that SNP treatment significantly prevented the reduction in capillary density which occurred 2 days after induction of ischemia (Fig. 4B). At this time-point, the presence of an active apoptotic process was monitored by detection of the active 17-kDa fragment of caspase-3 which was present in the adductor muscles of ischemic animals 2 days after FAL but it was not detectable in the presence of SNP (see inset in Fig. 4B).


Figure 4
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  		Fig. 4 Effect of NO on capillary density and endothelial cell apoptosis in ischemic adductor muscles. (A) The graph shows that as early as 8 h after FAL SNP-treated mice (black bar) exhibited a marked decrease in capillary apoptotic nuclei vs. saline-treated controls (open bar). The inset shows that in adductor muscle sections of SNP-treated mice (8 h SNP) TUNEL-positive nuclei are undetectable compared to saline-treated (8 h saline) mice. Results represent three animals per group and pictures are representative of a total of 10 different sections. **P<0.001. (B) Capillary density was evaluated in the adductor muscle of ischemic animals treated with saline solution (open bars) or chronically exposed for 2 and 7 days after surgery to SNP (10 µg/kg/min–1) (black bars) delivered by subcutaneous osmotic micropumps. The graph shows that SNP prevented the reduction of capillary density occurring at day 2 after surgery. Results represent six animals per group. The inset shows a representative picture of Western blotting analyses on caspase-3 protein expression in total lysate obtained from normoperfused and ischemic adductor muscles evaluated at 2 and 7 days after FAL. The presence of pro-caspase and active caspase-3 form, detectable at day 3 after ischemia, is indicated. Samples were normalized by tubulin (T) content. §P<0.05.

 
The effect of NO on the expression of p21 in vivo was evaluated by Western blotting and immunohistochemical analyses of adductor muscle samples from animals treated with saline solution or SNP. In normoperfused tissues, p21 immunoreactivity was detectable in about 40% of muscle fibers and 10% of whole endothelial and smooth muscle cell nuclei examined. However, at 2 days after the induction of ischemia, a marked reduction of p21 staining was observed in untreated animals (Fig. 5A). p21 signal progressively increased in animals treated with SNP (Fig. 5A). Quantitative analysis showed, in fact, that at day 2 the number of p21-positive nuclei in ischemic animals treated with SNP was similar to that observed in non-ischemic tissues and about 2.5-fold higher than that in untreated ischemic animals remaining still 2-fold higher at 7 days after ischemia (Fig. 5B). At this time-point in samples treated with SNP, total p21 protein level was 5-fold higher than in ischemic or normoperfused controls (see inset in Fig. 5B).


Figure 5
Figure 5
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  		Fig. 5 Effect of NO on p21 expression and apoptosis. (A) Expression of p21 was evaluated 8 h, 2 and 7 days after surgery. Representative panels of adductor muscle's hematoxylin-stained sections from ischemic mice treated with saline solution or SNP (0.5 mM) were immunodeveloped with a p21 specific antibody. In response to SNP treatment, p21 staining was positive at all considered time-points in muscle and endothelial cells' nuclei (indicated by arrows). Results represent six animals per group. (B) The graph shows that in the adductor muscle of control ischemic animals the number of p21-positive nuclei decreased between 8 h and 2 days after ischemia partially recovering at day 7. In contrast, in response to NO, p21 downregulation was prevented and, at days 2 and 7, the number of p21-positive nuclei was significantly higher than in controls (45±5 and 65±7 vs. 17±3 and 35±6). Results represent three animals per group. **P<0.001. The inset shows a representative picture of Western blotting analyses on p21 protein expression in total lysate obtained from normoperfused and ischemic adductor muscles at 8 h, 2 and 7 days after ischemia in the presence or absence of SNP. Samples were normalized by tubulin (T). Densitometric analysis shows that SNP prevents p21 reduction at day 2 and induces p21 expression about 5-fold above control at day 7. (C) p21 was co-expressed with LacZ (1 x 108 pfu/muscle for each vector) by adenovirus-mediated gene transfer 48 h before surgical induction of ischemia (black bars). Normal (white bars) and ischemic (striped bars) control animals were infected with adenovirus vector carrying LacZ (2 x 108 pfu/muscle). The graph shows that in response to overexpressed p21 the number of TUNEL-positive nuclei present in the adductor muscle of ischemic animals was reduced about 50% compared to ischemic animals infected with AdCMV.LacZ alone. The inset shows representative sections of adductor muscle from ischemic or normoperfused animals injected with AdCMV.LacZ and AdCMV.p21. The presence of β-galactosidase (blue) and TUNEL (brown) reactivity it is shown. Immunoreactivity to p21 in normal and ischemic tissues injected with saline solution, AdCMV.LacZ and AdCMV.LacZ/AdCMV.p21 is shown. These results represent six animals per group. *P<0.01; §P<0.05.

 
3.6 Adenovirus-mediated p21 expression reduces apoptosis in ischemic tissues
To address the possible role of p21 in vivo AdCMV.LacZ alone (2 x 108 pfu/muscle) or in combination with AdCMV.p21 (1 x 108 pfu/muscle for each vector) were injected in the right hindlimb of normoperfused nude mice 48 h before FAL. These animals were chosen to reduce the inflammatory response to the adenovirus vector. Animals were sacrificed 8 h after ischemia and apoptosis and p21 expression were evaluated by TUNEL and immunohistochemistry, respectively (see inset of Fig. 5C). Quantitative analysis showed that in ischemic muscles, in which p21 was overexposed, the number of apoptotic nuclei was reduced about 2.5-fold (Fig. 5C) compared to ischemic mice injected with saline solution and AdCMV.LacZ. Taken together, these results, in agreement with the antiapoptotic effect of AdCMV.p21 observed in vitro (Figs. 2 and 3)Go, show that elevated p21 levels may have a protective effect, in vivo, decreasing the post-ischemic apoptotic damage consequent to a reduction in blood flow.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, it was found that p21 induction, which is modulated by SS and NO [23], is required for the antiapoptotic effects of both these agents. Notably, the inhibition of endogenous NO production elicited by ECs treatment with L-NMA reduced but not abolished the protective effect of p21, indicating that additional mechanisms mediated by NO may cooperate with p21 ensuring optimal levels of cell survival in stress conditions. Prior studies have shown that SS may prevent cell death by the functional inhibition of pro-apoptotic BCL2 family members or the upregulation of scavenger molecules and NO production [24]; however, these observations have not been yet associated to a function of p21. The role played by p21 in the antiapoptotic effect of NO and SS, therefore, represents a novel finding and assigns to this molecule an important role in the antiapoptotic signaling activated by the biomechanical stimulation of endothelial cells.

In the present study, to investigate the protective role of p21 in vivo, a mouse model of surgically induced hindlimb ischemia was used. Clear signs of apoptosis were detected as early as 8 h after ischemia followed by the onset of a macrophage inflammatory infiltrate and tissue degeneration that reached a peak 48 h after femoral artery occlusion. Monitoring post-ischemic regeneration revealed the presence of an angiogenic process occurring, at the level of capillaries, between 2 and 7 days after surgery. Interestingly, at these time-points, no signs of arteriolar degeneration and/or arteriogenesis were detected (not shown) thus paralleling similar observations reported in other systems [22]. Administration of NO was per se beneficial reducing apoptosis as early as 8 h after ischemia preventing tissue degeneration and reduction of capillary density observed at day 2 after ischemia. Notably, at this time-point, p21 immunostaining was markedly reduced in untreated tissues; however, in the presence of NO, p21 was upregulated in the nuclei of capillary ECs, in smooth muscle cells as well as in the nuclei of muscle fibers. p21 expression was recently found to be regulated by NO in vitro [21]; however, its in vivo function, as a putative antiapoptotic molecule, is so far unexplored. Mice, in which p21 expression has been genetically inactivated, develop normally and do not present gross cardiovascular alterations [25]. Therefore, it is noteworthy that forced expression of p21 by means of direct adenovirus injection in ischemic adductor muscles significantly reduced the number of TUNEL-positive nuclei partially reproducing NO effects. In light of these observations, the flow-dependent antiapoptotic effects of p21, observed in vitro, were paralleled in vivo in a condition characterized by acute interruption of blood flow.

Hence, p21, which is capable of modulating cell growth and death, may also play an important role during remodeling of vascular structures that follows ischemic damage. In conclusion, the identification of p21 as a molecule important in the modulation of in vitro- and in vivo flow-dependent antiapoptotic effects may be relevant to design preventive interventions aimed at reducing apoptosis in acutely ischemic tissues.


   Acknowledgements
 
The authors thank Miss Susanna Rulli and Mrs. Gabriella Ricci for editorial assistance during the manuscript preparation. The authors would like to thank Paul B. Fisher, Department of Pathology of Urology, Columbia University, College of Physicians and Surgeons, for kindly providing the p21 antisense adenovirus. This work has been partly supported by grants of the Ministero della Salute (ICS-120.4/RA00-90 and ICS 120.1,1/RF00.208, ICS-190, ICS228, RF01/188), Associazione Italiana per la Ricerca sul Cancro (AIRC) grant #266/01 to M.C.C.


   Notes
 
1 Equal contributors. Back

Time for primary review 16 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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