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Am J Physiol Heart Circ Physiol 290: H1988-H1996, 2006. First published December 16, 2005; doi:10.1152/ajpheart.00344.2005
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ATP stimulates MMP-2 release from human aortic smooth muscle cells via JNK signaling pathway

William P. Robinson, III,1 Christelle D. Douillet,1 Peter M. Milano,1 Richard C. Boucher,2 Cam Patterson,2,3 and Preston B. Rich1

1Department of Surgery, 2Department of Medicine, and 3Carolina Cardiovascular Biology Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina

Submitted 6 April 2005 ; accepted in final form 8 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aortic smooth muscle cell release of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) has been implicated in aortic aneurysm pathogenesis, but proximal modulation of release is poorly understood. Extracellular nucleotides regulate vascular smooth muscle cell metabolism in response to physiochemical stresses, but nucleotide modulation of MMP and/or TIMP release has not been reported. We hypothesized that nucleotides modulate MMP-2 and TIMP-2 release from human aortic smooth muscle cells (HASMCs) via distinct purinergic receptors and signaling pathways. We exposed HASMCs to exogenous ATP and other nucleotides with and without interleukin-1beta (IL-1beta). HASMCs were pretreated in some experiments with apyrase, which degrades ATP, and inhibitors of ERK1/2, JNK, and p38 MAPK. MMP-2 and TIMP-2 released into supernatant were assessed using ELISA and Western blotting. ATP, adenosine, and UTP significantly stimulated MMP-2 release in the presence of IL-1beta (300 nM ATP: 181 ± 22%, P = 0.003; 30 µm adenosine: 244 ± 150%, P = 0.001; and 200 µm UTP: 153 ± 40%, P = 0.015; vs. 100% constitutive). ATP also stimulated MMP-2 release in the absence of IL-1beta (100 µm ATP: 148 ± 38% vs. 100% constitutive). Apyrase significantly reduced ATP-stimulated MMP-2 release (apyrase + 500 nM ATP: 59 ± 3% vs. 124 ± 7% with 500 nM ATP). Rank-order agonist potency for MMP-2 release was consistent with ATP activation of PAY and PAY receptors. ATP induced phosphorylation of intracellular JNK, and inhibition of the JNK pathway blocked ATP-stimulated MMP-2 release, indicating signaling via this pathway. Nucleotides are thus novel stimulants of MMP-2 release from HASMCs and may provide a mechanistic link between physiochemical stress in the aorta and aneurysms, especially in the context of inflammation.

abdominal aortic aneurysm; adenosine 5'-triphosphate; matrix metalloproteinase; extracellular nucleotides; mitogen-activated protein kinase; purines

MATRIX METALLOPROTEINASES (MMPs) secreted by vascular smooth muscle cells play an important role in physiological and pathophysiological degradation of elastin and collagen in vasculature (17, 61). Matrix metalloproteinase-2 (MMP-2), which degrades elastin and type IV basement membrane collagen (17, 33), has been strongly implicated in the development of abdominal aortic aneurysms (AAAs), which are characterized by destruction of elastin and collagen in the aortic media and adventitia (1, 2, 20, 37). Increased MMP-2 is found in aneurysmal vasculature and is likely necessary for AAA development (1, 9, 10, 19, 25, 35, 41, 49, 59). The tissue inhibitors of metalloproteinases (TIMPs), also produced by smooth muscle cells, bind and inhibit MMPs. TIMP-2 specifically inhibits MMP-2 and thus helps maintain physiological integrity of vessel extracellular matrix (7, 29). Increase in MMP-2 release above constitutive levels without increased TIMP-2 favors the medial and adventitial disruption that occurs in AAA development. Despite this understanding of the functional role of MMP-2 and TIMP-2 in aneurysm pathogenesis, the proximal stimuli of MMP-2 and TIMP-2 release are not well understood.

In addition to proteolysis, inflammation has been identified as a prominent etiologic factor in AAA development. A paradigm for AAA pathogenesis has emerged in which inflammatory cell infiltration and subsequent cytokine release stimulate MMP production and release, thereby initiating or enhancing proteolysis (54). Paracrine cytokine regulation of MMP-2 is evidenced by the finding that increased expression of MMP-2 in AAAs is most prominent in tissue where mesenchymal cells were surrounded by inflammatory infiltrate (10). However, definitive cytokine modulation of MMP-2 and TIMP-2 has not been identified (16, 31). Further investigation into the proximal modulators of MMP-2 and TIMP-2 release by both cytokines and noncytokine molecules is required. Given the interplay of proteolysis and inflammation, appropriate models investigating control of MMP-2 and TIMP-2 release from human aortic smooth muscle cells (HASMCs) should mimic the inflammatory milieu surrounding HASMCs in aneurysm development. Interleukin-1beta (IL-1beta) has been shown to be increased in aneurysms compared with normal aorta and is believed to be an important influence on some vascular smooth muscle cell functions, including MMP production (1, 36, 42). Inclusion of IL-1beta in ex vivo models to further investigate MMP-2 and TIMP-2 modulation is a simple yet useful way to mimic inflammatory conditions.

In addition to proteolysis and inflammation, biomechanical stress has been identified as a crucial factor in abdominal aortic aneurysm pathogenesis (59). However, alterations in metabolism at the level of the vascular smooth muscle cell as a result of the pericellular environment created by biomechanical stress has received minimal attention. Shear stress has been shown to cause cellular release of extracellular nucleotides, which act locally to impact numerous physiological and pathophysiological functions in a variety of tissues (6, 24, 34, 39, 47, 48, 62). In vasculature, the economy of nucleotides in the pericellular space and their cellular receptors is quite complex (44). Recent evidence indicates that nucleotides contribute to vascular smooth muscle cell proliferation, migration, and apoptosis. Acting on their receptors in this paracrine fashion, locally released nucleotides are believed to alter smooth muscle cell metabolism in pathological states such as atherosclerosis and restenosis after angioplasty (4, 8, 14, 26, 51–53).

There is thus a growing body of evidence implicating nucleotides in alterations of vascular smooth muscle cell metabolism in vascular disease. We theorized that extracellular nucleotides may also influence physiological and pathophysiological vessel matrix degradation via regulation of vascular smooth muscle MMP and TIMP release. A role for the purine nucleotide metabolite adenosine in MMP-2 secretion from trabecular network cells in the eye has been reported in one study (55). However, the relationship of nucleotides, nucleosides, MMPs, and TIMPs has not been examined in vasculature. Stress-induced nucleotides, stimulating MMP-2 in a paracrine fashion, may provide a mechanistic link between the shear stress thought to contribute to localized aneurysm development and matrix destruction implicated in aortic tissue becoming aneurysmal.

We hypothesized that extracellular nucleotides modulate MMP-2 and TIMP-2 release from HASMCs. Nucleotide stimulation of HASMCs was performed in the presence of IL-1beta to mimic an inflammatory pericellular milieu. We also examined the independent effects of ATP, adenosine, and UTP on MMP-2 release in the absence of IL-1beta to investigate the mechanism of nucleotide-induced changes. Stimulation of HASMCs with a variety of agonists was used to identify the purinergic receptors most prominently involved in MMP-2 release. In addition, we sought to identify the signaling pathway(s) mediating the nucleotide-induced MMP-2 release through inhibition of the ERK1/ERK2, JNK, and p38 MAPK pathways and through measurement of phosphorylation of these kinases in response to nucleotide stimulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Except where noted, all chemicals were obtained from Sigma (St. Louis, MO).

Cell culture. Primary normal HASMCs were obtained from Clonetics (Cambrex, Walkersville, MD). Cells in passages 4–6 were cultivated until 80–90% confluent in Clonetics medium (SmGM2). Before each experiment, cells were changed to medium without fetal bovine serum. Cultured HASMCs were exposed every 12 h for 72 h to a range of doses of ATP (100 nM–100 µM), adenosine (300 nM–300 µM), UTP (300 nM–200 µM), ADP (500 nM), adenosine 5'-[{gamma}-thio]triphosphate (ATP{gamma}S, 500 nM; Roche Diagnostics, Indianapolis, IN), and 2-(methylthio)adenosine 5'-diphosphate (2-MeS-ADP, 500 nM) in the presence and absence of IL-1beta. Cells were exposed to a single dose of IL-1beta (2 ng/ml) in experiments involving IL-1beta. In some experiments, HASMCs were pretreated with specific inhibitors of ERK1/ERK2 (U0126; Biosource, Camarillo, CA), JNK (JNK inhibitor I; Calbiochem, San Diego, CA), and p38 MAPK (SB203580) as well as apyrase (1 U/ml; Sigma) 20 min before stimulation. Conditioned media were collected after 72 h and centrifuged (1,000 g for 10 min at 4°C), and the supernatants were frozen at –80°C until analysis.

Protein determination. Cell culture supernatant protein content was measured spectrophotometrically by the commercially available Coomassie Plus protein assay (Pierce, Rockford, IL) following the manufacturer’s instructions.

Western blots. Proteins from the conditioned media were resolved on SDS-PAGE, and the separated proteins in the gel were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and immunoblotted with mouse monoclonal anti-human MMP-2 antibody (recognizing proenzyme, active, and bound moieties, 1:100 dilution; Oncogene, San Diego, CA). The blots were revealed using goat anti-mouse IgG antibody conjugated with horseradish peroxidase (1:10,000 dilution; Calbiochem). Immunoreactive bands were visualized using the ECL-plus chemiluminescence system (Amersham, Little Chalfont, UK) and Biomax MR film (Kodak, Rochester, NY). Films were scanned with a Multi-Image light cabinet (Alpha Innotech, San Leandro, CA), and the intensities of the bands were analyzed with AlphaEase (version 3.3a; Alpha Innotech).

ELISA assay. Cell culture supernatants were analyzed with ELISA for total human MMP-2 (proMMP-2, active MMP-2, and MMP-2:TIMP-2 complex) (Calbiochem) and TIMP-2 (R&D Systems, Minneapolis, MN) according to the manufacturers’ directions. Results were normalized per microgram of protein released into the supernatant and expressed as percentages of constitutive release.

ERK and JNK phosphorylation assays. In some experiments, cells were stimulated as described with 500 nM ATP for times ranging from 1 min to 3 h. Cells were then washed twice with cold PBS and lysed with cell extraction buffer. Cell extraction buffer for JNK contained 6 M urea, 0.5% Triton X-100, and protease inhibitor cocktail (according to the manufacturer’s guidelines; Sigma). Cell extraction buffer for ERK contained 100 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF (all from Sigma), and protease inhibitor cocktail (according to the manufacturer’s guidelines; Sigma). Cell lysates were centrifuged (13,000 g for 10 min at 4°C), and the supernatants were frozen at –80°C until ERK1/2 or JNK analysis. Samples were boiled before phosphorylated ERK1/2 and total ERK1/2 assay. Both phosphorylated ERK and total ERK as well as phosphorylated JNK and total JNK were analyzed on the same day using ELISA (Biosource, Camarillo, CA). The ratios of phosphorylated to total ERK and JNK were then calculated, and results were expressed as percentages of the ratio calculated for unstimulated control cells.

Statistical analysis. Data are presented as means ± SE. Data were analyzed using ANOVA and Fisher’s protected least significant difference tests where appropriate. Significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Nucleotide stimulation of HASMC in presence of IL-1beta. In conditions mimicking the inflammatory milieu of aneurysm development, nucleotides stimulated increased MMP-2 release from HASMCs. ATP stimulated increased MMP-2 release compared with baseline constitutive release over a range of physiological extracellular concentrations from 100 to 500 nM with a maximal effect at 300 nM (Fig. 1A). Adenosine maximally stimulated MMP-2 release at 30 µM (P = 0.001; Fig. 1B). UTP (200 µM) stimulation increased MMP-2 expression as well, although more moderately (P = 0.015; Fig. 1C). ATP-, adenosine-, and UTP-stimulated MMP-2 release was confirmed with Western blot analysis (Fig. 2). There was no significant change in TIMP-2 release with ATP, adenosine, or UTP, although adenosine tended to increase TIMP-2 release at 30 µM (P = 0.07; Fig. 1B).


Figure 1
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  		Fig. 1. Nucleotide stimulation of human aortic smooth muscle cells (HASMCs) in the presence of interleukin-1beta (IL-1beta). ELISA results demonstrate percent constitutive matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) release after ATP stimulation (A; n ≥ 3 at each dose), adenosine stimulation (B; n ≥ 2 at each dose), and UTP stimulation (C; n ≥ 2 at each dose). ADO, adenosine. *P < 0.05 compared with 100% constitutive release (C).

 

Figure 2
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  		Fig. 2. Confirmation of nucleotide-induced MMP-2 release with Western blot analysis. Maximal MMP-2 release is observed at 300 nM ATP (A), 30 µM adenosine (B), and 200 µM UTP (C). rMMP-2, recombinant MMP-2.

 
Nucleotide stimulation of HASMC in absence of IL-1beta. To determine the effects of nucleotides independent of other stimuli, we exposed HASMCs to increasing doses of nucleotides in the absence of IL-1beta. Stimulation of HASMCs with ATP alone resulted in an increase in MMP-2 release with a maximal effect at 100 µM (P = 0.019; Fig. 3A). This effect was confirmed with Western blot analysis (Fig. 3B). Adenosine and UTP stimulation did not alter MMP-2 release (data not shown).


Figure 3
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  		Fig. 3. ATP stimulation of HASMCs in absence of IL-1beta. A: ELISA results demonstrate percent constitutive release of MMP-2 with maximal response at 100 µM ATP (n ≥4 at each dose). *P < 0.05. B: representative Western blot confirming maximum MMP-2 release at 100 µM ATP.

 
Confirmation of ATP stimulation of MMP-2 release. To determine whether the enhanced MMP-2 release seen with exogenous ATP stimulation was due to ATP or metabolites of ATP (i.e., ADP, AMP, adenosine), we pretreated the HASMCs with apyrase, which quickly degrades added ATP to ADP and AMP. Apyrase eliminated the ATP-induced MMP-2 release (Fig. 4), underscoring the importance of ATP itself in stimulating MMP-2 release. Moreover, apyrase decreased constitutive MMP-2 release occurring without exogenous ATP addition (Fig. 4). The ATP analog ATP{gamma}S significantly increased MMP-2 release (Fig. 5), again confirming ATP as a stimulant of MMP-2 release.


Figure 4
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  		Fig. 4. ATP stimulation of HASMCs in the absence and presence of apyrase. ELISA results demonstrate MMP-2 release observed with no stimulation, 500 nM ATP stimulation alone, 500 nM ATP stimulation after pretreatment with apyrase, and apyrase pretreatment alone (n ≥ 4 for all groups). *P < 0.005 compared with constitutive release. {ddagger}P < 0.001 compared with ATP alone.

 

Figure 5
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  		Fig. 5. Adenosine 5'-[{gamma}-thio]triphosphate (ATP{gamma}S) stimulation of HASMCs. MMP-2 release was observed after ATP{gamma}S stimulation of HASMC. MMP-2 release was significantly increased at 500 nM ATP{gamma}S (n = 7). *P < 0.008 compared with constitutive release.

 
Agonist profile. To attempt to identify the nucleotide receptor mediating ATP-induced MMP-2 release, we compared the potency of stimulation of MMP-2 release with a variety of P2Y agonists at an identical physiological dose (500 nM) with each other and with the control. For potency of stimulation of MMP-2 release, the rank order was ATP > 2-MeS-ADP > ATP{gamma}S > ADP > UTP > constitutive (Fig. 6).


Figure 6
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  		Fig. 6. Stimulation of HASMCs with multiple P2Y agonists. ELISA results demonstrate MMP-2 release after stimulation with 500 nM ATP, 500 nM 2-(methylthio)adenosine 5'-diphosphate (2-MeS-ADP), 500 nM ATP{gamma}S, 500 nM ADP, and 500 nM UTP (n ≥ 3 for all groups). *P < 0.003 compared with constitutive release.

 
Signal transduction pathways. To determine whether ERK1/ERK2, JNK, and p38 MAPK were involved in ATP-induced MMP-2 release, we pretreated HASMCs with inhibitors of the ERK1/ERK2 (U0126), JNK (JNK inhibitor I), and p38 MAPK pathways (SB203580) 20 min before stimulation with ATP (Fig. 7). ATP-induced MMP-2 release was significantly inhibited by pretreatment with U0126 (P = 0.013) as well as JNK inhibitor I (P = 0.0004). Pretreatment with SB203580 did not alter ATP-induced MMP-2 release.


Figure 7
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  		Fig. 7. Stimulation of HASMCs after second messenger pathway inhibition. ELISA results demonstrate MMP-2 release after stimulation with 500 nM ATP, with the specific kinase inhibitors U0126 (–ERK), JNK inhibitor I (–JNK), and SB203580 (–p38), and with 500 nM ATP after pretreatment of cells with U0126 (ATP+ –ERK), JNK inhibitor I (ATP+ –JNK), and SB203580 (ATP+ –p38) (n ≥ 4 for all groups except JNK, where n ≥ 2). *P < 0.02 compared with constitutive release. {dagger}P < 0.02 compared with 500 nM ATP alone.

 
ATP and JNK and ERK phosphorylation. To support our findings that ATP stimulates MMP-1 release via a JNK-dependent signaling mechanism, we measured time-dependent phosphorylation of JNK after stimulation with 500 nM ATP (Fig. 8). The percentage of phosphorylated JNK increased substantially compared with control within 5 min of stimulation of HASMCs with ATP (P = 0.004). On the other hand, ATP did not significantly induce ERK phosphorylation (data not shown). The JNK signaling mechanism is therefore more strongly implicated in ATP-induced MMP-2 release.


Figure 8
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  		Fig. 8. ATP stimulation of phosphorylation of JNK. ELISA results demonstrate the ratio of phosphorylated JNK to total intracellular JNK at various times after stimulation with 500 nM ATP (n ≥ 3 for all groups ), expressed as a percentage of control. *P < 0.05 compared with constitutive phosphorylation.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although MMP-2 is known to play an important role in vascular pathophysiology, including abdominal aortic aneurysm development, the proximal stimuli of MMP-2 release are not understood. Our results demonstrate for the first time that extracellular nucleotides modulate MMP-2 release from HASMCs. Although the greatest MMP-2 stimulation was observed in the context of inflammation, ATP also independently stimulates MMP-2 release in the absence of cytokines. Based on the agonist potency profile, this effect is consistent with activation of the purinoceptors known to be abundantly expressed on vascular smooth muscle cells, P2Y1 and P2Y2. Furthermore, the JNK signaling pathway mediates ATP-induced MMP-2 release.

Abdominal aortic aneurysms are increasing in frequency in our aging population. Little progress has been made in reducing the considerable morbidity and mortality associated with aneurysms, given that the mortality rate of a ruptured AAA remains 40–50% even among those patients who reach hospitalization (1). Treatment focuses on the complications of aneurysm development. With our relatively limited understanding of aneurysm pathophysiology, the ability to attenuate the underlying disease process is extremely limited.

MMP-modulated proteolysis of aortic wall connective tissue has been identified as one key component in aneurysm pathogenesis (59). In particular, aortic smooth muscle cell release of MMP-2 is believed to be a critical etiologic factor. Compared with those in normal and atherosclerotic aorta, MMP-2 mRNA and protein levels in the aortic media are observed to be elevated in AAAs, particularly in small AAAs (10, 19). This suggests an early role in aneurysm initiation. Importantly, elevated MMP-2 in AAAs was found to exist largely in its active form tightly bound to tissue matrix, suggesting a causative role in aneurysm formation (10). Smooth muscle cells harvested from AAAs demonstrate increased MMP-2 mRNA and protein levels and enhanced proteolytic activity compared with cells cultured from normal aorta (9, 41). It is believed that the overproduction of MMP-2, and subsequent extracellular matrix and basement membrane digestion, leads to increased migration of smooth muscle cells into the intima and further weakening of the medial layer (22). The crucial role of MMP-2 in aneurysm pathogenesis has been confirmed in an in vivo model. Longo et al. (35) found that MMP-2 knockout mice resist experimental aneurysm induction, suggesting that smooth muscle cell-derived MMP-2 is required to produce AAA in their murine model.

MMPs are bound and inactivated by TIMPs. Because it specifically binds to MMP-2, TIMP-2 plays a homeostatic role in vessel remodeling. A relative paucity of TIMP-2 in relation to MMP-2 favors extracellular matrix degradation (17). TIMP-2 levels in AAAs have been found to be unchanged compared with controls (9, 21). Furthermore, increased TIMP-2 production by vascular smooth muscle cells that were implanted into aortic xenografts in rats was associated with resistance to aneurysm formation by elastase infusion (3).

Little is known about the proximal regulators of MMP-2 and TIMP-2 transcription and production, particularly in the inflammatory conditions contributing to aneurysm development. As discussed previously, IL-1beta was included in our culture conditions to replicate the cytokine milieu surrounding aortic smooth muscle cells in aneurysm development. IL-1beta alone did not significantly stimulate MMP-2 or TIMP-2 release from HASMCs. This finding is consistent with a previous report that IL-1beta did not influence MMP-2 mRNA expression but is inconsistent with a previous study indicating that IL-1beta induced MMP-2 expression in cardiac myofibroblasts (30, 31).

However, in the presence of IL-1beta, exogenous ATP, adenosine, and UTP were able to significantly increase MMP-2 release. There were no significant changes in TIMP-2 release. When one compares the maximal ATP stimulation in the presence of IL-1beta (181 ± 22% of control) with that in the absence of IL-1beta (148 ± 38% of control), there appears to be some additivity between ATP and IL-1beta. It is known that IL-1beta upregulates P2Y2 receptors on vascular smooth muscle cells (27). By making more receptors available for activation by ATP, IL-1beta may enhance ATP stimulation of MMP-2 release. Alternatively, IL-1beta and nucleotides may synergistically activate the same downstream second messenger pathways for MMP-2 release. Further experiments are necessary to determine the ways in which nucleotide and IL-1beta stimuli might interact and to understand why the maximal MMP-2 stimulation was observed at 300 nM with IL-1beta rather than at 100 µM ATP in the absence of IL-1beta.

Although the stimulation of HASMCs with IL-1beta creates a model mimicking in vivo conditions, we examined nucleotide stimulation of HASMCs in the absence of IL-1beta to more clearly examine the mechanism by which nucleotides modulate MMP-2 release. Our data demonstrate that ATP, but not adenosine or UTP, independently stimulates increased MMP-2 release from HASMCs. We were able to observe increased MMP-2 even at basal physiological concentrations of ATP (300 nM), although the maximal MMP-2 release was observed at 100 µM, a concentration that may be achieved in protected intercellular spaces.

To determine whether the increased MMP-2 was due to ATP itself or its downstream metabolites, we performed ATP stimulation in the presence and absence of the ATP-degrading enzyme apyrase. Addition of apyrase eliminated the increased MMP-2 seen in response to ATP (apyrase + 500 nM ATP: 59 ± 3% vs. 124 ± 7% with 500 nM ATP), confirming ATP itself as the primary stimulant of MMP-2 release. Importantly, the addition of apyrase significantly reduced baseline constitutive MMP-2 release (apyrase: 59 ± 6% vs. 100% constitutive release). This result indicates that constitutive MMP-2 release may be dependent on autocrine activation of purinergic receptors by ATP. Identification of ATP as the primary stimulant of MMP-2 release also is supported by the significant efficacy of ATP{gamma}S, a stable ATP analog, on MMP-2 release. The extracellular concentrations of ATP, adenosine, and UTP noted to stimulate MMP-2 release both independently and in the context of inflammation are similar to those required to regulate vascular smooth muscle cell proliferation and collagen and protein synthesis (13, 26, 58, 60). Nucleotide modulation of MMP-2 release in the absence of inflammatory conditions may have important implications for the initiation of vessel proteolysis before inflammatory cells have infiltrated aneurysmal tissue.

The nucleotide receptors present on vascular smooth muscle cells have been well characterized, primarily via studies examining the impact of nucleotides on vascular smooth muscle cell proliferation. Evidence suggests that P2Y1 and P2Y2 are the dominant receptors on vascular smooth muscle cells. ATP and ADP induce vascular smooth muscle cell proliferation, effects attributed to P2Y1 and P2Y2 activation (14, 58). UTP also stimulates vascular smooth muscle cell mitogenesis, which, according to some authors (26, 28), implicates P2Y2 and/or P2Y4 receptor involvement. However, Erlinge et al. (15) confirmed that P2Y1 and P2Y2 mRNAs were the most abundantly expressed P2Y receptors in the proliferative (synthetic) phenotype of rat aortic smooth muscle cells. P2Y1 and P2Y2 were upregulated 342- and 8-fold, respectively compared with expression in the contractile phenotype. On the other hand, P2Y4 and P2Y6 receptors were unchanged and P2X1 was completely downregulated to undetectable levels in the proliferative phenotype (15). We therefore created an agonist potency profile to determine whether nucleotide-induced MMP-2 release was consistent with activation of these receptors prominent in vascular smooth muscle cell metabolism.

The P2 receptor agonist potency profile (ATP > 2-MeS-ADP > ATP{gamma}S > ADP > UTP) on MMP-2 release in our experiments is consistent with activation of P2Y1 receptor and perhaps P2Y2 receptors. The agonist receptor profile, including natural and synthetic agonists, of the P2Y1 receptor is 2-MeS-ADP > 2-MeS-ATP > ADP > ATP, with no activation by UTP. The P2Y2 agonist profile is ATP = UTP > ATP{gamma}S. UTP is the most potent agonist at the P2Y4 and P2Y6 receptors, at which ATP is only modestly active (4). In our experiments, the potency of ATP, and to a lesser extent ADP (including its stable analog 2-MeS-ADP), compared with UTP is most consistent with P2Y1 activation. The very modest UTP effect is consistent with the diminished expression of P2Y4 and P2Y6 in proliferative vascular smooth muscle cells previously described. Because ATP and UTP are not equipotent in stimulating MMP-2 release, activation of P2Y2 receptor would appear to be modest. Conversion of exogenous UTP to ATP via nucleoside diphosphokinase lining the cell membranes may mediate limited receptor activation and explain the modest impact of UTP on MMP-2 release (43). Finally, a role for P2X activation is unlikely given the evidence demonstrating undetectable P2X expression in the proliferative phenotype taken on by HASMCs in culture. P2X receptors on vascular smooth muscle cells are generally believed to mediate fast actions such as second-to-second vasomotor tone rather than long-term metabolic functions of the cell (4, 14). Definitive characterization of receptor activation may be aided in the future by the development of receptor-specific P2Y antagonists.

In the present study we report the novel finding that the JNK pathway mediates the ATP-induced release of MMP-2 from HASMCs. It is known that purinergic receptors are coupled to activation of tyrosine kinases and downstream MAPK cascades (4, 8, 14). Shearer and Crosson (55) reported that the ERK1/ERK2 pathway mediates adenosine stimulation of MMP-2 secretion by trabecular network cells of the eye. However, there has been little previous investigation of these pathways in MMP-2 release from vascular smooth muscle cells. ATP stimulation of HASMCs induced JNK phosphorylation (Fig. 8), and JNK inhibition reduces ATP-induced MMP-2 release (Fig. 7). ERK1/2 does not seem to be implicated in ATP-induced MMP-2 release in HASMCs, because ATP does not induce ERK1/2 phosphorylation. ATP and adenosine are known to modulate different effects via distinct receptors and different second messenger cascades in a variety of cell types. We attribute the decreased MMP-2 release in the presence of the ERK1/ERK2 inhibitor U0126 to either to nonspecific metabolic inhibition of HASMCs by this agent or to indirect activation of ERK1/ERK2 via complex second messenger cascades, which was not measured in our phosphorylation experiments. Although p38 MAPK is involved in the induction of MMP-2 in nonvascular cells (32, 40), this pathway does not appear to mediate ATP-induced MMP-2 release in HASMCs.

These data suggest that MMP-2 levels and the potential balance between MMP-2 and TIMP-2 release from HASMCs in vivo may be dependent on the local concentrations of nucleotides surrounding HASMCs. ATP and UTP are known to be released into the extracellular space surrounding vascular smooth muscle cells from endothelium under liquid flow, hypoxia, and shear stresses (24, 50). ATP and adenosine are also released from stimulated platelets, damaged smooth muscle cells, and local sympathetic nerves (5, 6, 24, 50). ATP is subsequently degraded by extracellular ectoATPases into ADP, AMP, and adenosine (23). Cells are thus bathed in a dynamic pool of nucleotides that may vary from moment to moment in a given tissue depending on the balance of nucleotide release and degradation. By stimulating MMP-2 release from aortic smooth muscle cells, nucleotides potentially contribute to the modulation of vessel matrix proteolysis. Improvements in the ability to characterize ATP, adenosine, and UTP concentrations in vasculature in ex vivo and in vivo models under both normal and pathological conditions may allow assessment of the potential for matrix destruction based on nucleotide-stimulated MMP-2 release.

As stress-responsive molecules, nucleotide involvement in AAA pathogenesis via paracrine regulation of proteolysis is particularly plausible. Nucleotides modulate pathophysiological responses to biomechanical stress in a variety of diseases, including restenosis after angioplasty, neointimal hyperplasia of arterial bypass grafts, and ventilator-induced lung injury (4, 8, 11, 12, 14, 26, 45, 46, 51–53). Biomechanical stresses, including both cyclic circumferential strain and shear stress, are thought to be etiologic in AAA development, growth, and rupture (18, 38, 56, 57).

However, the cellular mechanisms by which wall stress promotes AAA development and expansion are unknown. Nucleotide-induced MMP-2 release from HASMCs provides a potential mechanistic link between biomechanical stress and the increased degradation of medial elastin and collagen seen in abdominal aortic aneurysms. Future experiments are necessary to examine the impact of biomechanical stress on nucleotide release, purinergic receptor activation, and MMP-2 release from smooth muscle cells.

In the present study, we have measured MMP-2 and TIMP-2 in cell culture supernatant. This method may underestimate the impact of nucleotide stimulation, because there may be intercellular and intracellular (but not released) MMP-2 and TIMP-2 that were not measured in this model. Furthermore, aortic smooth muscle cells in vivo are continuously exposed to a spectrum of nucleotides released from a multitude of surrounding cells. We speculate that the release of MMP-2 and TIMP-2 observed after intermittent nucleotide exposure over 3 days in our model may be substantially increased in vivo as a result of continuous nucleotide exposure over longer periods of time. The potential impact of significantly increased MMP-2 on aortic medial degradation over the life of the tissue is substantial.

In summary, this report describes a novel role for nucleotides in the modulation of MMP-2 release from HASMCs. ATP, adenosine, and UTP stimulate MMP-2 release in the context of the inflammation thought to effect aortic aneurysm development. Furthermore, ATP independently increases MMP-2 release via the JNK signaling pathways. Nucleotides potentially represent powerful proximal modulators of the extracellular matrix remodeling central to AAA pathogenesis, and their signaling cascades may provide targets for future therapeutic intervention. Nucleotide-induced MMP-2 release from HASMCs provides a potential mechanistic link between biomechanical stress and the increased aortic proteolysis crucial to development of AAAs.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants T32 GM-008450 and 1K08 HL-072836-01.


   ACKNOWLEDGMENTS
 
We thank the Immunotechnology Core Laboratory (University of North Carolina, Chapel Hill) for invaluable assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. B. Rich, Dept. of Surgery, Section of Trauma and Critical Care, 186 Medical School Wing D, CB# 7228, Chapel Hill, NC 27599-7228 (e-mail: prich{at}med.unc.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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