JOURNAL OF CELLULAR PHYSIOLOGY 208:229–237 (2006)
Nitric Oxide and p38 MAP Kinase Mediate Shear Stress-Dependent Inhibition of MMP-2 Production in Microvascular Endothelial Cells MALGORZATA MILKIEWICZ,1,2 CASSANDRA KELLAND,1 STEPHEN COLGAN,1 AND TARA L. HAAS1* 1 School of Kinesiology and Health Sciences, York University, Toronto, Ontario, Canada 2 Department of Laboratory Diagnostics and Molecular Medicine, Pomeranian Medical University, Szczecin, Poland Chronic exposure of the skeletal muscle microcirculation to elevated shear stress-induces angiogenesis. Previous studies observed that shear stress-induced capillary growth involves luminal sprouting, or internal division, of the capillaries, which is characterized by a minimal proliferative response and the retention of an intact basement membrane. Matrix metalloproteinases (MMPs) are associated with the process of abluminal sprouting angiogenesis, but may not be required for the process of luminal division during capillary growth. We analyzed the production of MMP-2, using both the in vivo model of prazosin-induced angiogenesis in rat skeletal muscle, and cultured microvascular endothelial cells exposed to laminar shear stress. We found that MMP-2 was not elevated in capillaries of shear stress-stimulated skeletal muscle, despite a significant increase in capillary number in response to a shear stress stimulus. In cultured microvascular endothelial cells, MMP-2 mRNA and protein levels were attenuated significantly in response to shear stress exposure. This effect on MMP-2 was reversed by nitric oxide (NO) synthase inhibition using LNNA. In contrast, exposure of static cultures of endothelial cells to NO donors significantly reduced MMP-2 production. Shear stress exposure and NO donors both modified phosphorylation levels of several members of the MAPK family. Treatment of shear stressexposed cells with the p38 MAPK inhibitor, SB203580, abolished the shear stress-mediated reduction in MMP-2 mRNA. Thus, our data provide strong evidence that elevated shear stress inhibits MMP-2 production in microvascular endothelial cells, an effect that is mediated by signal pathways involving both production of NO and activation of p38 MAPK. J. Cell. Physiol. 208: 229–237, 2006. ß 2006 Wiley-Liss, Inc.
Microvascular endothelial cells experience continuous exposure to fluid shear stress, the magnitude of which varies dependent on location within the vascular network, and physiological stressors. Long duration exposure to elevated shear stress is a potent stimulus for capillary growth in skeletal muscle (Dawson and Hudlicka, 1989). Typically, the angiogenesis process involves augmentation in cell proliferation accompanied by increased production and activation of proteases, such as matrix metalloproteinases (MMPs), in order to facilitate abluminal sprouting. However, angiogenesis in response to shear stress is not associated with a substantial increase in the rate of endothelial cell proliferation or basement membrane degradation (Egginton et al., 2001). The absence of observable basement membrane degradation during shear stressmediated capillary growth implies that shear stress is not a positive regulator of MMP production. The increased activity and expression of endothelial cell nitric oxide synthase (eNOS), which leads to elevated levels of the potent vasodilator nitric oxide (NO), is a well-defined response to both transient and chronic increases in shear stress (Kuchan et al., 1994; Koller and Kaley, 1998; Fleming and Busse, 2003). Endothelial NO production is credited with constitutive maintenance of appropriate vascular tone, as well as facilitating shear stress-dependent vasodilation to provide homeostatic regulation of shear forces on the endothelial surface (Koller and Kaley, 1998). NO is not simply a vasomotive regulator, it also provides cellular protection through anti-thrombotic, anti-oxidative, and anti-inflammatory functions that are both cyclic guanosine monophosphate (cGMP) dependent and independent (Traub and Berk, 1998). It is feasible that shear ß 2006 WILEY-LISS, INC.
stress-induced NO production is a critical component in shear stress-induced angiogenesis. NO involvement in capillary growth is somewhat controversial, as there are reports of it both eliciting and inhibiting angiogenesis (Papapetropoulos et al., 1997; Jones et al., 2004). NO donors reduced angiogenesis in both CAM and matrigel models of angiogenesis, whereas NOS inhibitors stimulated angiogenesis (Pipilisynetos et al., 1994). In gastric microvascular endothelial cells, low concentrations of the NO donor SNAP (0.1–0.03 mM) stimulated capillary-like tube formation and cell migration through activation of PKC and ERK and c-Jun phosphorylation, whereas high concentrations of SNAP (>2 mM) inhibited these angiogenic responses (Jones et al., 2004). Inhibition of endogenous NOS with LNAME blocked VEGF-induced angiogenesis in rabbit cornea (Ziche et al., 1997). L-NNA treatment prevented angiogenesis in EDL muscle undergoing chronic electrical stimulation (Hudlicka et al., 2000), whereas treatment with the NO-releasing aspirin derivative (NCX4016) augmented capillary density in ischemic hindlimbs of mice (Emanueli et al., 2004).
Contract grant sponsor: Canadian Institutes of Health Research; Contract grant sponsor: Premiers Research Excellence Award. *Correspondence to: Tara L. Haas, School of Kinesiology and Health Sciences, York University, 4700 Keele St., Toronto, Ont., Canada M3J 1P3. E-mail:
[email protected] Received 18 November 2005; Accepted 24 February 2006 DOI: 10.1002/jcp.20658
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In large arteries, increased shear stress causes extensive remodeling of the vascular wall, and correlates with increased production of MMP-2 and MMP-9 (Tronc et al., 2000; Sho et al., 2002). However, elevated shear stress in rat EDL capillaries does not evoke an increase in MMP-2, as assessed by immunohistochemistry or by Western blotting of whole muscle homogenates (Rivilis et al., 2002). Further, chronic exposure to shear stress decreases MT1-MMP expression in cultured microvascular endothelial cells (Yun et al., 2002). Given the apparent contradictions in the regulation of endothelial cell synthesis of MMPs in response to shear stress, we clarified these relationships and further investigated the role of specific NO production in the shear stress mediated regulation of MMP-2. MATERIALS AND METHODS Materials
All chemicals for cellular lysis, electrophoresis and Western blotting were purchased from Sigma Aldrich Canada (Oakville, ON), as was No-nitro-L-arginine (LNNA), prazosin hydrochloride, and anisomycin. Cell culture media components were purchased from Invitrogen Canada (Burlington, ON). NO donors (NOC18, SNAP) and p38 MAPK inhibitor SB203580 were obtained from EMD Biosciences (San Diego, CA). Phospho-specific and total antibodies for ERK1/2, c-jun, p38, and beta-actin were purchased from New England Biolabs (Pickering, ON). Rat studies
Male Sprague–Dawley rats (300 g) (Charles River Labs, Montreal, QE) were treated for 4 days with LNNA (100 mg/L) (Hudlicka et al., 2000), or with prazosin (50 mg/L) (Dawson and Hudlicka, 1989). Both LNNA and prazosin were administered daily in the drinking water. After 4 days, extensor digitorum longus muscles (EDL) from treated and age-matched control animals were removed and frozen in liquid nitrogen for subsequent analyses. Cell culture
Microvascular endothelial cells were isolated from rat EDL (Han et al., 2003) and propagated in culture for a maximum of 10 passages in gelatin-coated flasks with appropriate growth media (DMEM supplemented with 10% FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin, 0.3 mg/ml L-glutamine, and 0.11 mg/ml sodium pyruvate) at 378C with 7% CO2. For shear stress experiments, cells were plated onto glass coverslips coated with gelatin, allowed to adhere and reach confluency (18 h). Coverslips then were mounted in a laminar flow chamber (FCS2; Bioptechs). Two flow chambers were operated in parallel, with flow controlled by a dual-head micropump (Ismatec, Harvard Apparatus, St. Laurent, QE), calibrated to deliver 3.5 ml media per minute per flow chamber. Shear stress (t, in dyne/cm2), calculated according to the formula: g ¼ 6Q/ab2 and t ¼ mg, where Q is volume flow (in ml/sec), a and b are the width and height (in cm) of the flow path, and m is the viscosity of the perfusate (in poise), was 16 dyne/cm2. This value is identical to the calculated shear stress present within EDL capillaries during prazosin-induced vasodilation (Milkiewicz et al., 2001). After specific time intervals, cells were processed for RNA or protein analysis. Cells plated on coverslips and not subjected to fluid flow served as static controls. For drug treatments, one of the two flow chambers was exposed to drug while the other flow chamber served as the vehicle-only shear stress condition. Drugs were administered at final concentrations of 30 mM for LNNA (Bolz and Pohl, 1997) or, 40 mM for SB203580 (Boyd et al., 2005). For static cultures, cells (1 million cells/well) were plated on 35 mm dishes coated with 2.5 mg type I collagen/ml coating buffer (0.1 M NaHCO3, pH 9.4). Cells were treated with glycoSNAP (10 or 100 mM) or NOC-18 to elicit a sustained production of NO. In some experiments, cells were pre-incubated for Journal of Cellular Physiology DOI 10.1002/jcp
15 min with anisomycin (0.01 mM) prior to treatment with NO donors. Laser capture microdissection (LCM)
As described in detail earlier (Milkiewicz and Haas, 2005), LCM was performed on cryosections of rat EDL muscle (8 mmthick) that were stained with isolectin GS-IB4 from Griffonia simplicifolia Alexa Fluor1 488 conjugate (1 mg/ml) diluted 1:200 in PBS (Molecular Probes, Eugene, OR) in order to detect capillaries. Capillary laser microdissection was performed on freshly deyhydrated sections using an Arcturus PixCellTM II system with a power setting of 100 mW, a 7.5-mm spot size and a pulse duration ranging from 100 to 200 ms (Arcturus Engineering, Inc., Palo Alto, CA). Capillaries distinguished by the size ( 0.01 vs. control) and p42 (*P > 0.01 vs. control levels) (n ¼ 5).
occurs independently of (or upstream to) activation of NO production. Recently, p38 MAPK-dependent activation of eNOS was observed in endothelial cells (Anter et al., 2004, 2005). In their study, p38 MAPK activated PI3K, which in turn phosphorylated its substrate Akt. Finally, Akt-mediated phosphorylation of eNOS enhanced the synthesis of NO. This relationship remains to be determined in our model system. Importantly, we found that p38 activation during shear stress exposure contributes to the shear stress-induced decrease in MMP-2 production, as p38 inhibitor treatment resulted in a shear stress-mediated increase in MMP-2 mRNA. Activation of p38 MAPK may modulate MMP-2 expression through two independent mechanisms. Firstly, phosphorylation of p38 MAPK via cGMPdependent protein kinase activation (Browning et al.,
2000) leads to the activation of numerous transcription factors, including activating transcription factor (ATF) and MEF2 family members (Bogatcheva et al., 2003; Harper et al., 2005). ATF3 is induced rapidly in response to environmental stress in both endothelial and smooth muscle cells (Hai et al., 1999). Recently, ATF3 was reported to mediate the inhibitory effect of NO on MMP2 expression in static cultures of endothelial cells (Chen and Wang, 2004). Although ATF3 was shown to downregulate p53-dependent MMP-2 transcription (Yan et al., 2002), a more recent study from the same researchers demonstrated ATF3-dependent activation of p53 (Yan et al., 2005). Further studies are required to define specific involvement of ATF and/or MEF family transcription factors with the MMP-2 promoter in response to shear stress.
Fig. 7. Effect of shear stress and nitric oxide on phosphorylation of p38 levels. A: Phospho-p38 levels increased significantly in response to 2 h exposure to shear stress (SS). However, cells treated with LNNA and exposed to shear stress (SS þ LNNA) also maintained a significant increase in phospho-p38 relative to control (n ¼ 3; *P < 0.05 vs. control). Total p38 signal was used to normalize for loading. B:
Cultured microvascular endothelial cells were analyzed to determine the effect on NO donor SNAP (100 mM; 15 min) on phospho-p38. SNAP treatment induced a significant increase in phospho-p38, although this increase was not as large as that seen in response to anisomycin treatment (n ¼ 3; *P < 0.05 vs. control). Beta actin levels were used to normalize for loading.
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Fig. 8. p38 inhibition blocks shear stress reduction in MMP-2 mRNA and protein A: MMP-2 mRNA, detected by real-time PCR, was not significantly decreased following 2 h of shear stress exposure (SS), or treatment of static cultures with the p38 inhibitor SB203580 (SB; 40 mM). However, MMP-2 mRNA increased significantly in cells exposed to 2 h of shear stress in the presence of the p38 inhibitor
(SS þ SB) (n ¼ 3; *P < 0.05 vs. control, SB alone; #P < 0.01 vs. 2 h shear stress). B: MMP-2 protein, detected by gelatin zymography, was not affected by 2 h of shear stress exposure (SS), but did increase significantly above control in response to treatment with SB, or with SB and 2 h SS (n ¼ 3; *P < 0.05 vs. control).
Secondly, activated p38 MAPK may exert negative effects on MMP-2 production indirectly through inhibitory crosstalk with the ERK1/2 pathway (Singh et al., 1999; Westermarck et al., 2001). This effect appears to be mediated by p38 MAPK activation of protein phosphatase 2A (PP2A), which dephosphorylates both MEK1/2 and ERK1/2 (Liu and Hofmann, 2004). Conversely, inhibition of the p38 MAPK pathway induces activation of the ERK1/2 pathway (Aguirre-Ghiso et al., 2003) in numerous cell types, including rat microvascular endothelial cells (Boyd et al., 2005). Because ERK1/2 promotes MMP-2 transcription (Boyd et al., 2005), signals that affect ERK1/2 activity may impact on MMP-2 production. Thus, shear stress may inhibit transcription of MMP-2 mRNA via p38-mediated suppression of ERK1/2 activity. As well as controlling MMP2 mRNA levels, p38 MAPK activity may exert translational or post-translational control of MMP-2 protein, as a significant increase in MMP-2 protein was observed in static cultures treated with the p38 inhibitor. Consistent with our observation, it is known that p38 MAPK phosphorylates Mnk1, which in turn can negatively regulate cap-dependent mRNA translation via its effects on eukaryotic initiation factor 4E (eIF4E) (Knauf et al., 2001). In contrast to the effects seen in capillaries, carotid or coronary arteries exposed either to decreased or increased shear rates are reported to increase MMP-2 expression (Tronc et al., 2000; Sho et al., 2002, 2005; Nanjo et al., 2006). While smooth muscle cells may contribute significantly to this effect, at least one study showed that MMP-2 expression was upregulated in the endothelial cell layer, based on immunohistochemistry (Sho et al., 2002). The magnitude of wall shear stress used as a stimulus differed considerably between the large artery and the microvascular studies, which may explain the observed differences in MMP-2 production. In the model of arteriovenous fistula, as utilized by Sho et al. (2002), control artery shear stress was calculated at 10 dyne/cm2 while shear stress values in the experimental arteries ranged from 40 to 85 dyne/cm2. Histological images showed evidence of significant cellular injury to the endothelial cell layer in response to this level of shear stress (Sho et al., 2002), implying the occurrence of pathological wounding responses. In contrast, exposure of skeletal muscle capillary networks, or isolated microvascular endothelial cells, to shear stress values of 16 dyne/cm2 (as calculated in EDL muscle during prazosin treatment (Milkiewicz et al.,
2001)), does not induce endothelial cell damage. In further support of magnitude-dependent shear stress effects on MMP-2 production, we found that 5 dyne/cm2 shear stress had no effect on MMP-2 production in cultured endothelial cells. Several studies point to a critical role of p38 MAPK in the formation and maintenance of a mature vascular network. Genetic deletion of p38a in mice resulted in homozygous embryonic lethality, due to inadequate vascular remodeling into a mature vascular network (Mudgett et al., 2000). Interestingly, deletion of the gene encoding the transcription factor MEF2C, a major target of p38, also results in severe vascular deformities within the embryo (Bi et al., 1999), strengthening the hypothesis that p38 signaling is inherent to maintenance of a stable vascular network. MEF2C also may be activated by big MAPK (ERK5) (Kamakura et al., 1999). In an elegant conditional knockout approach, researchers showed that adult BMK / mice died within 4 weeks of inducing the knockdown, as a result of compromised vascular integrity (Hayashi et al., 2004). Thus, both p38 and ERK5 pathways may contribute to vascular stabilization, and may play roles in promoting shear stress-induced capillary remodeling. A recent study clearly demonstrated increased Snitrosylation of many proteins in endothelial cells exposed to shear stress for 24 h (Hoffmann et al., 2003). S-nitrosylation, which results from NO reacting with cysteine thiols to produce S-nitrosothiol, modifies protein conformation and function and is now recognized as a physiological regulatory activity of NO (Stamler et al., 2001; Hess et al., 2005). JNK proteins, but not other members of the MAPK family, are Snitrosylated at Cys116 and this modification inhibits JNK activity (Park et al., 2000). Our data do not support this effect on JNK within the first 2 h of shear stress stimulation, because we observed increases in both phospho-JNK and phospho c-jun, which were not affected by concurrent treatment with the NOS inhibitor, LNNA. In summary, our results support a stabilizing influence of NO production and p38 MAPK activation in shear stress-exposed endothelial cells. Both NO and p38 MAPK are involved in the downregulation of MMP-2 following microvascular endothelial cell exposure to elevated shear stress. The data suggest that within the skeletal muscle microcirculation in vivo, resting levels of shear stress maintains MMP-2 expression at a low level through constitutive NO production. Thus, an
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SHEAR STRESS-INDUCED INHIBITION OF MMP-2
implication for the data collected in the current study is that impaired endothelial cell function (through impaired NO bioavailability) may challenge not only acute vasodilatory responses to hemodynamic challenges but also may impair the process by which long term remodeling of capillary networks occurs in response to chronic alterations in hemodynamic forces. ACKNOWLEDGMENTS
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