Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

Nadia A. Haseneen, Gayle G. Vaday, Stanley Zucker, and Hussein D. Foda

Department of Medicine and Research, Northport Veterans Affairs Medical Center, Northport 11768; and The State University of New York at Stony Brook, Stony Brook, New York 11794


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.

matrix metalloproteinase; ventilator-induced lung injury; extracellular matrix metalloproteinase inducer; human lung microvascular endothelial cell; mechanical ventilation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MECHANICAL VENTILATION (MV) has become an indispensable therapeutic modality for patients with respiratory failure. However, it has been recognized that MV per se can lead to a number of serious complications. The most serious of these complications is the newly described ventilator-induced lung injury (VILI) (5). The concept has emerged that stretching of the lung by MV with high tidal volumes may lead to acute lung injury with severe damage to the alveolar-capillary barrier and pulmonary edema (5, 6). This type of lung injury is characterized by an increased endothelial and epithelial barrier permeability in the lung. The mechanism of such an increase in permeability is not well understood. It was recently reported that MV might play a role in initiating and propagating an inflammatory response in the lung by increasing the release of cytokines (TNF-alpha and IL-1beta ) from the lung (27).

Matrix metalloproteinases (MMPs) are a family of enzymes that degrade components of the extracellular matrix (ECM) (23, 24). The 72-kDa gelatinase A (MMP-2) is the most widely distributed of all the MMPs (2) and is expressed constitutively by a number of cells, including endothelial and epithelial cells. The 92-kDa gelatinase B (MMP-9) is produced by several types of inflammatory cells, including polymorphonuclear neutrophils and alveolar macrophages, as well as stimulated connective tissue cells. MMP-2 and MMP-9 play an important role in pericellular basement membrane turnover by degrading the main components of the basement membrane. The extracellular matrix metalloproteinase inducer (EMMPRIN) is a 58-kDa, membrane-bound protein that has been identified in both normal (4) and diseased human tissues (1, 14, 21). Exposure of human fibroblast to recombinant EMMPRIN caused induction of MMP-1, MMP-2, and MMP-3 (4, 14).

We recently reported that in a rat model of VILI there was an upregulation and increased release and activation of MMPs, especially MMP-2, MMP-9, and membrane type-1 MMP (MT1-MMP). This increase in MMP-2 and MT1-MMP was preceded by the upregulation of EMMPRIN. We further found that the synthetic MMP inhibitor Prinomastat (AG3340) protected rat lungs from VILI (9). One possible mechanism leading to MMP upregulation and activation may be the effect of cell stretching caused by MV. Pulmonary microvascular endothelial cells are the cells forming the alveolar-capillary barrier (25) and are most likely to be subjected to stretch stresses during MV (16). Therefore, we sought in this study to systematically examine the effect of cell stretching in vitro on the release and activation of MMPs from pulmonary microvascular endothelial cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Human lung microvascular endothelial cells (HMVEC-L) were purchased from Clonetics (San Diego, CA). HMVEC-L cells were grown in EGM-2 MV media (human endothelial cell media supplemented with 5% fetal bovine serum, 0.04% hydrocortisone, 0.4% human fibroblast growth factor-beta , 0.1% vascular endothelial growth factor, 0.1% insulin-like growth factor, 0.1% ascorbic acid, 0.1% human epidermal growth factor, and 1% GA-1000) (Clonetics). The cells were grown in a humidified atmosphere of 95% air-5% CO2 at 37°C and passaged every 5-7 days. Cells from the second to the fifth passage were employed in this study.

Application of cyclical mechanical strain. HMVEC-L (5 × 105 cells/ well) were propagated in six-well plates coated with type 1 collagen (BioFlex collagen 1 culture plate; Flexcell International, Hillsborough, NC). We exposed the cells grown to confluence on the flexible surface of the BioFlex plates to cycles of stretch and relaxation using a computer-driven, vacuum-operated, stress-providing instrument (Flexercell Strain Unit FX-4000 Tension plus; Flexcell International). The vacuum induced 17-18.5% elongation in the diameter of the flexible surface. We used a "Flexstop," which is a rubber stopper inserted into the underside of the BioFlex culture plate wells of the control nonstretched cells to prevent the vacuum-induced flexing of the BioFlex growth surface. The cells were exposed to stretch for 1, 2, 3, and 4 days. In the 3- and 4-day stretch experiments, serum-enriched medium was added for 2 days and then replaced with serum-free medium (endothelial cell basic medium; Invitrogen) for the following two days. In the 1- to 2-day stretch experiments, serum-free medium was added from the start. Cells were examined by phase-contrast microscopy and trypan blue exclusion to verify cell attachment and viability after mechanical stretch. The conditioned medium collected after 1, 2, 3, and 4 days of stretch was centrifuged at 1,500 g for 5 min to remove particulate matter and nonadherent cells and then stored at -80°C until assayed. At the end of each experiment, either cell lysate or total cellular RNA was obtained. We obtained cell lysate by incubating the cells with lysis buffer containing 0.1% SDS, 0.5% sodium deoxycholate, and 1% Nonidet P-40 in PBS containing protease inhibitor (Sigma, St. Louis, MO) for 1 h at 4°C. The cell lysate was then pelleted by centrifuging at 1,500 g for 15 min to remove cellular debris and collagen. Total RNA from the cells was isolated with Tri reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer's instructions. All stretch experiments were repeated 3-5 times.

Lactate dehydrogenase cytotoxicity assay. Conditioned media of HMVEC-L exposed to stretch and nonstretch conditions were assayed for lactate dehydrogenase (LDH) release. This test provides a method for quantitating cytotoxicity based on the measurement of the activity of LDH released from damaged cells. The LDH assay was performed according the manufacturer's instructions (LDH Cytotoxicity Detection Kit; Biovision). Briefly, 100 µl of each of the following were examined: 1) serum-free medium as background control, 2) conditioned medium of HMVEC-L cells as low control, 3) conditioned medium of HMVEC-L cells incubated with serum-free medium containing 1% Triton X-100 as high control, and 4) test samples of conditioned medium of HMVEC-L cells exposed to stretch and nonstretch conditions. Each sample was then mixed with 100 µl of reaction mixture (catalyst and dye solution) in a 96-well plate and incubated for 30 min at room temperature in the dark. Absorbance was measured at 490 nm. The mean of the background value was subtracted from all other values. Cytotoxicity was calculated with the following equation
cytotoxicity (<IT>%</IT>)<IT>=</IT>(test sample<IT>−</IT>low control)

<IT>÷</IT>(high control<IT>−</IT>low control)<IT>×</IT>100

Gelatin zymography. Conditioned media of both stretched and nonstretched cells were diluted 1:1 in nonreducing sample buffer and separated on 10% SDS-PAGE gels containing 0.1% gelatin (precast gels from Invitrogen) for 150 min at 125 V. SDS was removed by incubation with renaturing buffer (Triton X-100, 25% in water) for 30 min at room temperature. The gels were washed for 30 min in developing buffer (Tris base, Tris · HCl, NaCl, CaCl2, and Brij 35 in distilled water) and then incubated for 24 h at 37°C in developing buffer as previously described (7). Finally, gels were stained with Coomassie brilliant blue r-250. Zones of enzymatic activity were characterized by the absence of Coomassie blue staining. Gelatinolytic bands were quantified by gel scanning and densitometry with an Alpha Imager (Alpha Innotech, San Leandro, CA).

Treatment with the MMP inhibitor Prinomastat. Prinomastat (AG3340; Agouron Pharmaceuticals, Pfizer) is a potent inhibitor of MMP-2, MMP-9, MMP-13, and MT1-MMP activity (20). An aqueous stock solution was prepared by dissolving Prinomastat in DMSO and used at a final concentration of 300 ng/ml in sterile PBS. This nontoxic concentration of Prinomastat is sufficient to inhibit MMP-2 and MMP-9 activation (20). DMSO at a final concentration of 0.5% in PBS was used as a vehicle control.

Casein gel. Equal amounts of conditioned media from both stretched and nonstretched cells were mixed with an equal volume of nonreducing Laemmli sample buffer and electrophoresed in SDS-7.5% polyacrylamide gels containing 1 mg/ml casein. After electrophoresis, we cleared the gels of SDS by incubating them for 1 h with incubation buffer (50 mM Tris · HCl, 2.5% Triton X-100, 5 mM CaCl2, and 1 µM ZnCl2 in distilled water). Gels were then incubated overnight in developing buffer at 37°C. The gels were then stained with Coomassie brilliant blue. Caseinolytic bands were quantified by gel scanning and densitometry with an Alpha Imager.

Reverse zymography. Reverse zymogram was performed as described previously (8). Briefly, equal amounts of conditioned media of both stretched and nonstretched cells were mixed with an equal volume of nonreducing Laemmli sample buffer and electrophoresed in SDS-7.5% polyacrylamide gels impregnated with both 0.05% gelatin and excess activated MMP-2. Tissue inhibitors of metalloproteinase (TIMPs) were identified by Coomassie blue staining bands on a clear background. Semiquantitative analysis of the amount of TIMP was performed with gel scanning and densitometry with the Alpha Imager.

Immunoblotting. Immunoblotting for MT1-MMP and EMMPRIN of cell lysates of stretched and nonstretched cells was performed with affinity-purified rabbit antibodies directed against MT1-MMP (hinge region) (Chemicon International, Temecula, CA), a mouse monoclonal antibody against the hemopexin-like domain of MT1-MMP (Oncogene, Cambridge, MA), and a mouse monoclonal antibody directed against TIMP-2 and EMMPRIN (Santa Cruz Biotechnology), respectively. Cell lysates were run in 8-16% SDS-polyacrylamide gel (Invitrogen). Gels were blotted onto polyvinylidene fluoride membranes (Invitrogen). Membranes were blocked for 1 h in PBS containing 0.1% Tween 20 and 5% milk. Primary anti-MT1-MMP, -TIMP-2, and -EMMPRIN antibodies diluted to a final concentration of 20 ng/ml, 2 µg/ml, and 200 ng/ml, respectively, in blocking solution were added and incubated overnight at 4°C. Secondary goat anti-rabbit and sheep anti-mouse immunoglobulin horseradish peroxidase-conjugate (Amersham) were used at 1:5,000 dilution (7, 9). Enhanced chemiluminescence (ECL) detection was performed per manufacturer's instruction (Amersham).

Immunoblotting of cell conditioned media was performed using rabbit antibodies directed against human MMP-3 and MMP-1 (Biogenesis) or mouse antibodies directed against human TIMP-2 (Oncogene). Conditioned medium with sample buffer was sized fractionated in 8-16% SDS-polyacrylamide gel. Gels were blotted onto nitrocellulose membranes. Membranes were blocked for 1 h in PBS containing 0.1% Tween 20 and 5% milk and incubated overnight at 4°C with primary antibodies. The final concentrations of the primary antibodies employed were as follows: anti-MMP-1 (2.6 µg/ml), anti-MMP-3 (1.36 µg/ml), and anti-TIMP-2 antibodies (2 µg/ml). Secondary antibodies, goat anti-rabbit or sheep anti-mouse immunoglobulin, and horseradish peroxidase-conjugate (Amersham) were used at 1:5,000 dilution. ECL detection was performed per manufacturer's instruction. Bands were identified, analyzed and photographed using alpha imaging.

RT-PCR for MMP-2, MMP-1, MT1-MMP, EMMPRIN, and TIMP-2. After 4 days of mechanical stretch, total RNA was isolated from HMVEC-L by lysis and extraction in Tri reagent (Molecular Research Center) per manufacturer's instructions. We performed reverse transcription by boiling 1 µg RNA with 1 µM oligo(dT12-18) (Amersham Pharmacia) for 1 min, followed by incubation for 1 h at 37°C with the following reagents: 1× PCR buffer II (Sigma), 2.5 mM MgCl2 (Sigma), 1 mM dNTPs (Epicentre, Madison, MI), 800 U/ml ribonuclease inhibitor (Sigma), and 20 units Moloney murine leukemia virus-RT. Reactions were terminated by incubation at 95°C for 10 min. PCR was performed with 200 ng cDNA, 1× PCR buffer II, 1.5 mM MgCl2, 0.5 unit Tfl DNA polymerase (Epicentre), and the following oligonucleotide primers used at 0.4 µM: MMP-2 (+), 5' CGCCGTCGCCCATCATCAAGT 3'; MMP-2 (-), 5' TGGATTCGAGAAAACCGCAGTGG 3'; EMMPRIN (+), 5' GTGAAGGCTGTGAAGTCGTCA 3'; EMMPRIN (-), 5' TTCCGGCGCTTCTCGTAGATGAA 3'; MT1-MMP (+), 5' ATTCGCAAGGCGTTCCGCGTGTG 3'; MT1-MMP (-), 5' TGATGGCCGAGGGGTCACTGGA 3'; TIMP-2 (+), 5'TGCAGCTGCTCCCCGGTGCAC 3'; TIMP-2 (-), 5'TTATGGGTCCTCGATGTCGAG 3'; MMP-1 (+), 5'GAAATCTTGCTCATGCTTTTCAACC 3'; MMP-1 (-), 5'AAGGTTAGCTTACTGTCACATGCTT 3'.

Amplification conditions for each reaction were as follows: MMP-2 and TIMP-2, 35 cycles of 96°C for 30 s, 60°C for 30 s, 72°C for 45 s, followed by 72°C for 10 min; EMMPRIN, 30 cycles of 94°C for 1 min, 48°C for 1 min, 72°C for 1.5 min, followed by 72°C for 10 min; MT1-MMP, 40 cycles of 92°C for 30 s, 60°C for 30 s, 72°C for 30 s, followed by 72°C for 10 min; MMP-1, 35 cycles, 94°C for 30 s, 55°C for 30 s, 72°C for 30 s; GAPDH, 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min. The expected sizes of each PCR product were: MMP-2, 400 bp; EMMPRIN, 382 bp; MT1-MMP, 339 bp; TIMP-2, 585 bp; MMP-1, 315 bp; and GAPDH, 900 bp. We analyzed PCR samples by electrophoresis on 2% agarose gels (Invitrogen) and photographed them using alpha imaging.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MMP-2 is both induced and activated by mechanical stretch. Both static and stretched cells produced a constitutive 72-kDa gelatinolytic band consistent with MMP-2. The size of the 72-kDa band increased progressively with time and was much greater in the conditioned media from stretched cells compared with nonstretched cells. There was a time-dependent increase in 64- and 62-kDa gelatinolytic bands in conditioned media of stretched cells consistent with intermediate and active forms of MMP-2 (Fig. 1). The conditioned media of stretched cells treated with concanavalin A (ConA) showed the same activated gelatinolytic band (62 kDa) as conditioned media from stretched cells without ConA treatment (data not shown). Both static and cyclically stretched HMVEC-L cell cultures had >90% cell viability at the end of the experiment, as assessed by trypan blue and LDH cytotoxicity assay, indicating that growth on collagen 1-coated BioFlex membrane and cell stretching did not interfere with essential cell functions.


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Fig. 1.   Induction and activation of matrix metalloproteinase (MMP)-2 by mechanical stretch of human lung microvascular endothelial cells (HMVEC-L). Conditioned medium of HMVEC-L exposed to stretch and no-stretch was collected after 1, 2, 3, and 4 days and analyzed by gelatin zymography. Top: a constitutive, low level of pro-MMP-2 (72 kDa) in nonstretched cells and a significant increase in total amount of MMP-2 (72 kDa) and activated MMP-2 (62 kDa) in the stretched cells. Secretion and activation of pro-MMP-2 increased with time, especially in stretched cells. Bottom: densitometric analysis of pro-MMP-2 (72 kDa) and activated MMP-2 (62 kDa) from the zymogram of HMVEC-L conditioned media with and without stretch.

MT1-MMP is upregulated and activated by stretch. Immunoblotting of cell lysates using the polyclonal rabbit anti-MT1-MMP antibodies (against the hinge region) detected a 63-kDa band representing pro-MT1-MMP, a 54-kDa band representing activated MT1-MMP, and a 45-kDa band representing a degradation product of MT1-MMP in cells exposed to stretch (12) (Fig. 2A). The intensity of the MT1-MMP bands increased progressively over 4 days in stretched cells but not in nonstretched cells (Fig. 2B). When the monoclonal mouse anti-MT1-MMP antibody was used against the hemopexin-like domain, only the active form of MT1-MMP at 54 kDa was most prominently displayed (18) (Fig. 2B). Immunoblotting of cell lysates of cells exposed to stretch for 1, 3, and 4 days showed progressive increase in activated MT1-MMP compared with cell lysates from nonstretched cells (Fig. 2C).


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Fig. 2.   Membrane type-1 (MT1) MMP is activated by stretch. Immunoblotting of cell lysate of HMVEC-L exposed to stretch and no-stretch for 4 days (using a rabbit polyclonal antibody) directed against the hinge domain detected a 63-kDa band representing pro-MT1-MMP, a 54-kDa band representing activated MT1-MMP, and a 45-kDa band representing a degradation product of MT1-MMP; the intensity of MT1-MMP bands was greater in cells exposed to stretch compared with nonstretched cells. B: cell lysate of HMVEC-L exposed to stretch (+) and no-stretch (-) obtained after 1, 3, 4 days and immunoblotted using a monoclonal mouse anti-MT1-MMP antibody directed against a hemopexin-like domain revealed an increase in the 54-kDa band representing active MT1-MMP in cell lysates from stretched cells compared with lysates from nonstretched cells; this antibody is efficient in detecting activated MT1-MMP (catalog no. IM57L; Oncogene Research Products). C: densitometric analysis of MT1-MMP from immunoblot in B.

MMP inhibitor Prinomastat attenuates the activation of MMP-2 caused by stretch. Continuous treatment of stretched cells with Prinomastat (300 ng/ml) blocked the cleavage of the 72-kDa pro-MMP-2 to the active 62-kDa form. However, the MMP inhibitor had no effect on pro-MMP-2 secretion (Fig. 3). These results suggest that the activation of MMP-2 by mechanical stretch may be MT1-MMP dependent through a signal transduction pathway.


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Fig. 3.   Prinomastat (AG3340) inhibited the activation of MMP-2 in response to stretch. Gelatin zymography of the conditioned media of HMVEC-L exposed to stretch and no-stretch in the presence and absence of Prinomastat for 4 days. Prinomastat inhibited the activation of pro-MMP-2 (72 kDa) to 62 kDa resulting from stretch.

TIMP-2 is upregulated by stretch. Reverse zymography and immunoblotting of conditioned media showed a single 21-kDa band representing TIMP-2 in the conditioned media from stretched cells; this band became evident in the conditioned media only after 4 days of stretch. No bands were evident in the conditioned media collected on day 1, 2, or 3. No TIMP-2 bands were evident in conditioned media from nonstretched cells (Fig. 4A). Immunoblotting of cell lysates from stretched and nonstretched HMVEC-L for TIMP-2 using mouse monoclonal anti-TIMP-2 showed a band at 57 kDa representing TIMP-2 [most possibly bound to active MT1-MMP (13) or representing a multimer of TIMP-2]. These bands were present after one day of stretch. Stretch increased the density of the bands at each time point (Fig. 4B).


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Fig. 4.   A: tissue inhibitor of metalloproteinase (TIMP)-2 is induced on 4th day of stretch. Reverse zymography of 5× concentrated conditioned media of HMVEC-L exposed to stretch and nonstretch conditions after 1, 3, and 4 days shows that TIMP-2 increased in conditioned media of stretch cells on the 4th day only. Lane 1 (Control) contains recombinant TIMP-2 (31). B: TIMP-2 in cell lysate after stretch. Western blot using a mouse monoclonal anti TIMP-2 antibody on cell lysate of HMVEC-L exposed to stretch and nonstretch conditions after 1, 2, 3, and 4 days shows that TIMP-2 increased in cell lysate of stretched cells compared with nonstretched cells.

MMP-1 but not MMP-3 is induced and activated by stretch. Casein gel zymography revealed a band at 52 kDa, consistent with either MMP-3 or MMP-1, which was more prominent in the conditioned media of cells exposed to stretch than in the conditioned media of nonstretched cells (data not shown). Immunoblotting of conditioned media using antibodies directed against MMP-1 confirmed the result of the casein gel and showed two bands at 52 and 43 kDa, consistent with pro-MMP-1 and its activation product MMP-1 (Fig. 5). No bands consistent with MMP-3 were observed when the antibody directed against MMP-3 was used. In the time-course experiments, immunoblotting of conditioned media of cells exposed to stretch and nonstretch for 1, 2, 3, and 4 days showed a progressive increase in MMP-1 in the conditioned media of stretched cells, which was significantly more than what was observed with nonstretched cells (data not shown).


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Fig. 5.   MMP-1 is induced and activated by stretch. Western blot using a polyclonal rabbit anti-human MMP-1 antibody of 5× concentrated conditioned media of HMVEC-L exposed to stretch and nonstretch conditions for 4 days. MMP-1 (latent 52 kDa and activated 43 kDa) was increased in the conditioned media of stretched cells compared with nonstretched cells.

EMMPRIN is upregulated by stretch. Immunoblotting of lysate of cells exposed to stretch and nonstretch using the mouse antibodies directed against EMMPRIN showed a 58-kDa band representing EMMPRIN; EMMPRIN was significantly increased in stretched cells compared with nonstretched cells. EMMPRIN increased in a time-dependent fashion beginning on day 1 in cell lysate from stretched cells compared with nonstretched cells (Fig. 6).


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Fig. 6.   Extracellular matrix metalloproteinase inducer (EMMPRIN) is induced by mechanical stretch. Western blot of HMVEC-L cell lysates exposed to stretch and nonstretch conditions after 1, 3, and 4 days using a monoclonal mouse anti-EMMPRIN antibody. EMMPRIN increased in cell lysates of cells exposed to stretch compared with nonstretched cells.

MMPs, TIMP-2, and EMMPRIN mRNA are induced by mechanical stretch. Utilizing RT-PCR on RNA extracted from stretched and nonstretched cells at the end of 4 days, we found a marked induction of mRNA for MMP-1, MMP-2, MT1-MMP, TIMP-2, and EMMPRIN in stretched cells compared with nonstretched cells (Fig. 7). The expression patterns of these genes was consistent with our findings at the protein level in our experiments described above using gelatin zymography, casein gel zymography, reverse zymography, and immunoblotting.


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Fig. 7.   Cell expression of MMPs, TIMP-2, and EMMPRIN mRNA is increased by stretch. Increased HMVEC-L mRNA expression of MMP-1, MMP-2, MT1-MMP, TIMP-2, and EMMPRIN demonstrated by RT-PCR in response to stretch.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We recently reported that in a rat model of VILI there was an upregulation and increased release and activation of MMPs, especially MMP-2, MMP-9, and MT1-MMP (9). One of the possible mechanisms leading to MMP upregulation in these experiments is the effect of cell stretching caused by MV. Pulmonary microvascular endothelial cells are likely to be subjected to stretch stresses during MV. Therefore, we sought in this study to systematically examine the effects of cell stretching in vitro on the release and activation of MMPs from HMVEC-L. We used a degree of stretch (17% elongation) that is well above the expected stretch with normal breathing (15, 29) in an attempt to mimic the type of stretch that may be seen with high-volume lung MV. Our results show that cyclic mechanical stretch of HMVEC-L leads to an increase in the release and activation of MMP-2. This increase was accompanied by an increase in MT1-MMP. The activation of pro-MMP-2 was inhibited by the MMP inhibitor Prinomastat (AG3340). We also found that cell stretching caused an increase in the release and activation of MMP-1. EMMPRIN synthesis was also increased by cell stretching. Together, our results suggest that cyclic mechanical stretch caused by MV can cause lung injury by upregulating and increasing the release and activation of MMPs, especially MMP-2, MT1-MMP, and MMP-1.

MMP-2 is not generally regulated at the level of transcription (22) but, rather, is constitutively expressed and controlled through a MT1-MMP-, TIMP-2-specific mechanism of activation (13, 26, 31). However, there are data to indicate that the basal expression of MMP-2, MT1-MMP, and TIMP-2 is coregulated (22), consistent with their cooperation in MMP-2 activation. Our data support these conclusions in showing that MMP-2 upregulation was accompanied by a similar increase in MT1-MMP and TIMP-2. Our results support the role of MT1-MMP in the activation of pro-MMP-2, especially the data demonstrating that MT1-MMP levels increased in a time course that paralleled the increased activation of pro-MMP-2. Furthermore, treatment with Prinomastat prevented MMP-2 activation. These data strongly suggest a direct relationship between MT1-MMP expression and MMP-2 activation, consistent with the currently accepted mechanism of MMP-2 activation (3, 31). The results presented here are consistent with those reported in cardiac fibroblasts showing increased production of MMP-2 and MT1-MMP in response to stretch (28). Mechanical strain has also been reported to increase MMP-2 production and activity in vascular smooth muscle cells (17) and chondrocytes (10). Recently it has been shown that cyclic strain upregulates the early growth response gene product-mediated expression of MT1-MMP in rat microvascular endothelial cells (30).

In our study, reverse zymograms and Western blots established that TIMP-2 levels did not increase in the conditioned media during the initial 3 days of stretch, but TIMP-2 increased in response to stretch on the 4th day. However, in cell lysate, TIMP-2, which appears to be present in complex [possibly bound to active MT1-MMP (13) or as a multimer of TIMP-2 (8)], was enhanced by stretch beginning on day 1. The increase in the amount of activated MMP-2, with no change in stoichiometric amounts of TIMP-2 in the conditioned media during the initial 3 days of stretch, favors an environment where MMP-2, MT1-MMP, and TIMP-2 function together, leading to the degradation of ECM. The appearance of excess TIMP-2 on the 4th day of stretch in conditioned media is consistent with an inhibitory effect of increased TIMP-2 production.

Mechanical stretch increased EMMPRIN gene expression and protein synthesis in endothelial cells. This increase started early in the course of stretch (day 1). EMMPRIN is known to induce MMP-1, MMP-2, and MMP-3 expression in human fibroblasts (4, 14). EMMPRIN also causes increased expression of MT1-MMP and MT2-MMP as well as increased production and activation of MMP-2 in brain-derived fibroblasts (19). Increased EMMPRIN levels have also been shown to be associated with increased production and activation of MMP-2 and MT1-MMP in dilated cardiomyopathy (21). Recently, we reported that EMMPRIN mRNA was increased in rat lung tissues subjected to VILI; this increased EMMPRIN mRNA preceded the increase in MMP-2 and MT1-MMP mRNA seen in this type of injury (9). Together, our results suggest that mechanical stretch causes an increased upregulation of EMMPRIN that in turn leads to the increased production of MMP-1, MMP-2, and MT1-MMP seen in our study.

In conclusion, our results suggest that mechanical stress in the form of cell stretching may be an important stimulus to increased production and activation of MMP-2 through MT1-MMP- and EMMPRIN-dependent mechanisms. The clinical use of pharmacological MMP inhibitors to prevent early activation of MMPs leading to VILI should be considered. A limitation to the use of MMP inhibitors in inflammation, however, is in the difficulty distinguishing between the negative and positive effects of activated MMPs in the tissue injury versus the repair process (11).


    ACKNOWLEDGEMENTS

We thank Dr. David Shalinsky (Agouron Pharmaceuticals, Pfizer) for generously providing Prinomastat.


    FOOTNOTES

This work was supported by funds from the Veterans Affairs (V. A.) Research Enhancement Award Program REAP, National Heart, Lung, and Blood Institute Grant HL-646340 (H. D. Foda), and a V. A. Merit Review grant (S. Zucker).

Address for reprint requests and other correspondence: H. D. Foda, Pulmonary and Critical Care Medicine, SUNY at Stony Brook, Health Science Center, Stony Brook, NY, 11794-8172 (E-mail: hfoda{at}mail.som.sunysb.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.

First published November 27, 2002;10.1152/ajplung.00290.2002

Received 22 August 2002; accepted in final form 25 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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