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(Circulation Research. 2004;95:364.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Minocycline Exerts Multiple Inhibitory Effects on Vascular Endothelial Growth Factor–Induced Smooth Muscle Cell Migration

The Role of ERK1/2, PI3K, and Matrix Metalloproteinases

Jianhua S. Yao, Yongmei Chen, Wenwu Zhai, Kaiyan Xu, William L. Young, Guo-Yuan Yang

From the The Center for Cerebrovascular Research, Departments of Anesthesia and Perioperative Care (J.S.Y., Y.C., W.L.Y., G.-Y.Y.), Neurological Surgery (W.L.Y., G.-Y.Y.), and Neurology (W.L.Y.), Lung Biology (W.Z.), and Gladstone Institute (K.X.), University of California, San Francisco.

Correspondence to Guo-Yuan Yang, MD, PhD, The Center for Cerebrovascular Research, Departments of Anesthesia and Neurosurgery, UCSF, 1001 Potrero Ave, Box 1371, San Francisco, CA 94110. E-mail gyyang{at}anesthesia.ucsf.edu

	Abstract

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Widely used tetracycline antibiotics affect many cellular functions relevant to human vascular disease including cell proliferation, migration, and matrix remodeling. We examined whether minocycline inhibited human aortic smooth muscle cell (HASMC) migration induced by vascular endothelial growth factor (VEGF).

After the establishment of an optimal dose, minocycline treated HASMC were exposed to VEGF. HASMC migration, matrix metalloproteinase (MMP)-2 and MMP-9 activities, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) phosphorylation were determined by smooth muscle cell (SMC) invasion assay, real-time polymerase chain reaction, zymograms, and Western blot analysis, respectively.

We demonstrated that VEGF and platelet-derived growth factor (PDGF)-induced SMC migration in a dose-dependent manner. MMP-9, but not MMP-2, mRNA was increased during VEGF stimulation. MMP-9 activity was increased from 1.5- to 2.5-fold in a dose-dependent manner (P<0.05). Both ERK1/2 and PI3K/AKt pathways were activated during VEGF-induced HASMCs migration. We then demonstrated that minocycline can inhibit VEGF-induced HASMC migration (P<0.05). The effects may be through the inhibition of MMP-9 mRNA transcription, protein activities and downregulation of ERK1/2 and PI3K/Akt pathway phosphorylation.

Our results indicated that minocycline exerts multiple effects on VEGF-induced SMC migration, including inhibition of MMP-9 mRNA transcription and protein activities and downregulating ERK1/2 and PI3K signal pathways, suggesting minocycline may be a potentially therapeutic approach to inhibit disease process induced angiogenesis.

Key Words: matrix metalloproteinase • minocycline • migration • smooth muscle cell • vascular endothelial growth factor

	Introduction

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Minocycline, a widely used antibiotic,¹ is known to possess other potentially therapeutic effects that is distinct from its antimicrobial action.^2,3 For example, tetracycline derivatives such as doxycycline inhibit the activity of collagenase, gelatinase, and stromelysin in vitro,^4,5 and they have been used to reduce tissue degradation in aortic aneurysms, arthritis, and to inhibit tumor cell invasion and metastasis.^6–8 In in vitro studies, investigators found that minocycline and doxycycline can decrease human umbilical vascular endothelial cell proliferation and tube formation, tumor cell migration, inducible nitric oxide synthetase expression, and induce macrophage apoptosis.^9,10 In balloon catheter denudation of the rat carotid artery, doxycycline inhibited SMC migration through the reduction of MMP-2 and MMP-9 activity in the arterial wall.¹¹ The mechanisms of minocycline on cell proliferation, migration, and MMP activity processes, especially on the signal pathway, are still largely unknown.

Smooth muscle cell (SMC) migration plays an important role in normal angiogenesis and is relevant to disease-related vascular remodeling in conditions such as brain arteriovenous malformations, pulmonary hypertension, arteriosclerosis, and restenosis after angioplasty.^12–14 This physiopathological process also closely relates to the angiogenic changes in in vitro studies. Experimental results demonstrate that vascular endothelial growth factor (VEGF) receptors existed in the SMCs and that VEGF can induce SMC migration in vitro.^15,16 SMC migration was regulated by insulin-like growth factor-I,¹⁷ PDGF,¹⁸ basic fibroblast growth factor,¹⁹ etc. However, the precise molecular mechanisms of regulating SMC migration are still unknown.

Activation of VEGF is mediated, in part, by mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKt signal pathways. VEGF binds its receptors, flk-1 and flt-1, activates the extracellular signal-regulated kinase 1/2 (ERK 1/2) signal pathway, upregulates MMP expression, and subsequently promotes SMC growth and migration.²⁰ This is critical for the initiation and progression of vascular lesions.²¹ Similarly, experimental evidence has also shown that PI3K/Akt pathway activation may play an important role for migration of cultured SMCs.²²

VEGF is of particular interest because of its ability to induce angiogenesis in the normal developmental and abnormal pathological condition.²³ Cerebrovascular diseases such as aneurysms, arteriovenous malformations, ischemia, and hemorrhage could increase VEGF expression.^24,25 To identify the action of VEGF during these disease processes, we chose VEGF as a stimulator to study the effect of minocycline during SMC migration in vitro. Our previous studies demonstrated that VEGF hyperstimulation could induce human brain SMC migration. The present study was to further examine: (1) whether minocycline inhibits VEGF-induced human aortic SMC (HASMC) migration in vitro; (2) whether minocycline inhibits HASMC migration through reduced MMP-2 and MMP-9; and (3) if so, whether minocycline exerts multiple inhibitory effects via downregulating ERK1/2 and PI3K/AKt pathways.

	Materials and Methods

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HASMC Culture
HASMCs were purchased from Cell Systems (St. Katharinen, Germany). Their homogenous nature was confirmed through immunostaining with anti-SM--actin (Chemicon, Temecule, Calif). The HASMCs were incubated in DMEM (GIBCO, Grand Island, NY) and maintained at 37°C in 5% CO₂/95% ambient mixed air. The culture media were changed every 2 days. HASMC experiments were performed on 5 to 8 passages.

During the VEGF and minocycline experiments, HASMCs were grown in 10% fetal bovine serum in 35-mm polystyrene plates and rinsed 3 times with a serum free medium, then arrested after 24 hours. HASMCs were then incubated with minocycline and VEGF in specified conditions. HASMCs were grown to 80% to 90% confluence and made quiescent by serum starvation (0.2% bovine serum albumin[BSA]) for at least 24 hours.

HASMC Invasion Assay
HASMC migration was evaluated using 24-well Transwell cell culture chambers with 8.0-µm pore polycarbonate filter inserts. The filters were coated with matrigel (BD Biosciences, Bedford, Mass) suitable for SMC invasion assay.^26–28 The stock solution of matrigel was diluted to 300 µg/mL using serum free DMEM. An aliquot of 75 µL matrigel was added into each filter insert and incubated overnight at room temperature under a laminar flow hood. The next day the coated insert was rehydrated with 0.5 mL of serum free DMEM for 2 hours. Cultured HASMCs were trypsinized and suspended in 0.2% BSA/DMEM at a concentration of 2.5x10⁵/mL. A total of 500 µL of 0.2% BSA/DMEM was added to the lower chamber and 100 µL cell suspension was applied to coated insert filters. Chamber was incubated at 37°C/5% CO₂. HASMCs were treated with minocycline (7.5 to 150 µmol/L, St Louis, Mo) and GM6001 (1 to 10 µmol/L) at increasing concentrations. A concentration of 20 ng/mL of VEGF (R&D Systems, Minn) was added to the lower compartment of each chamber. Negative control was treated by 0.2% BSA/DMEM and positive control was treated by 30 µmol/L of PD98059. The chamber was incubated for 18 hours at 37°C/5% CO₂ to allow cell migration, the insert was then removed, and the membrane was washed with 0.1 mol/L phosphate-buffered saline. No migrated cells on the upper side of filter were scraped; migrated cells on the lower side of chamber were fixed and stained with hematoxylin. Membrane was mounted on a slide and then examined under a microscope. Migration was quantified by measuring the stained cells in five random areas per membrane.

HASMC Viability
The cytotoxicity of minocycline on HASMCs was determined using a MTT assay.²⁹ HASMCs were grown in 96-well microtiter plates for 24 hours, then they were treated with minocycline in increasing concentrations in 0.2% BSA/DMEM. The culture plates were incubated at 37°C/5% CO₂ and VEGF (20 ng/mL) was added. The plates were incubated for 36 hours and the medium was replaced with 100 µL of fresh DMEM containing 0.2% BSA. Next, 2 mg/mL MTT solution was added and the plates were incubated again for 3 hours at 37°C/5%CO_2. Finally, MTT-containing medium was aspirated off and 150 µL of DMSO solution was added. Absorbance was measured at 570 nM using an enzyme-linked immunosorbent assay reader.

Real-Time Polymerase Chain Reaction
Total RNA was extracted using Trizol reagent (Gibco Life Technologies). Reverse-transcription was performed using a RETROscript kit (Ambion, Austin, Tex). Oligonucleotide primers were as follows: forward: 5'-CCGCAGGGCCCCTTCCTTAT-3' and reverse 5'-GCCCACTTGGTCCACCTGGTT-3' for MMP-9 (Maxim Biotech); 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3' for GAPDH. Real-time polymerase chain reaction (PCR) were performed by use of an ABI-prism 7000 sequence detector using 2.5 µL (5 ng) cDNA, 12.5 µL SYBR Green PCR Master Mix (2x), 2.5 µL primer pair mix (5 pmol each primer), and water to a 25-µL final volume. Thermocycler conditions were comprised an initial holding at 50°C for 2 minutes, then 95°C for 3 minutes. This was followed by 94°C for 15 seconds, 56°C for 45 seconds, and 72°C for 30 seconds for 45 cycles. Results were analyzed by use of Sequence Detection Software (Applied Biosystems, Foster City, Calif), and level of MMP-9 expression of mRNA was normalized to GAPDH (18 S rRNA endogenous control). The products of real-time quantitative PCR were verified on agarose gels.

MMP Zymograms
Zymograms are electrophoresis gels with embedded gelatin.³⁰ MMP-2 and MMP-9 activity was detected through zymograms.³¹ The cultured HASMCs grew to 80% confluence in 35-mm plates (concentration of 2.5x10⁵/mL cells) in a 10% fetal bovine serum media. These cells were incubated in serum free medium for 24 hours. HASMCs were then treated with minocycline and with/without VEGF at the same time for 24 hours. Aliquots of conditional medium were mixed with 2X sample buffer and loaded on a 10% polyacrylamide gel incorporated with 0.1% gelatin for electrophoresis. MMP-2 and MMP-9 zymographic standards were used as the standards (Chemicon, Temecula, Calif). Gels were renatured for 30 minutes, developed overnight, stained with 0.25% Coomassie brilliant blue (Bio-Rad, Richmont, Calif), and destained to visualize the MMP-2 and MMP-9 bands.

Phospho-ERK1/2 and Phospho-AKt Expression
Phospho-ERK1/2 and phospho-AKt expression were semi-quantified by Western blot analysis. HASMCs grew to 80% confluence and were made quiescent with serum free medium for 24 hours. After being pretreated with minocycline, VEGF (20 ng/mL) was added. Plates were then incubated for 15 minutes for ERK 1/2 and 20 minutes for PI3K/AKt expression according to our previous study. Cells were washed twice with phosphate-buffered saline and scraped into a lysis buffer. Protein concentrations were analyzed with a Bio-Rad system. Equal amounts of protein (20 µg/lane) for each sample were electrophoresed through a 10% SDS-PAGF gel and blotted onto a hybond nitrocellulose membrane (Amersham, Piscataway, NJ). The membrane was blocked by 5% nonfat milk solution in Tris-buffered saline/0.1%Tween-20 for 1 hour at room temperature. Phospho-ERK1/2 and phospho-AKt were detected by incubating membranes with rabbit polyclonal phospho-ERK1/2 and phospho-AKt antibodies overnight (1:1000 dilution; Cell Signaling, Beverly, Mass); secondary horseradish peroxidase–labeled antirabbit IgG antibody (1:2000 dilution, Amersham) was added for 1 hour. The membrane was plastic wrapped and exposed to Kodak film (Eastman Kodak Co, Rochester, NY). Bands were scanned and semi-quantified by densitometry.

Statistical Analysis
Results of the dose-dependent migration and inhibition experiments were analyzed using an ANOVA with post-hoc multiple comparison test. All data represent mean±SD. Statistical significance was determined with an ANOVA. A random P<0.05 or 0.01 is considered statistically significant for the comparisons.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Minocycline on VEGF-Induced SMC Invasion Into Extracellular Matrix
Biological activity associated with MMP upregulation in HASMCs was examined using a migration assay with modified Boyden chamber. The polycarbonate filter was coated with matrigel. VEGF enhances HASMC invasion into matrix barriers in a dose dependent manner (Figure 1A). The optimal doses of VEGF that significantly promote SMC migration appeared between 20 and 40 ng/mL. As a positive control, PDGF showed a similar effect in promoting HASMC migration (Figure 1B). Treatment with minocycline inhibited VEGF or PDGF induced HASMC migration in a dose-dependent fashion (Figures 1B and 2A ). Minocycline at 15 and 30 µmol/L reduced HASMC migration to 17% to 46% of the control (P<0.05). We further examined the effects of GM6001 on HASMC migration. GM6001, a MMP inhibitor, could inhibit HASMC invasion through matrigel in a dose-dependent manner (Figure 2B). These results support that MMPs may play a role in the HASMC invasion process into a "thick" matrix layer.

View larger version (21K): [in this window] [in a new window]   Figure 1. VEGF and PDGF induce HASMC migration. A, Bar graph shows the increase of HASMC migration after 18 hours of incubation with VEGF. HASMC migration was determined by using a cell invasion assay. B, Bar graph shows that PDGF induces HASMC migration after 18 hours of incubation with PDGF, and minocycline inhibits PDGF (10 ng)-induced HASMC migration. The numbers of migrated cells were counted in 5 random areas per membrane. C indicates control; N, negative control pretreated with anti-VEGF antibody; 10, 20, 40, and 100 ng/mL=doses of VEGF. Data are shown as mean±SD; n=3. *P<0.05; P<0.01, control versus VEGF-treated and/or PDGF-treated HASMCs.

View larger version (18K): [in this window] [in a new window]   Figure 2. Minocycline inhibits VEGF-induced HASMC migration. Bar graphs show that the effects of minocycline (A) and GM6001 (B) on VEGF-induced HASMC migration. Confluent HASMCs were arrested in 0.2% BSA/DME medium. HASMCs were incubated with VEGF and either minocycline or GM6001 for 30 minutes. The effect of minocycline (A) and GM6001 (B) on VEGF-induced HASMC migration was determined in these experiments. C, The 0.001% concentration of DMSO does not affect SMC migration. Data are shown as mean±SD; n=3. *P<0.05 and P<0.01, VEGF alone versus minocycline, PD98059, and Wortmannin. GM6001, PD98059, and Wortmannin serve as control groups.

Effect of Minocycline on HASMC Viability
To rule out HASMC death as the cause for the inhibition of migration, we evaluated minocycline ability to induce HASMC death using a MTT test. The viability of cells incubated for 24 hours with the indicated concentration of minocycline is presented in Figure 3A. Our results demonstrated that minocycline at the concentration of 60 µmol/L was not cytotoxic to confluent HASMCs. Increasing the concentration of minocycline to 150 µmol/L resulted in a 50% reduction of viability. This result suggests that the inhibitory effect of minocycline on HASMC migration and MMP activity was not caused by the cytotoxicity caused by minocycline. Because DMSO was used to dissolve GM6001, we further examined the effect of different concentrations of DMSO in HASMC migration. The results confirmed that the concentration of DMSO did not affect HASMC migration (Figure 2C) or cell viability (Figure 3B).

View larger version (19K): [in this window] [in a new window]   Figure 3. MTT assay for minocycline cytotoxicity. A, Bar graph shows that the viability of HASMCs incubated with indicated concentration of minocycline. HASMCs were incubated with minocycline and VEGF for 36 hours; and 2 mg/mL MTT solution was added and the plates were incubated again for 3 hours. The final solution was measured at 570 nM using a plate reader. B, Bar graph shows that the viability of HASMCs incubated with DMSO alone, indicating that up to 0.002% concentration of DSMO do not affect HASMC viability. Data are shown as mean±SD; n=3. *P<0.05, control group versus treated groups.

Effects of Minocycline on MMP-2 and MMP-9 mRNA Expression
To detect whether minocycline inhibits MMP occurring at the mRNA transcription levels, we performed real-time PCR. We confirmed that VEGF could stimulate HASMC MMP-9 mRNA expression in a dose-dependent manner (Figure 4A). Further study demonstrated that minocycline at the concentration of 15 and 30 µmol/L could inhibit VEGF-induced MMP-9 mRNA expression (P<0.05; Figure 4B). These results suggest minocycline can inhibit MMP-9 at the transcription step.

View larger version (19K): [in this window] [in a new window]   Figure 4. VEGF increased MMP-9 mRNA in HASMCs. A, Bar graph shows the increase of MMP-9 mRNA expression after 18 hours of incubation with VEGF, which was quantitatively determined by using real-time PCR. Real-time PCR densitometry from 3 independent experiments. *P<0.05, P<0.01, compared with control group. B,. Bar graphs shows the effect of minocycline in VEGF-induced MMP-9 mRNA expression. Data are shown as mean±SD; n=3 per group, *P<0.05 and =P<0.01, control group versus treated groups.

Effects of Minocycline on MMP-2 and MMP-9 Activity
Recent studies show that VEGF upregulates MMP-9 expression and activation in HASMCs in vivo and in vitro.²⁶ Further studies demonstrate that MMPs play an important role in regulating HASMC migration.²⁰ To identify the relationship between VEGF stimulation and MMP activation, we therefore measured MMP activity in VEGF-treated HASMCs using zymograms. VEGF stimulation did not enhance MMP-2 activity at the dose that induced HASMC migration (Figure 5A and 5B). However, VEGF stimulated latent MMP-9 (MMP-9L) and active MMP-9 (MMP-9A) in the HASMCs compared with the controls (Figure 5C and 5D); 20 ng/mL of VEGF appeared to be the optimal dose to increase MMP-9 activity.

View larger version (38K): [in this window] [in a new window]   Figure 5. VEGF increased MMP-9 activities in HASMCs. The effects of VEGF on MMP-2 and MMP-9 activation in HASMCs were determined by MMP activity in zymographic assay. HASMCs were stimulated with VEGF. Upper panel, Zymographic picture shows MMP-2 and MMP-9 activities are expressed after VEGF stimulation. Standard=MMP-2/MMP-9 zymographic standards. Lane 1 shows the control (without VEGF); lanes 2 and 3 show the HASMCs treated with PD98059 and Wortmannin, considered negative controls. Lanes 3 to 7 show the treatment of HASMCs with different doses of VEGF. Lower panel, Bar graphs represent the semi-quantitative zymograms. MMP activity levels are analyzed by latent MMP-2 (A), active MMP-2 (B), latent MMP-9 (C), and active MMP-9 (D), separately. PD indicates PD98059; Wort, Wortmannin. Data are shown as mean±SD; n=5.*P<0.05 and =P<0.01, control group versus treated groups.

To determine whether minocycline blocked MMP activity in VEGF-treated HASMCs, we further examined the doses of minocycline in VEGF-treated HASMCs. The results showed that there was no MMP-9L or MMP-9A detected in the control HVSMC media. Minocycline could inhibit MMP-9 activation in a dose-dependent manner (P<0.05, Figure 6C and 6D). Interestingly, minocycline could also inhibit activity in VEGF-treated HASMCs because VEGF did not stimulate MMP-2 (P<0.05; Figure 6B).

View larger version (33K): [in this window] [in a new window]   Figure 6. Minocycline inhibits VEGF-induced MMP-2 and MMP-9 activities in HASMCs. Effects of minocycline on VEGF-induced MMP activities in HASMCs were determined through MMP zymographic assay. HASMCs were treated with minocycline, PD98059, and Wortmannin for 30 minutes, and then treated with VEGF for 24 hours. Upper panel, Zymogram shows the effects of minocycline on MMP-2 and MMP-9 expression after VEGF stimulation. Standard=MMP-2 and MMP-9 zymographic standards. Lane 1 shows the control; lanes 2 and 3 show the HASMCs treated with PD98059 and Wortmannin, which are considered negative controls. Lanes 3 to 7 show the treatment with minocycline in the HASMCs. Lower panel, Bar graphs represent the semi-quantitative zymograms. MMP activity levels are analyzed by latent MMP-2 (A), active MMP-2 (B), latent MMP-9 (C), and active MMP-9 (D), separately. Data are shown as mean±SD; n=5. *P<0.05 and P<0.01, control group versus treated groups.

Minocycline Inhibits VEGF-Induced HASMC Migration Through ERK1/2 and PI3K/Akt Phosphorylation Inhibition
We next explored the mechanism that minocycline regulates VEGF-induced migration and MMP activation in HASMCs. Two distinct signaling pathways, ERK1/2 and PI3K/AKt, were assessed. Western blots showed that VEGF could induce ERK1/2 and AKt phosphorylation. Minocycline in the doses of 15 to 30 µm significantly downregulated ERK1/2 and AKt phosphorylation in VEGF-treated HASMCs compared with the control (P<0.05; Figure 7). These results suggest that minocycline regulates VEGF-induced HASMC migration and MMP activity through the ERK1/2 and PI3K signaling pathways.

View larger version (22K): [in this window] [in a new window]   Figure 7. Minocycline inhibits ERK1/2 and PI3K/AKt pathways in VEGF-induced SMC migration. Effects of minocycline on VEGF-induced ERK1/2 and PI3k/Akt expression in HASMCs were determined by Western blot analysis. After pretreatment of serum free HASMCs for 24 hours, HASMCs were treated with minocycline for 30 minutes, and then treated with VEGF for 24 hours. Bar graphs show Western blotting results. Minocycline inhibits VEGF-induced ERK1/2 (A) and Akt/PKB (B) phosphorylation in the HASMCs. Cont indicates control group; PD98, PD98059; Wort, wortmannin; M15, minocycline 15 µm; M30, minocycline 30 µm. Data are shown as mean±SD; n=5. *P<0.05 and P<0.01, control group versus VEGF-treated groups.

	Discussion

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SMC migration plays an important role in angiogenesis during disease processes. Our study demonstrates that: (1) minocycline, at a nontoxic dose, inhibits VEGF-induced HASMC migration in vitro; (2) VEGF upregulates MMP-9 but not MMP-2 mRNA transcription and protein activation and minocycline directly inhibit MMP-2 and MMP-9 activity during VEGF stimulation in a dose-dependent manner; (3) minocycline inhibits MMP-9 through downregulation of ERK1/2 activation; and (4) minocycline directly inhibits VEGF-induced SMC migration through downregulation of the PI3K/AKt signal pathway. Our results provide evidence that minocycline has multiple effects in the inhibition of SMC migration, suggesting downregulating MMP-9 production and activity and/or inhibiting ERK1/2 and PI3K/AKt phosphorylation may offer a therapeutic approach to block HASMC migration.

VEGF and VEGF receptors play a prominent role in regulating normal and abnormal angiogenesis. Administration of VEGF causes angiogenic responses in vitro and in vivo. Our previous experiment demonstrated that VEGF could induce human brain SMC migration. However, further investigation is necessary to determine which downstream molecules and signal pathways are involved in SMC migration. Using minocycline, we demonstrated that MMP-9 is involved in the VEGF-accelerated SMC migration.

SMC migration presumably requires degradation of the basement membrane and extracellular matrix surrounding the cell.³² One family of such enzymes is MMPs. MMPs are zinc proteases that cleave components of the extracellular matrix. There are 3 main types of MMPs: collagenase, stromelysins, and gelatinase.³³ Gelatinases such as MMP-2 and MMP-9 have been implicated in removing the first extracellular matrix barrier to migrating SMCs.^19,27 They have also been thought as a mediator of lesion development in response to vascular injury. MMP-9 was expressed within 6 hours after carotid artery injury in rats and continues to be expressed for up to 6 days.^19,34 Treatment with MMP-2 antibody can inhibit SMC migration across a synthetic extracellular matrix membrane.²⁷ Although MMP-2 and MMP-9 have similar substrate specificities,^35,36 regulation of their expression is different. For example, MMP-2 is constitutively expressed in SMCs, and cytokines or growth factors could not induce its expression.^37,38 In contrast, the basal levels of MMP-9 in SMCs are usually low and cytokines or growth factors could induce its expression.^37,38 It was demonstrated that MMP-2 could increase MMP-9 activity.³⁹ In our experiment, minocycline could inhibit MMP-2 activity, suggesting it may be through downregulation of MMP-9 and then inhibition of SMC migration. Many studies identify increased expression of MMP-2 and MMP-9 coincident with SMC migration after vascular injury in vitro and in vivo.⁴⁰ We found that VEGF can induce MMP-9 but not MMP-2 phosphorylation. Interestingly, minocycline can inhibit MMP-2 and MMP-9 activation in VEGF-treated HASMCs.

The effect of minocycline in VEGF-treated SMCs is unknown. We found that minocycline markedly inhibited VEGF-induced SMC migration, and it also inhibited MMP-2 and MMP-9 activities. This effect was not likely caused by the drug toxicity, because cell viability results showed that there was no difference in cell death between minocycline treated and control SMC culture. Similar results were obtained when systemic administration of MMP inhibitors or overexpression of tissue inhibitors of metalloproteines significantly reduced SMC migration after injury.⁴¹ The mechanisms by which minocycline inhibits VEGF-induced SMC migration are not completely understood. One potential explanation is thought to be downregulation of MMP-2 and MMP-9 activities via direct and indirect mechanisms. Minocycline binds to Zn⁺⁺ or Ca⁺⁺ associated with MMPs, blocking the active site or inducing conformational changes that render the proenzyme susceptible to fragmentation during activation.^41,42 The extent of this inhibition is probably underestimated by zymogram analysis because minocycline is expected to dissociate away from MMPs during electrophoresis. Another explanation for the decreases of active MMP-2 on zymograms after minocycline treatment is that minocycline prevents pro-MMP-2 activation through the MT1–MMP or reactive oxygen species.⁴³ In addition, minocycline-inhibited inducible nitric oxide synthase is known to activate MMPs.³⁹

Published data show that MAPKs are involved in regulation of MMP-9 expression in SMC culture and injured vessels. Therefore, we examined the effect of minocycline on translating extracellular stimuli to intracellular molecular signals, which is thought to regulate cell growth, differentiation, survival, and death.⁴⁴ Currently, 3 mammalian cytoplasmic MAPK pathways have been characterized in detail. Of these, the ERK1/2 pathway is especially activated by growth factor, whereas JNK/SAPK and P38 signal pathways are mainly activated by inflammatory cytokines and stress stimuli.^44,45 Phosphorylation of the conserved threonine and tyrosine residues of MAPKs by their specific upstream dual-specificity kinases (MEK1/2) results in the activation and subsequent translocation to the nucleus. Activated ERK1/2 then phosphorylated protein kinases 1, 2, and 3, which in turn induced activator protein-1 (AP-1) complex expression.⁴⁶ At the transcriptional level, AP-1 complex plays an essential role in the regulation of several MMPs including MMP-9. Studies also show that AP-1, nuclear factor (NF-b), stimulatory protein-1, and retinoblastoma binding elements are involved in the regulation of the human MMP-9 gene.^47,48

Our study demonstrates that VEGF-induced MMP-9 expression is mediated by ERK1/2 activation. Selectively blocking ERK1/2 using MEK1/2 inhibitor PD 98059 abrogates MMP-9 phosphorylation. This is in agreement with previous studies performed in SMCs, in which activation of ERK1/2 signaling pathway by tumor necrosis factor- correlated with increased expression of MMP-9.²⁰ In contrast VEGF induced ERK1/2 activation had no effect on MMP-2 enhancement, suggesting that alternative signaling pathways may be involved, including no expression at the AP-1 site in MMP-2 promoters. Importantly, we found that consistent with cell migration studies, minocycline treatment inhibited ERK1/2 signaling pathway in VEGF-stimulated SMCs. These results suggest that minocycline may have an antimigration effect on SMCs through inhibiting VEGF induced ERK1/2 phosphorylation.

Our present study first demonstrated that the inhibitory effects of minocycline in VEGF-induced SMC migration is through downregulation of the PI3K signal pathway. Previous studies showed that PI3K is indispensable for cell migration induced by growth factors such as VEGF in several cell types.⁴⁹ When VEGF binds its receptors, it leads to activate PI3K and downstream signaling.⁵⁰ PI3K catalyzes inositol phospholipids at the D3 position to generate PI3, 4, 5-trisphosphate and PI3, 4-bisphosphate. These 3-phosphoinositides act as potent signaling molecules to regulate many cellular responses that are important for angiogenesis.^22,51 The role of PI3K in VEGF-mediated signal transduction and angiogenic responses is established.^52,53 In our present study, we confirmed that phospho-AKt increased rapidly in HASMC response to VEGF stimulation, accompanied by increased HASMC migration. Inhibition of VEGF-induced migration using Wortmannin indicated that early activation of PI3K was necessary for the SMC migration. As we expected, minocycline also inhibited VEGF-induced AKt activation and then inhibited HASMC migration, suggesting that minocycline has multiple inhibitory effects on VEGF-induced SMC migration. Because Wortmannin had no effect on MMP-2 and MMP-9 activation during VEGF stimulation, this suggests that a different signal pathway is used in the regulation of HASMC migration stimulated by VEGF.

In conclusion, our results allow a better understanding of intracellular events activated by VEGF stimulation and shed new light on the mechanisms by which minocycline inhibits VEGF-induced SMC migration. This warrants further exploration of the possible therapeutic uses of minocycline and other tetracycline derivatives in regulation of VEGF-mediated pathological angiogenesis.

	Acknowledgments

 
These studies were supported by a grant from the National Institutes of Health P01 NS44144 (G.Y.Y., W.L.Y.), R01 NS27713 (W.L.Y.), and R21 NS45123 (G.Y.Y.). The authors thank Carroll Schreibman and Broderick Belenson for editorial assistance, and the collaborative support of the staff of the Center for Cerebrovascular Research (http://avm.ucsf.edu/).

	Footnotes

 
Original received December 9, 2003; revision received June 29, 2004; accepted June 30, 2004.

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