Study Of Shear Stress-Induced Matrix-Metalloproteinase 9 Expression And Signal Transduction Pathway In Endothelial Cells

BackgroundHemodynamics plays an important role in many various atheropathogenesis. Pober and Cotran summarized numerous researches about the relation of hemodynamics and atherosclerosis (AS) and raised shear stress hypothesis. They thought that the abnormal fluid shear stress was a significant factor to promote the formation of AS lesion.Hemodynamics has a visible impact on the morphous of endothelial cells (EC). The fluid in tubular aortas is laminar and ECs are elliptic that align in a direction to blood flow. There is complex turbulent flow in the bifurcation and curvature of vessel where the shape of ECs are polygonal lack of specific direction. Chronic changes of flow induce the remodel of aortas, alter the proliferation and death of cells, and change the balance of synthesis and degradation of extra cellular matrix. Shear stress hypothesis thinks that steady laminar fluid shear stress could selectively induce ECs atheroprotective gene expression, therefore, counteract harmful action of systemic dangerous factors to location.Matrix metalloproteinase 9 (MMP-9) is also named gelatinase that could be secreted by many kinds of cells such as ECs, vascular smooth muscle cells (VSMC), monocytes, macrophages, fibroblasts, neutrophil and so on. It is essential to degrade ECM and degrade intimal ECM, which results in leukocyte infiltration into the vessel wall and smooth muscle cell migration into the developing neointima. These two processes are keys to the development of atheroma. The expression and activity of MMP-9 are closely related to the generation and development of AS.Integrins are main receptors connecting ECM and intracellular skeleton system. The study demonstrates that integrins play a key role in the process of mechanical signal transduction.At present, it is not very clear about the relationship between shear stress and MMP-9 expression in ECs. The regulation of signal transduction is known little. So, we raised this hypothesis that low and oscillatory shear stress induced MMP-9 expression in HUVECs and physiological shear stress suppressed this expression.Objectives1. To identify if varied fluid shear stresses impact on MMP-9 expression in HUVECs.2. To identify if fluid shear stress induces MMP-9 expression in HUVECs through integrins by using founctional blocking-antibodies of integrins.Methods1. Cell CultureEthical approval was obtained from Shandong University Research and the Ethics Committee for the procurement of human umbilical veins from healthy term pregnant women.HUVECs were freshly isolated from human umbilical cord veins with 0.1% Collagenase II (Sigma, St Louis, MO, USA), and grown in T25 flasks in M199 medium supplemented with 20% fetal calf serum, 100 μg/ml streptomycin, 100 U/ml penicillin, and 20 ng/ml VEGF. Confluent primary cultures were harvested with use of 0.25% trypsin solution (Sigma, St Louis, MO), seeded onto slides pre-coated with 1% gelatin (Sigma). After 5 to 8 h, complete medium was added to flasks. The cells reached confluence in 3 to 4 days. HUVECs were grown in a humidified incubator in an atmosphere of 5% CO₂/95% O₂. 2. Shear stress intervention3 to 9 passage cells were used and seeded onto slides and then cultured in sterile flasks. Culture media were added in after 8 h. These cells will be divided into various groups when they are confluent to 80%.2.1 The effect of low fluid shear stress on MMP-9 expression in different time point1) Static control;2) Exposure to low fluid shear stress for 1, 3, 6, 8 h, respectively2.2 The effect of varied fluid shear stress on MMP-9 expression1) Static control;2) Exposure to low fluid shear stress (4dyn/cm²) for 6 h;3) Exposure to physiological shear stress (12dyn/cm²) for 6 h;4) Exposure to oscillatory shear stress (±5 dyn/cm² 1HZ) for 6 h.2.3 Induced expression of MMP-9 via integrins under fluid shear stress1) Static control;2) The group of Anti-integrinpi body: Pretreatment HUVECs with anti-integrinpM body for 2 h;3) The group of anti-integrin β3 body: Pretreatment HUVECs with anti-integrin β3 body for 2 h;4) The group of anti-integrinαvβ3 body: Pretreatment HUVECs with anti-integrin αvβ3 body for 2 h. 3. Taqman Real-time Quantitative RT-PCR AnalysesTotal RNA from HUVECs was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. The reverse transcription was performed at 42℃ for 1 h using the MLV Kit (Promega). Real-time PCR was performed on a Light Cycler (Roche Applied Science, USA). Three technical replicates were run for each gene in each sample. The primers used for MMP-9 (GenBank NM004994) amplification were 5'-cctggagacctgagaaccaatc-3' (upper strand) and 5'-gatttcgactctccac gcatc-3' (lower strand). The probe was5'-taccgctatggttacactcgggtggc-3'. The primers used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank M33197) were 5'-ggaaggactcatgaccacagt-3' (upper strand) and 5'-gccat cacgccacagtttc-3' (lower strand). The probe was 5'-tgccatcactgccacccag aagac-3'. Amplification was performed with 50 cycles and annealing at 62℃ for 5 s, extension at 72℃ for 10 s. The data was analyzed with Light Cycler software 4.0 (Roche Applied Science, USA). MMP-9 mRNA expression was normalized to the expressed housekeeping gene GAPDH.4. SDS-PAGE ZymographyThe conditioned media from stressed or static HUVECs culture was concentrated 30-fold using bag filter. Protein was separated by SDS-PAGE under the non-reducing condition on 8% polyacrylamide gels containing 1mg/ml gelatin. After electrophoresis, the gels were washed at room temperature for 1h in wash buffer (50 mM Tris-Cl, pH 7.4 and 2.5% Triton X-100), which were then incubated overnight at room temperature in 50 mM Tris-Cl, pH 7.4, 75 mM NaCl and 2.5 mM CaC12. The gels were stained with Coomassie Brilliant Blue R-250 and distained in 45% methanol and 10% acetic. After gel staining, MMP-9 was identified based on gelatin lysis at molecular masses 92 kDa for MMP-9. Gelatinolytic bands were quantified using Multi-Analyst densitometry software.5. Western Blot AnalysisThe medium of stressed HUVECs was condensed 30-fold using bag filters. Protein was boiled for 5 min. Equal amounts of protein were separated by 14% SDS-PAGE and transferred to a nitrocellulose membrane (BioRad, Hercules, CA). Following blocking with 5% non-fat milk, the blots were washed with PBS containing 0.1% Tween 20 and incubated with an appropriate primary antibody at 4℃ overnight. The blots were probed with antibodies against β-actin (rabbit, 1:1000 dilution) and TIMP-1 (mouse, 1:100 dilution). After overnight incubation, the blots were washed with TBST and incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:2000 dilution), then washed again. The blots were then visualized by use of enhanced chemiluminescence.6. Statistical analysisData were presented as mean ± SEM. For each condition, data from at least three independent experiments were quantified and analyzed by one-way ANOVA. A value of p<0.05 was considered statistically significant.Results1. The effect of low fluid shear stress on MMP-9 expression indifferent time pointTo explore the effect of mechanical stress on the expression of MMP-9, HUVECs were exposed to 4dyn/cm² shear stress at various time points. The level of MMP-9 mRNA maintained unchanged following 1 h of exposure. When HUVECs were subjected to shear stress for 3 h, 6 h or 8 h, there was a significant time-dependent increase in the MMP-9 mRNA level. The medium of stressed-HUVECs was condensed 30-fold using bag filters. The MMP-9 protein activity was measured by SDS-PAGE zymography. The level of active MMP-9 protein had no obvious change after 3 h exposure to 4dyn/cm² shear stress. After 6 h exposure, the level was 3.3-fold higher than that in static control (p < 0.01), but lower than that after 8 h. MMP-9 activity is known to be restricted by the presence of TIMP-1, a specific inhibitor of MMP-9. TIMP-1 protein levels were therefore measured by Western blot. The increased MMP-9 levels were not accompanied by a corresponding increase in TIMP-1 protein levels. 2. The effect of varied fluid shear stress on MMP-9 expressionThe MMP-9 mRNA level in HUVECs under varied fluid shear stress was measured by quantitative real time RT-PCR. Under low fluid shear stress (4 dyn/cm²), the mRNA level was comparable to that under static conditions after 1-h exposure but showed a significant time-dependent increase (p<0.05). After 6-h oscillatory flow (±5 dyn/cm², 1 HZ), the level increased about 6-fold as compared with that under static conditions, and 1.6-fold as compared with that after 6-h low fluid shear stress. However, the level decreased 3-fold after 6-h exposure to physiological (steady laminar) stress (12 dyn/cm²) as compared with that under static conditions and approximately 10-fold as compared with that under low fluid shear stress.We measured the levels of active MMP-9 in HUVECs by SDS-PAGE zymography. Under 6-h oscillatory stress , the protein level increased 1.7-fold as compared with that under static conditions (p < 0.01) and 1.3-fold as compared with that after 6-h exposure to low fluid shear stress (p < 0.01). However, under 6-h physiological stress, the protein level decreased 4-fold as compared with that under static conditions and 7-fold as compared with that under low fluid shear stress (p < 0.01).Since MMP-9 activity is restricted in the presence of TIMP-1, a specific inhibitor of MMP-9, we used western blot analysis to measure TIMP-1 protein levels and found increased MMP-9 levels not accompanied by a corresponding increase in TIMP-1 protein levels. 3. Induced expression of MMP-9 via integrins under fluid shear stressTo investigate the relation between integrins and stress-induced MMP-9 expression, HUVECs were co-incubated for 2 h with function-blocking antibodies to the integrins β1 β3 and αvβ3 and then subjected to varied fluid shear stress. Low and oscillatory shear-induced MMP-9 mRNA expression was inhibited by 2.7- and 2.5-fold, respectively (p < 0.01) on treatment with the antibody to integrin β1; 2.3- and 2.7-fold, respectively, with the antibody to integrin β3 (p<0.01); and 3.6- and 2.8-fold, respectively, with the antibody to integrin αvβ3, as compared with no antibody treatment (p < 0.01). Low and oscillatory shear-induced MMP-9 protein levels were reduced 4.2- and 2.6-fold, respectively, with the antibody to integrin β1 (p < 0.01); 3.4- and 3.1-fold, respectively, with the antibody to integrin β3 (p < 0.01) and 2.8- and 3.1-fold, respectively, with the antibody to integrin αvβ3, as compared with no antibody treatment (p < 0.01).Conclusion1. Both low and oscillatory shear stress could induce MMP-9 expression in HUVECs in vitro, however, physiological shear stress could suppress this expression.2. Varied fluid shear stress induced MMP-9 expression in HUVECs through integrins and integrinsβ1, β3 and αvβ3 played important roles in this progress. BackgroundPober and Cotran have raised shear stress hypothesis about atherogenesis after summarizing numerous studies related to the relationship between fluid shear stress and atherosclerosis (AS). The hypothesis thinks that abnormal flow shear stress is a critical factor resulting in AS lesion formation.Vascular endothelial cells (VEC) line inner vessel wall experiencing shear stress resulting from blood flow. Abnormal hemodynamic factors induce unsteady laminar shear stress and decrease of atheroprotective molecular secreted by VEC. Therefore, some risk factors persistenting, such as hypercholesteremia and high cysteine, promoted AS. There is disturbed flow model in bifurcation and high curvature, such as low magnitude wall shear stress, split stream and reversal. These sites are the the most impaired region. Obviously, the characterization of atherosclerotic local distribution mainly is related to the vessel impair degree, unrelated to risk factors and other related factors.Matrix metalloproteinase 9 (MMP-9) is one kind of gelatinases and also named gelatimase B. It is secreted by many kind cell types, such as endothelial cells, vascular smooth cells, monocytes, macrophage, fibroblast and neutrophil. MMP-9 can degrade the intimal extracellular matrix, which results in leukocyte infiltration into the vessel wall and smooth muscle cell migration into the developing neointima. These two processes are crucial in atheroma development. Genetic studies show that variations in the MMP-9 gene are related to the presence and severity of atherosclerosis.Integrins are a group of transmembrane glycoprotein which could connect extracelluar matrix and intracellular skeleton protein. They transmit bidirectional signals in cytomebrane: the intracellulr signals can regulate the connection activity between integrins and extracellular Hgands (inside-out signaling), at the same time, extracellular signals can transmit into cells through integrins (outside-in signaling) and change the biological functions of cells. Some studies demonstrated that integrin is a mechanical sensor and integrins-dependent signal pathway take part in the reaction of VEC to shear stress. Integrin could active many kinds of protein kinases in cytoplasm, such as MAPKs, and result in intacellular signal transmitting processe.Mitogen activated protein kinase (MAPK) is an important signal system mediating cellular reactions, which play a critical role in the development and progression of inflammatory and the generation of cytokines. Up to now, four MAPK signal molecules have been found in mammals, they are ERK1/2, JNK, p38 and ERK5. Studies demonstrated that MAPK participated in the mechanical signal transduction in cells.Now, there are at least four kinds of transcription factors on VECs that could be activated by shear stress, including NF-kB. Shear stress stimulates phosphorylaion and degradation of IkB in VEC and promotes subunits p50 and p65 to translocate into nucleus binding specific DNA sequence, finally, modulates the expression of target genes.However, at present the signal transduction pathway related to low shear stress-induced MMP-9 expression in VEC is unclear. ObjectivesIn our first part, we have proofed that low fluid shear stress could induce MMP-9 expression in HUVECs through integrins. In this part, we will demonstrate these questions as follow:Low fluid shear stress can induce MMP-9 expression via integrins/MAPKs/ NF-kB.Methods1. Cell CultureEthical approval was obtained from Shandong University Research and the Ethics Committee for the procurement of human umbilical veins from healthy term pregnant women.We improved the method that cultured HUVECs in according to Jaffe's. Please refer to the first part' intimate operating methods.2. Shear stress intervention3 to 9 passage cells were used and seeded onto slides and then cultured in sterile flasks. Culture media were added in after 8 h. These cells will be divided into various groups when they are confluent to 80%.2.1 NF-kB Regulates stress-induced MMP-9 expression in HUVECs1) Static control: no shear stress, no any inhibitors;2) Exposing HUVECs to low shear stress for 0, 15, 30 minutes, 1, 6 h, repectively;3) Exposing HUVECs to low shear stress for 6 h following 2 h pretreatment with SN50.2.2 ERK1/2 or p38 MAPK leads to stress-induced MMP-9 expression in a NF-KB-dependent manner in HUVECs1) static control: no shear stress and no any inhibitors;2) Exposing HUVECs to low shear stress for 0, 5, 10, 15, 30 min,1, 6 h; 3) Exposing HUVECs to low shear stress for 5, 15 min, 1, 6 h, respectively following 2 h pretreatment with PD98059;4) Exposing HUVECs to low shear stress for 5, 15 min, 1, 6 h, respectively following 2 h pretreatment with SB203580;5) Exposing HUVECs to low shear stress for 15 min, 1, 6 h, respectively following 2 h pretreatment with SP600125.2.3 Integrins mediate stress-induced MMP-9 expression via MAPK -NF-kB signaling pathways in HUVECs1) Static control: no shear stress and no any inhibitors;2) Exposing HUVECs to low shear stress for 0, 5, 10, 15, 30 min,1, 6 h;3) Exposing HUVECs to low shear stress for 5, 15 min, 1, 6 h following 2 h pretreatment with GRGDNP;MMP-9 mRNA expression in HUVECs was detected with probe real time RT-PCR, Active MMP-9 protein was detected with SDS-PAGE Zymography. MAPKs phorsphorylation level and IicBα were measured with western bolt. NF-kB p65 DNA-binding activity was measured with TransAM^TM assay kit. 3. Taqman Real-time Quantitative RT-PCR AnalysesTotal RNA from HUVECs was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. The reverse transcription was performed at 42 °C for 1 h using the MLV Kit (Promega). Real-time PCR was performed on a Light Cycler (Roche Applied Science, USA). Three technical replicates were run for each gene in each sample. The primers used for MMP-9 (GenBank NM004994) amplification were 5'-cctggagacctgagaaccaatc-3' (upper strand) and 5'-gatttcgactctccac gcatc-3' (lower strand). The probe was5'-taccgctatggttacactcgggtggc-3'. The primers used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank M33197) were 5'-ggaaggactcatgaccacagt-3' (upper strand) and 5'-gccat cacgccacagtttc-3' (lower strand). The probe was 5'-tgccatcactgccacccag aagac-3'. Amplification was performed with 50 cycles and annealing 62℃ 5 s, extension at 72℃ for 10 s. The data was analyzed with Light Cycler software 4.0 (Roche Applied Science, USA). MMP-9 mRNA expression was normalized to the expressed housekeeping gene GAPDH.4. SDS-PAGE ZymographyThe conditioned media from stressed or static HUVECs culture was concentrated 30 -fold using bag filter. Protein was separated by SDS-PAGE under the non-reducing condition on 8% polyacrylamide gels containing 1mg/ml gelatin. After electrophoresis, the gel was washed at room temperature for 1h in wash buffer (50 mM Tris-Cl, pH 7.4 and 2.5% Triton X-100) which were then incubated overnight at room temperature in 50 mM Tris-Cl, pH 7.4, 75 mM NaCl and 2.5 mM CaC12. The gels were stained with Coomassie Brilliant Blue R-250 and distained in 45% methanol and 10% acetic. After gel staining, MMP-9 was identified based on gelatin lysis at molecular masses 92 kDa for MMP-9. Gelatinolytic bands were quantified using Multi-Analyst densitometry software.5. Western Blot AnalysisProtein was boiled for 5 min. Equal amounts of protein were separated with a 14% SDS-PAGE and transferred to nitrocellulose membrane (BioRad, Hercules, CA). Following blocking with 5% non-fat milk, the blots were washed with PBS containing 0.1% Tween 20 and incubated with an appropriate primary antibody at 4°C overnight. The blots were probed with antibodies against β-actin (rabbit, 1:1000 dilution) IkBα (mouse, 1:100 dilution), phosphor-p38 MAPK (mouse, 1:100 dilution), phosphor- ERK1/2 (mouse, 1:300 dilution), phospho JNK1/2 (rabbit, 1:100 dilution). After overnight incubation, the blots were washed with TBST and incubated with secondary antibody conjugated to HRP (Santa Cruz. 1:2000 dilution), and then washed again. The blots were then visualized with enhanced chemiluminescence (ECL).6. Detection of NF-kB p65 activityThe DNA-binding activity of NF-kB p65 was detected with TransAMTM NF-kB p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA, USA) according to the manufacture's instructions. Positive controls, blanks and samples in duplicate were tested to measure the activity of NF-kB p65. Nuclear extracts containing 20 μg of protein were incubated with binding buffer and complete lysis buffer for 1 h at 100 rpm on a rocking platform at room temperature. Then wells were washed three times and incubated with primary (1:1000 dilution for NF-kB p65) and secondary antibody for 1 h, respectively. The developing solution was added to the wells to incubate for 10 min and stop solution was added when blue color of the samples turned dark blue. NF-kB p65 was evaluated with absorbance (A) value by spectrophotometer within 5 minutes at 450 nm with a reference wavelength of 655 nm.7. Statistical analysisFor each condition, data from at least three independent experiments were quantified and analyzed by one-way ANOVA. A value of p<0.05 was considered statistically significant. Data were presented as mean ± SEM. Results1. NF-kB Regulates stress-induced MMP-9 expression in HUVECs HUVECs were exposed to 4 dyn/cm² shear stress at various time points, and then total protein extracts were analyzed by Western blot. The levels of IkBα were significantly reduced after 15 and 30 min exposure to 4dyn/cm2 shear stress. To define the role of NF-kB in the induction of MMP-9 in stressed-HUVECs, transcription factor assay was performed. NF-kB DNA-binding activity was stronger after 1 h exposure to 4dyn/cm² shear stress than that in static control (p<0.01). HUVECs were pretreated for 2 h with 18μM SN50, a cell-permeant peptide that interrupted translocation of NF-kB, and then subjected to 4dyn/cm² shear stress for 1 h. The pretreatment with SN50 efficiently inhibited NF-kB DNA-binding activity (p<0.05). In the presence of the inhibitor, both MMP-9 mRNA expression (p<0.01) and protein activity in the supernatant (p<0.01) were significantly attenuated. These results indicated that NF-kB played a crucial role in the expression of this cytokine in stressed-HUVECs.2. ERK1/2 or p38 MAPK leads to stress-induced MMP-9 expression in a NF-kB-dependent manner in HUVECsHUVECs were exposed to 4dyn/cm² stress at various time points. A rapid activation of p38 MAPK, ERK1/2, and JNKl/2, as determined by phosphorylation levels, occurred after low fluid shear stress exposure in HUVECs. Phosphorylations of ERK1/2 and p38 MAPK peaked at 5 min, as did Phosphorylation of JNKl/2 at 15 min. Thereafter HUVECs were pretreated for 2 h with 20μM PD98059, a specific ERK1/2 inhibitor, 5μM SB203580, a specific p38 MAPK inhibitor and 18μM SP600125, a specific JNKl/2 inhibitor, respectively. Then HUVECs were exposed to 4dyn/cm2 shear stress. Stress-induced ERK1/2, p38 MAPK and JNKl/2 phosphorylations were abolished by PD98059, SB203580 and SP600125 respectively. MMP-9 mRNA expression and protein activity were evidently inhibited by PD98059 and SB203580, not by SP600125. However, neither SB203580 nor PD98059 was able to abrogate MMP-9 induction completely.HUVECs were incubated with PD98059 or SB203580 for 2 h before being stressed. The IkBα. levels increased significantly after 15 min exposure to 4 dyn/cm² shear stress and the NF-kB DNA-binding activity was depressed obviously after 1 h. 3. Integrins mediate stress-induced MMP-9 expression via MAPK -NF-kB signaling pathways in HUVECsHUVECs were preincubated for 2 h with 50μM GRGDNP, a synthetic peptide that could competitively inhibit integrins binding to extracellular matrix proteins containing RGD peptide, and then given 4dyn/cm² shear stress for 6 h. The shear stress-induced increases in MMP-9 mRNA (p<0.01) and protein activity (p<0.01) were both significantly inhibited.HUVECs were pretreated for 2 h with 50μM GRGDNP before being projected to shear stress for 5 min or 15 min. Total protein was extracted and measured by western blot. Shear stress-induced ERK1/2 and p38 MAPK phosphorylations were inhibited after 5 min exposure. JNK phosphorylation was inhibited and the degradation of IkBα was reduced after 15 min.To examine NF-kB DNA-binding activity, HUVECs were pretreated for 2 h with 50μM GRGDNP before being exposed to 4dyn/cm2 shear stress for 1 h. Nuclear protein was extracted from the collected HUVECs and transcription factor assay was performed. NF-kB DNA-binding activity was depressed significantly (p<0.01). Conclusion1. Integrins were involved in Low shear stress-induced regulate MMP-9expression in HUVECs.2. ERK1/2 or p38 MAPK was involved in Low shear stress-inducedregulate MMP-9 expression in HUVECs.3. NF-kB was involved in Low shear stress-induced regulate MMP-9expression in HUVECs.