| 1. IntroductionCardiovascular disease has become the greatest danger to human life and health. Hypertension and hyperlipidemia, which can cause vascular structure and function abnormality, are major risk factors for cardiovascular disease. Hypertension can increase the strain environment on the vessel wall. And elevated mechanical stretch can change the form and function of smooth muscle cells. At the same time, atherosclerotic plaque can rupture under abnormal fluctuations of blood pressure.We know that the renin angiotensin system (RAS) is closely related to cardiovascular disease. ACE2 can convert angiotensin I (Ang I) to angiotensin II (Ang II). Angiotensin II, the principal effector of RAS, binds to two types of cell surface receptors, type 1 (AT1R) and type 2 (AT2R).ACE2 is a new component of the RAS. It is the first known human homologue of ACE but differs from ACE in substrate specificity. Like ACE, ACE2 is a zinc-dependent peptidase of the M2-metalloprotease family that is sensitive to chloride ion concentration. ACE2 is able to cleave Ang Ⅱ and produce the vasodilating peptide Ang-(1-7). At the same time, ACE2 can cleave Ang I to Ang (1-9), which can be converted by ACE into the heptapeptide Ang-(1-7). It has been established that Ang-(1-7) acting on the Mas receptor mediates apoptosis antiproliferation and vasodilation, therefore opposing the effects of Ang II. Mechanical stretch can regulate the expression of ACE2, and ACE2 has a protective effect on cardiovascular disease. So we hypothesized that ACE2 plays an important role in the regulation of vascular smooth muscle cells.In light of these previous researches, we speculate that pathologic mechanical stretch (18% elongation,1HZ) can modulate the expression of ACE2 and its enzyme activity. ACE2 may be involved in the effects of pathologic stretch (18% elongation, 1HZ) on the cellular functions of HASMCs. In order to verify the hypothesis, in vitro and in vivo experiments were used to examine the effect of pathologic mechanical stretch on the expression of ACE2 in HASMCs. At the same time, the proliferation and migration of HASMCs were detected under the stimulation of pathologic mechanical stretch (18% elongation,1HZ) with or without Ad-ACE2.2. Objectives:(1)To investigate whether pathologic mechanical stretch(18% elongation,1HZ) can modulate the expression of ACE2 in HASMCs.(2) To elucidate the role of ACE2 in pathologic mechanical stretch mediated cellular functions of HASMC.(3)To assess signaling pathway involved in ACE2 production caused by pathologic mechanical stretch.3. Methods3.1. Cell culture and treatment3.1.1. Cell cultureHuman aortic smooth muscle cells (HASMCs) were obtained from the ATCC (American Type Cell Collection) and cultured in SMCM (smooth muscle cell medium) with 5% CO2 at 37 ℃. When cells reached confluence, removed the medium and washed with PBS twice, and then added pre-heated 0.25% Trypsin, centrifuged for 5 minutes (1000 r/min),after that, removed the supernatant and added fresh complete medium to collect cells. To apply mechanical stretch on HASMCs, cells at passages 4 to 7 were seeded onto flexible bottomed 6-well culture plates.3.1.2. Cell treatmentTo apply mechanical stretch on smooth muscle cells, flexible bottomed 6-well culture plates from Flexcell International Corporation were used. To starve HASMCs for 24h with serum-free medium when they reached 80-90% confluence. Media were replaced, then cells were stimulated with equibiaxial cyclic stretch. A Flexercell Tension Plus FX-5000T system (Flexcell International Corp., Hillsborough, NC) was used to apply 10% cyclic stretch (physiologic) and 18% cyclic stretch (pathologic) at 1Hz.3.2. Abdominal aortic banding of ratsAortic banding was performed on rats to induce pressure overload. Twenty Wistar rats (male,200-250g) were obtained from Beijing University Animal Research Center.3.3. Quantitative real-time PCRTRIzol reagent was used to extract total RNA from HASMCs. We use spectrophotometry to quantify the concentration of RNA. After that, RNA was reverse-transcribed into cDNA. SYRB Premix Ex Taq kit (Takara Bio Inc.) was involved in Real-time PCR. The relative mRNA expression level of ACE2 and ACE were assessed by the 2-△△Ct method.3.4. Western blot analysisProtein from HASMCs and rat aortas was extracted. Total cell lysates were separated on 10% SDS-PAGE and transferred to PVDF membranes. The membrances were incubated with 5% nonfat milk for 2 h, then overnight at 4℃ with the antibodies. We wash the membrances with TBST one time, then put them in appropriate secondary antibodies for 2 h. Visualization involved enhanced chemiluminescence plus reagents. Band densities were analyzed by use of Adobe Photoshop CS3.3.5. ACE2 activity assay7-Mca-YVADAPK (Dnp, 1uM) was used to assay ACE2 activity.3.6. ELISAThe Ang (1-7) and Ang Ⅱ levels in culture media were measured by ELISA according to the manufacturer’s instructions.3.7. Construction of adenovirus vectorThe Adenovirus-ACE2 and empty Adenovirus (control) were constructed by Genechem, Shang hai.3.8. Bromodeoxyuridine (BrdU) Incorporation AssayBrdU incorporation assay was used to determine HASMCs proliferation. Cells at passages 4 to 7 were seeded onto 6-well Bioflex plates coated with collagen Ⅰ for different stimulation factors. Cells were treated with BrdU labeling medium for 6 hours and were fixed with Ethanol fixative at-20℃, incubated at 4℃ with Anti-BrdU working solution overnight, stained with Anti-mouse-Ig-fluorescein antibody for 30 minutes and DAPI for 8 minutes for the nucleus. Then, cells wre examined using a microscope.3.9. Cell scratch testCell scratch test was used to determine HASMCs migration.3.10. Transient TransfectionHASMCs were transfected with negative control siRNA or ATF3 siRNA (GenePharma, Shang hai). For miR-421 over-expression and inhibition, miR-421 mimics and inhibitor (GenePharma,Shang hai) were transfected into HASMCs. And Lipofectamine 2000 was used for their transfection.3.11. Construction of plasmid encoding ACE2-3’UTRPlasmids containing luciferase fused to the wild-type ACE2-3’UTR (ACE2-3’UTR-WT) or the mutated ACE2-3’UTR (ACE2-3’UTR-MUT) were constructed to demonstrate the direct targeting of ACE2-3’UTR by miR-421.3.12. HistopathologyImmunohistochemistry was used to test the expressions of ACE2 and ACE.3.13. Statistical analysisData are expressed as mean ±SD. Comparisons of two groups involved unpaired t test and more than two groups one-way ANOVA. p< 0.05 was considered statistically significant. SPSS v16.0 (SPSS, Chicago, IL, USA) was used for statistical analysis.4. Results4.1. Pathological stretch modulates ACE2, ACE, Ang(1-7)and Ang II expression in HASMCs in vitroIn the in vitro time-response study,18% mechanical stretch suppressed the mRNA and protein expression of ACE2, with peak effect at 12 h (p<0.01). ACE2 enzyme activity declined at 6h,12h and remained at 24h(p<0.05). However,18% mechanical stretch increased the expression of ACE (p<0.05). Ang II levels increased at 12h and remained at an increased level after 24h of 18% mechanical stretch(P<0.01), while Ang (1-7) levels decreased under 18% mechanical stretch at 12h and maintained at 24h(P<0.01).In the in vitro force-response study, the expression and activity of ACE2 declined under 18% mechanical stretch at 12h (p<0.05) compared with static control. At the same time, the expression and activity of ACE2 declined under 18% mechanical stretch at 12h (p<0.05) compared with physiologic mechanical stretch. Both 10% and 18% mechanical stretch increased the mRNA and protein expression of ACE (p< 0.01), with greater effect with 18% than 10% stretch (p<0.05).The levels of Ang II increased,while the levels of Ang (1-7) decreased under 18% mechanical stretch at 12h compared with static control and physiologic mechanical stretch(P<0.01).4.2. In vivo aortic banding in rats influences the expression of ACE2 and ACERats underwent aortic banding to explore whether ACE2 and ACE levels were changed during pressure overload. The expression of ACE2 decreased at 5 and 7 days after aortic banding (P<0.01). Rather, the expression of ACE increased at 7days after aortic banding (P<0.01).4.3. The effect of ACE2 on pathological stretch-induced HASMCs proliferation, migration and collagen metabolismBrdU incorporation method was used to investigate the effect of 18% mechanical stretch on HASMCs proliferation.18% mechanical stretch promoted proliferation of HASMCs (P<0.01). Overexpression of ACE2 partly rescued proliferation increase with pulsatile shear stress (P<0.05) Cell scratch test indicated that the migration distance of HASMCs was obviously stimulated after 12 hours of 18% mechanical stretch (P<0.01). The promotive effect of stretch on HASMCs migration was partly abolished by ACE2 overexpression (P<0.01)18% mechanical stretch significantly increased the expression of type I and III collagen in HASMCs (P<0.05). Overexpression of ACE2 had no effect on the promotion of collagen with 18% mechanical stretch (P> 0.05)Taken together, our results revealed that ACE2 was involved in pathological stretch-induced HASMCs proliferation and migration, but had no effect on collagen metabolism.4.4. p38 is involved in the expression of ACE2 under pathological stretchWe examined the effect of 18% mechanical stretch on the phosphorylation of p38 MAPK, JNK and ERK1/2. Stretch induced the phosphorylation of p38 MAPK and JNK (p<0.05) but had no effect on ERK1/2 (p> 0.05). To validate these results, HASMCs were pretreated with the p38 MAPK inhibitor SB203580, JNK inhibitor SP600125, ERK1/2 inhibitor PD98059 for 1 h, then underwent 18% mechanical stretch for 12 h. SB203580 significantly attenuated the stretch-induced regulation of ACE2 expression (p<0.05).4.4. ATF3 is involved in the expression of ACE2 under pathological stretchWe transfected HASMCs with ATF3 siRNA. We found that down-regulation of ATF3 blocked the ACE2 level induced by pathological stretch (P<0.01), which indicated that ATF3 was involved in the expression of ACE2 under pathological stretch.4.5. miR-421 is involved in the expression of ACE2 under pathological stretchWe transfected HASMCs with Dicer siRNA. With the down-regulation of Dicer, the expression of ACE2 was elevated, which indicated that microRNA was involved in the expression of ACE2. By using TargetScan and microcosm, we explored miR-421 binding sequences at the 3’UTR of ACE2 mRNA. HASMCs were transfected with miR-421 mimics, RT-PCR and Western blot confirmed decreased level of ACE2 compared with the control RNA-transfected cells. In complementary experiments, HASMCs transfected with miR-421 inhibitor exhibited higher levels of ACE2. The transfected miR-421 mimics decreased the luciferase activity of ACE2-3’-UTR-WT in HEK293 cells.The level of miR-421 in HASMCs decreased after exposure to mechanical stretch for 12 hours and laste for at least 24 hours. HASMCs were transfected with miR-421 mimics and then exposed to 18% mechanical stretch. miR-421 mimics transfection attenuated stretch induction of ACE2 at both mRNA and protein levels.5. Conclusions(1) Pathological stretch (18% elongation,1HZ) suppressed the expression of ACE2 and Ang (1-7) and increased the expression of ACE and Ang Ⅱ.(2) ACE2 was involved in pathological stretch-induced HASMCs proliferation and migration, but had no effect on collagen metabolism.(3) ATF3 and miR-421 were involved in the expression of ACE2 under pathological stretch (18% elongation,1HZ).1. IntroductionVasculars are continually exposed to mechanical forces that, if excessive, such as in arterial hypertension, lead to vascular remodeling. The complex process of vascular remodeling involves enhanced protein synthesis and extracellular matrix (ECM) reorganization. Collagens, the major structural proteins in the ECM, are deposited to accommodate increased biomechanical loading with increased blood pressure. The synthesis and degradation of collagens are closely related to vascular remodeling.Many investigations show that almost all cell functions of VSMCs can be affected by mechanical stretch, including differentiation, collagen metabolism, proliferation, migration and apoptosis. In general,9%-12% stretch is called physiological stretch, it maintains the vessel wall normal functions and its structure. However, the stretch more than 15% can promote pathological vascular remodeling. We call it pathological stretch, which occurred in hypertension and atherosclerosis.Prolyl-4-hydroxylase (P4H) plays a central role in the synthesis of all known types of collagens. It catalyzes proline located in repeating X-Pro-Gly triplets to hydroxyproline during posttranslational processing. P4H has α and β subunits, and P4Hα1 is rate-limiting and essential for collagen maturation and secretion. Inhibition of P4H produces unstable collagen assoeiated with collagen decrease. And overexpression of P4H leads to more collagen.Matrix metalloproteinases (MMPs) primarily degrade components of the ECM. Remodeling of the ECM by MMPs is significant in both pathological and physiological processes. The activities of MMPs are regulated at multiple levels:by gene transcription and synthesis of inactive proenzymes, posttranslational activation of proenzymes, or the interaction of secreted MMPs with their inhibitors called tissue inhibitors of metalloproteinases (TIMPs).To determine the adaptive remodeling process in arterial hypertension, we assessed the cross-talk among signaling pathways involved in MMP and P4Hα1 production caused by pathologic mechanical stretch.2. Objectives:(1)To investigate whether pathologic mechanical stretch can modulate the expression of P4Hα1, MMPs, TIMPs and colalgen in HASMCs.(2)To assess signaling pathway involved in P4Haland MMP-2 production caused by pathologic mechanical stretch.(3) To elucidate the role of ACE2 on P4Hα1 and MMP-2.3. Methods3.1. Cell cultureHuman aortic smooth muscle cells (HASMCs) were obtained from ATCC(The American Type Cell Collection) and cultured in SMC medium with 5% CO2 at 37℃. When cells overgrew the bottom of culture flask, threw away SMCM and washed with PBS twice, then the pre-heated 0.25% Trypsin was poured into the culture flask, centrifuged for 5 minutes (1000 r/min). By the end of the centrifugation, discarded the supernatant and added SMCM complete medium to collect cells. To apply mechanical stretch on smooth muscle cells, flexible bottomed 6-well culture plates from Flexcell International Corporation were used.3.2. Cell treatmentTo apply mechanical stretch on HASMCs, cells at passages 4 to 7 were seeded onto 6-well Bioflex plates coated with collagen I. When cells reached 80-90% confluence, they underwent serum-free starvation for 24 h. Media were replaced, then cells were stimulated with equibiaxial cyclic stretch. A Flexercell Tension Plus FX-5000T system (Flexcell International Corp., Hillsborough, NC) was used to apply 10% cyclic stretch (physiologic) and 18% cyclic stretch (pathologic) at 1 Hz.3.3. Abdominal aortic banding of ratsAortic banding was performed on rats to induce pressure overload. Twenty Wistar rats (male,200-250 g) were obtained from Beijing University Animal Research Center.3.4. Quantitative real-time PCRTRIzol reagent was used to extract total RNA from HASMCs. RNA concentration was quantified by spectrophotometry and reverse-transcribed into cDNA. The SYRB Premix Ex Taq kit (Takara Bio Inc.) was used in Real-time PCR. The relative mRNA expression level of P4Hα1, MMP-2, TIMP-1, TIMP-2 and collagen were assessed by the 2-△△Ct method.3.5. Western blot analysisProtein from HASMCs and rat aortas was extracted. Total cell lysates were separated on 10% SDS-PAGE and transferred to PVDF membranes, which were incubated with 5% nonfat milk for 2 h, then overnight at 4℃ with the antibodies. Being washed by TBST, membranes were incubated with appropriate secondary antibodies for 2 h. Enhanced chemiluminescence plus reagents were involved in visualization. Band densities were analyzed by use of Adobe Photoshop CS3.3.6. ELISAType Ⅰ and Ⅲ collagen levels in culture media were assayed by use of ELISA kits.3.7. Gelatin zymographyThe enzymatic activity of MMPs in HASMCs and rat abdominal aortas were assayed by gelatin zymography. We used 10% SDS polyacrylamide gel containing 1 mg/ml gelatin for electrophoresis. Samples were renatured at room temperature in washing buffer, followed by a substrate buffer incubation in a 37℃ incubator for 20 h. Gels were stained with Coomassie brilliant blue R-250 and destained.3.8. HistopathologyThe banding abdominal aortas were dissected and immediately fixed in 4% formalin. Tissue was paraffin-embedded. Successive transverse paraffin sections were cut at 5 um thickness and underwent Masson’s trichrome staining and immunohistochemistry incubation of antibodies for MMP-2, P4Hα1, TIMP-1, and TIMP-2 overnight, then appropriate secondary antibodies.3.9. Statistical analysisData are expressed as mean±SD. Comparisons of two groups involved unpaired t test and more than two groups one-way ANOVA. p< 0.05 was considered statistically significant. SPSS v16.0 (SPSS, Chicago, IL, USA) was used for statistical analysis.4. Results4.1. Pathological stretch modulates P4Hα1, MMPs and TIMPs expression in HASMCs in vitroIn the in vitro time-response study,18% mechanical stretch increased the mRNA and protein expression of P4Hal, with peak effect at 12 h(p<0.05). Gelatin zymography showed that mechanical stretch increased MMP-2 activity(p<0.05), but MMP-9 activity was not detected. In line with these results,18% mechanical stretch increased MMP-2 mRNA and protein levels(p<0.05). The mRNA and protein levels of TIMP-1 and -2 were unchanged in response to mechanical stretch (p>0.05).In the in vitro force-response study, the expression of P4Hal raised under 18% mechanical stretch at 12h (p<0.01). Gelatin zymography showed that mechanical stretch increased MMP-2 activity by force (10% and 18%) (p<0.01), with greater activity with 18% than 10% stretch (p<0.01), but MMP-9 activity was not detected. In line with these results, force increased MMP-2 mRNA and protein levels (p<0.05), more with 18% than 10% stretch (p<0.05).The mRNA and protein levels of TIMP-1 and -2 were unchanged in response to mechanical stretch.4.2. Pathological stretch modulates collagen expression in HASMCs in vitro18% mechanical stretch significantly increased the expression of type Ⅰ and Ⅲ collagen in HASMCs (p<0.05).4.3. In vivo aortic banding in rats increases the expression of P4Hal and MMP-2MMP-2 activity was increased significantly at 7 days after aortic banding (p< 0.05), MMP-9 activity was also not detected. Aortic banding increased the protein levels of MMP-2 and P4Hα1(p<0.05). In addition, TIMP-1 and -2 protein levels were unchanged after aortic banding (p> 0.05). As a result, collagen content was greater in banded than control aortas(p< 0.05).4.4. MMP-2 and P4Hal participate in collagen metabolism induced by pathologic mechanical stretchTo investigate the effect of MMP-2 and P4Hα1 on collagen metabolism with pathologic mechanical stretch, HASMCs were transfected with MMP-2 and P4Hα1 siRNA before stretch treatment. Silencing of MMP-2 or P4Hα1 significantly increased or decreased the expression of collagen Ⅰ and Ⅲ under 18% stretch (p< 0.01).4.5. PI3K/Akt pathway mediates the expression of MMP-2 by pathologic mechanical stretchStretch induced the phosphorylation of Akt lasting for 6 h (p<0.01). To further investigate whether the PI3K/Akt pathway participates in MMP-2 and P4Hα1 expression with 18% mechanical stretch in HASMCs, cells were pretreated with the PI3K inhibitor LY294002 for 1 h, then underwent 18% mechanical stretch for 12 h. Along with inhibition of phosphorylation of Akt, stretch-increased MMP-2 activity and expression was attenuated (p< 0.05), but P4Hα1 level was retained (p> 0.05). Therefore, the PI3K/Akt pathway mediated the expression of MMP-2 but not P4Hα1 with 18% mechanical stretch in HASMCs.4.6. p38 MAPK and JNK pathways were involved in pathologic stretch-induced upregulation of P4Hal expressionWe examined the effect of 18% mechanical stretch on the phosphorylation of p38 MAPK, JNK and ERK1/2. Stretch induced the phosphorylation of p38 MAPK and JNK (p< 0.05) but had no effect on ERK1/2 (p> 0.05). To validate these results, HASMCs were pretreated with the p38 MAPK inhibitor SB203580, JNK inhibitor SP600125, ERK1/2 inhibitor PD98059 for 1 h, then underwent 18% mechanical stretch for 12 h. SB203580 and SP600125 significantly attenuated the stretch-induced upregulation of P4Hα1 expression (p< 0.01), whereas blockade of the ERK1/2 pathway had no effect on P4Hα1 expression (p> 0.05). MAPK inhibitors had no effect on MMP-2 activity and expression (p> 0.05).4.7. The effect of ACE2 on P4Hα1 and MMP-2In our preliminary experiment, ACE2 had no effect on pathologic mechanical stretch induced collagen metabolism. But MMP-2 and P4Hα1 participate in collagen metabolism induced by pathologic mechanical stretch. So we want to know the effect of ACE2 on P4Hα1 and MMP-2. Silencing of ACE2 in HASMCs, had no influence on the expression of P4Hal and MMP-2(p> 0.05).5. Conclusions(1) Pathological stretch (18%) increased the expression of P4Hal, MMP-2 and collagen.(2) Pathological stretch promotes P4Hal and MMP-2 production in HASMCs via Akt-p38 MAPK-JNK signaling.(3) ACE2 had no influence on the expression of P4Hα1 and MMP-2. |
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