Mol. Cells 2019; 42(3): 218-227
Published online February 1, 2019
https://doi.org/10.14348/molcells.2018.0162
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: gangqiao2004@126.com
This study was designed to determine the effects of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (
Keywords abdominal aortic aneurysm, apoptosis, extracellular matrix, lncRNA PVT1, vascular smooth muscle cell
Abdominal aortic aneurysm (AAA) is defined as focal dilatations of the abdominal aorta that are either 50% greater than the proximal normal segment or when the aorta measures more than 30 mm in diameter (Kumar et al., 2017). AAA is a leading cause of sudden death in the elderly (Ren et al., 2015). The risk factors of AAA include male gender, age of more than 75 years, prior vascular disease, hypertension, hypercholesterolaemia, cigarette smoking, and family history (Pande and Beckman, 2008). Clinically, surgical intervention is only recommended when the aneurysms are prone to rupture (Kent, 2014). Unfortunately, there is a lack of effective therapeutic drugs to limit the progression of AAA (Zhou et al., 2017). Therefore, basic research concerning the molecular mechanism of AAA is urgently needed to identify new biomarkers and therapeutic targets.
AAA is characterized by extracellular matrix (ECM) degradation, loss of arterial wall integrity, apoptosis of vascular smooth muscle cells (VSMCs), and infiltration of inflammatory cells (Miyake and Morishita, 2009). Previous studies suggest that apoptosis and depletion of VSMCs make an important contribution to AAA by eliminating a cell population that promotes connective tissue repair (Sachdeva et al., 2017). In addition, studies of human AAA tissues have shown that extensive inflammatory infiltrates containing macrophages, lymphocytes, and mast cells occurred in both the media and adventitia and that increased aneurysm diameter was associated with a higher density of inflammatory cells in the adventitia (Freestone et al., 1995). These infiltrating cells secrete various inflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. These cytokines can induce the activation of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9 (Galis et al., 1995; Sakata et al., 2000). MMPs and their tissue inhibitors (TIMPs) play a crucial role in ECM metabolism (Kosmala et al., 2008). Increased expression of MMPs and decreased expression of TIMPs contribute to ECM disruption and VSMC depletion, leading to AAA progression and rupture (Li and Maegdefessel, 2017). In addition, AAA demonstrates VSMCs phenotypic modulation of VSMCs characterized by downregulation of contractile-phenotype VSMC biomarkers (for example, α-smooth muscle actin [α-SMA]), upregulation of synthetic biomarkers (for example, osteopontin [OPN]), and upregulation of MMPs (Ailawadi et al., 2009; Sachdeva et al., 2017). Collectively, targeting ECM degradation, vascular apoptosis, and vascular inflammation may be a potential therapeutic approach for AAA pathologies.
Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides that are transcribed by RNA polymerase II, polyadenylated, spliced, and capped but lack significant open reading frames and cannot encode proteins (Ng et al., 2013). Collective evidence indicates that lncRNAs exert a wide variety of biological functions through various mechanisms and are correlated with both normal developmental processes and diseases such as cancer (Kung et al., 2013). Nevertheless, studies of the involvement of lncRNAs in AAA remain scarce (Yang et al., 2016; Zhou et al., 2017). Plasmacytoma variant translocation 1 (
AAA patients (n = 20) and control subjects (n = 20, aged 55–80 years) were recruited from Henan Provincial People’s Hospital. AAA tissues were acquired by surgery, and normal abdominal aortic tissues were obtained from subjects who suffered physical trauma unrelated to AAA. AAA and normal aortic tissues from each participant were snap-frozen in liquid nitrogen immediately after resection and stored at −80°C. This study was approved by the Research Ethics Committee of Henan Provincial People’s Hospital, and a written informed consent was obtained from each participant (Approval Number: HNPPH-2016-23).
qRT-PCR was performed as previously described (Guo et al., 2018), with some modifications. Total RNA from VSMCs or tissues was isolated using TRIzol (Invitrogen, Canada) reagent according to the standard protocol. First-strand cDNA was synthesized using the Reverse Transcription System Kit (Takara, China). qRT-PCR was performed using SYBR Green Mixture (Takara) in the ABI StepOne-Plus System (Applied Biosystems, USA). Data were normalized to the internal control, GAPDH. Comparative quantification was determined using the 2−ΔΔCt method.
Mouse primary VSMCs were purchased from Procell Co., China (cat. no. CP-M076). VSMCs were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, USA), supplemented with 10% foetal bovine serum (FBS, Gibco BRL), penicillin (100 U/mL) and streptomycin (100 mg/mL) in humidified air with 5% CO2 at 37°C.
Generation of lncRNA-
Short hairpin RNAs (shRNAs) against lncRNA-
Apolipoprotein E-deficient (ApoE−/−) male mice (genetic C57BL/6J background, 6–8 weeks old, 20–25 g) were purchased from Shanghai Slac Laboratory Animal Co, Ltd (China). All mice were raised in a specific pathogen-free environment under a 12 h light/12 h dark cycle throughout the experimental period. All animal experiments were performed in strict accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Animal Care and Use Committee of Henan Provincial People’s Hospital (Approval Number: HNPPH-2017-13).
Angiotensin II (Ang II) was used to induce AAA model in ApoE−/− mice in this study. Male ApoE−/− mice were infused with 1000 ng/kg/min Ang II (Sigma-Aldrich) over the course of 28 days. Ang II was infused via a subcutaneous osmotic minipump (Alzet Osmotic Pump, Model 2004; Durect Corp, USA) as previously described (Qin et al., 2017). Mice were anaesthetized with isoflurane as previously described, and pumps were implanted subcutaneously in the back in the prone position through a small incision that was closed with sutures (Fu et al., 2013).
The ApoE−/− male mice were randomly divided into four groups: normal saline (NS), Ang II, Ang II + sh-Ctrl, and Ang II + sh-
To quantify rate of apoptosis, an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) cell apoptosis kit was used according to the manufacturer’s instructions (Invitrogen). In brief, 48 h post-transfection, VSMCs were incubated with 10 μM Ang II for an additional 48 h. The total cells were washed twice with PBS buffer (pH 7.4), and re-suspended in the staining buffer provided in the kit. Subsequently, 5 μL Annexin V/FITC and 5 μL PI were mixed with VSMCs. After 10 min of cultivation at room temperature, the mixtures were analysed using the FACScan flow cytometry (BD Biosciences, USA). The percentage of apoptotic cells is indicated by the sum of the numerical values represented in the upper right and lower right quadrants. The cell apoptosis rate is equivalent to the average apoptosis rate from five flow cytometry analyses.
Protein was isolated from VSMCs or aortic tissues using a RIPA lysis buffer kit (Santa Cruz Biotechnology, Inc., USA). The protein content was determined in the supernatants using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., USA). Protein lysates (30 μg/sample) were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore Corp., USA) membranes. The membranes were blocked in 5% fat-free milk overnight at 4°C. Membranes were then probed with the following primary antibodies purchased from Abcam (Cambridge, USA): anti-α-SMA (cat. no. ab5694, 1:1,000), anti-OPN (cat. no. ab8448, 1:1,000), anti-MMP-2 (cat. no. ab92536, 1:1,000), anti-MMP-9 (cat.no. ab38898, 1:1,000), and anti-TIMP-1 (cat. no. ab38978, 1:1,000) and incubated overnight at 4°C. Subsequently, protein bands were detected by incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG, cat. no. ab6789; 1:2,000; Abcam) at room temperature for 1 h. Signals were detected using an enhanced chemiluminescence kit (ECL kit, Wuhan Boster Biotechnology Co., Ltd, China) and exposed to Kodak X-OMAT film (Kodak, Rochester, USA). The band intensity was quantified with the Quantity One software. Each experiment was performed at least three times. GAPDH served as the loading control.
The abdominal aortas of the mice in each group were cut and fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin and cut into serial sections (6 μm thick). Sections were stained with haematoxylin and eosin (HE) and Elastica van Gieson (EVG) as previously described (Watanabe et al., 2014). Immunohistochemistry for MMP-2 and MMP-9 was performed as previously described (Martorell et al., 2016). In brief, a rabbit polyclonal anti-mouse MMP-2 (cat. no. ab37150, 1:100, Abcam) or an anti-mouse MMP-9 (cat. no. ab38898, 1:100, Abcam) antibody was used. Representative images were captured using a fluorescence microscope (Nikon Corporation, Japan).
Apoptotic cells in the aortic tissues were detected by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining, according to the manufacturer’s protocol for the
Cytokine (TNF-α, IL-1β, and IL-6) levels were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol for each ELISA kit (Bender MedSystems, Austria).
Statistical analyses were performed using SPSS statistical software package standard version 16.0 (SPSS, Inc., USA). Statistical differences between two independent groups were determined using Student’s
To investigate the correlation between
Next, we examined relative
To explore the effect of
To assess the potential role of
Four weeks after Ang II infusion, the abdominal aortas of the mice in each group were isolated and cut into serial sections. HE staining revealed modest thinning of the media and marked thickening of the adventitia in the Ang II group compared to the NS group; however, these changes were attenuated by treatment with sh-
Four weeks after Ang II infusion, aortic tissues were isolated from mice and then stored at −80°C for RNA and protein analysis. The data revealed that Ang II infusion significantly upregulated
In the present study, we have shown for the first time that treatment with sh-
AAA is a vascular disease with a high mortality rate. Due to a lack of effective medications to reverse the progression of AAA, surgery is the most commonly recommended treatment for AAA patients. The phenotypic shift and apoptosis of aortic smooth muscle cells (SMCs), degradation of elastin and collagen, accumulation of T lymphocytes, and neovascularization are the best-known features of AAA (Parvizi and Harmsen, 2015). There are many potential mechanisms involved in the pathogenesis of AAA, including inflammation, VSMCs apoptosis, ECM degradation, and oxidative stress (Wang et al., 2017; Zhang et al., 2018). Pro-inflammatory cytokines such as IL-1β, IL-10, IL-6, IL-13, TNF-α, and interferon (IFN)-γ in the diseased aortic wall promote AAA development by modulating MMP secretion (Parvizi and Harmsen, 2015). MMPs are a family of zinc-dependent endopeptidases that include collagenases and elastases. MMPs mainly target and degrade the components of the ECM, such as collagen and elastin (Diaz et al., 2011). It has been shown that the expression of MMPs is increased in the aortic wall (Morris et al., 2014). MMP activation is tightly regulated by the TIMP family of inhibitors, and downregulation of TIMPs in AAA tissue has also been documented (Defawe et al., 2003; Tamarina et al., 1997). Importantly, the increase in MMPs and decrease in TIMPs contribute to ECM disruption and VSMC apoptosis (Li and Maegdefessel, 2017). Apoptosis of VSMCs leads to the massive loss of these contractile cells and, thus, of dilation of the abdominal aortic wall. Meanwhile, the delicate balance of synthetic and contractile phenotype of SMC is tilted to a synthetic phenotype. In these phenotypically shifted SMCs, ECM turnover is also increased, promoting more ECM degradation than ECM production. Together, these results contribute to AAA progression.
To study the mechanism underlying AAA formation, several animal models have been developed in recent years (Sénémaud et al., 2017). Different animal models for AAA may have diverse pathological manifestations. For example, increased aortic dilation and inflammatory response, which are characterized by medial degeneration with accumulation of macrophages, were observed in a CaCl2-induced AAA model (Chiou et al., 2001). Medial degeneration, accumulation of macrophages and distribution of neutrophils were identified as the pathological characteristics of AAA in an elastase-induced model (Pyo et al., 2000). Indeed, one of the most commonly used mouse models of AAA is Ang II perfusion-induced AAA in ApoE−/− mice, which results in many features similar to those of human lesions, including luminal dilatation, leukocyte infiltration, VSMC apoptosis, and ECM deformation (Qin et al., 2017). In this study, we demonstrated that we successfully established the classical murine Ang II-induced AAA model, as evidenced by aortic dilation, marked adventitial thickening, aortic elastin loss, enhanced aortic cell apoptosis, elevated levels of MMP-2 and MMP-9, reduced levels of TIMP-1, and increased levels of pro-inflammatory cytokines.
Some potential therapeutic drugs have been proposed and tested in animal models and clinical trials, such as statins, angiotensin pathway inhibitors, and antiplatelet drugs (Parvizi and Harmsen, 2015). For example, doxycycline, an inhibitor of MMPs, inhibited the development of AAA in animal experiments (Curci et al., 1998). Macrophage chemotaxis protein-induced protein 3 (MCPIP3) may act as an endogenous inhibitor of the inflammatory signalling pathway in endothelial cells and may be a potential therapeutic target in AAA (Zhang et al., 2018). Gingival fibroblasts help prevent the development of experimental AAA and rupture by promoting TIMP-1 production (Giraud et al., 2017). However, studies on the involvement of lncRNAs in AAA remain scarce (Yang et al., 2016; Zhou et al., 2017).
Yang et al. (Yang et al., 2016) utilized microarray analysis and identified 3,688 lncRNAs that were differently expressed between AAA and normal aortic tissues, in which 1,582 lncRNAs were upregulated and 2,106 lncRNAs were downregulated. Among these lncRNAs,
Accumulating evidence has demonstrated that
In summary, we have shown in the present study that sh-
Mol. Cells 2019; 42(3): 218-227
Published online March 31, 2019 https://doi.org/10.14348/molcells.2018.0162
Copyright © The Korean Society for Molecular and Cellular Biology.
Zhidong Zhang1,2, Gangqiang Zou1,2, Xiaosan Chen1,2, Wei Lu1,2, Jianyang Liu1,2, Shuiting Zhai1,3, and Gang Qiao1,2,*
1Department of Vascular and Endovascular Surgery, Henan Provincial People’s Hospital, Henan, China, 2Department of Aortic Surgery, Fuwai Central China Cardiovascular Hospital, Henan, China, 3Department of Vascular and Endovascular Surgery, Fuwai Central China Cardiovascular Hospital, Henan, China
Correspondence to:*Correspondence: gangqiao2004@126.com
This study was designed to determine the effects of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (
Keywords: abdominal aortic aneurysm, apoptosis, extracellular matrix, lncRNA PVT1, vascular smooth muscle cell
Abdominal aortic aneurysm (AAA) is defined as focal dilatations of the abdominal aorta that are either 50% greater than the proximal normal segment or when the aorta measures more than 30 mm in diameter (Kumar et al., 2017). AAA is a leading cause of sudden death in the elderly (Ren et al., 2015). The risk factors of AAA include male gender, age of more than 75 years, prior vascular disease, hypertension, hypercholesterolaemia, cigarette smoking, and family history (Pande and Beckman, 2008). Clinically, surgical intervention is only recommended when the aneurysms are prone to rupture (Kent, 2014). Unfortunately, there is a lack of effective therapeutic drugs to limit the progression of AAA (Zhou et al., 2017). Therefore, basic research concerning the molecular mechanism of AAA is urgently needed to identify new biomarkers and therapeutic targets.
AAA is characterized by extracellular matrix (ECM) degradation, loss of arterial wall integrity, apoptosis of vascular smooth muscle cells (VSMCs), and infiltration of inflammatory cells (Miyake and Morishita, 2009). Previous studies suggest that apoptosis and depletion of VSMCs make an important contribution to AAA by eliminating a cell population that promotes connective tissue repair (Sachdeva et al., 2017). In addition, studies of human AAA tissues have shown that extensive inflammatory infiltrates containing macrophages, lymphocytes, and mast cells occurred in both the media and adventitia and that increased aneurysm diameter was associated with a higher density of inflammatory cells in the adventitia (Freestone et al., 1995). These infiltrating cells secrete various inflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. These cytokines can induce the activation of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9 (Galis et al., 1995; Sakata et al., 2000). MMPs and their tissue inhibitors (TIMPs) play a crucial role in ECM metabolism (Kosmala et al., 2008). Increased expression of MMPs and decreased expression of TIMPs contribute to ECM disruption and VSMC depletion, leading to AAA progression and rupture (Li and Maegdefessel, 2017). In addition, AAA demonstrates VSMCs phenotypic modulation of VSMCs characterized by downregulation of contractile-phenotype VSMC biomarkers (for example, α-smooth muscle actin [α-SMA]), upregulation of synthetic biomarkers (for example, osteopontin [OPN]), and upregulation of MMPs (Ailawadi et al., 2009; Sachdeva et al., 2017). Collectively, targeting ECM degradation, vascular apoptosis, and vascular inflammation may be a potential therapeutic approach for AAA pathologies.
Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides that are transcribed by RNA polymerase II, polyadenylated, spliced, and capped but lack significant open reading frames and cannot encode proteins (Ng et al., 2013). Collective evidence indicates that lncRNAs exert a wide variety of biological functions through various mechanisms and are correlated with both normal developmental processes and diseases such as cancer (Kung et al., 2013). Nevertheless, studies of the involvement of lncRNAs in AAA remain scarce (Yang et al., 2016; Zhou et al., 2017). Plasmacytoma variant translocation 1 (
AAA patients (n = 20) and control subjects (n = 20, aged 55–80 years) were recruited from Henan Provincial People’s Hospital. AAA tissues were acquired by surgery, and normal abdominal aortic tissues were obtained from subjects who suffered physical trauma unrelated to AAA. AAA and normal aortic tissues from each participant were snap-frozen in liquid nitrogen immediately after resection and stored at −80°C. This study was approved by the Research Ethics Committee of Henan Provincial People’s Hospital, and a written informed consent was obtained from each participant (Approval Number: HNPPH-2016-23).
qRT-PCR was performed as previously described (Guo et al., 2018), with some modifications. Total RNA from VSMCs or tissues was isolated using TRIzol (Invitrogen, Canada) reagent according to the standard protocol. First-strand cDNA was synthesized using the Reverse Transcription System Kit (Takara, China). qRT-PCR was performed using SYBR Green Mixture (Takara) in the ABI StepOne-Plus System (Applied Biosystems, USA). Data were normalized to the internal control, GAPDH. Comparative quantification was determined using the 2−ΔΔCt method.
Mouse primary VSMCs were purchased from Procell Co., China (cat. no. CP-M076). VSMCs were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, USA), supplemented with 10% foetal bovine serum (FBS, Gibco BRL), penicillin (100 U/mL) and streptomycin (100 mg/mL) in humidified air with 5% CO2 at 37°C.
Generation of lncRNA-
Short hairpin RNAs (shRNAs) against lncRNA-
Apolipoprotein E-deficient (ApoE−/−) male mice (genetic C57BL/6J background, 6–8 weeks old, 20–25 g) were purchased from Shanghai Slac Laboratory Animal Co, Ltd (China). All mice were raised in a specific pathogen-free environment under a 12 h light/12 h dark cycle throughout the experimental period. All animal experiments were performed in strict accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Animal Care and Use Committee of Henan Provincial People’s Hospital (Approval Number: HNPPH-2017-13).
Angiotensin II (Ang II) was used to induce AAA model in ApoE−/− mice in this study. Male ApoE−/− mice were infused with 1000 ng/kg/min Ang II (Sigma-Aldrich) over the course of 28 days. Ang II was infused via a subcutaneous osmotic minipump (Alzet Osmotic Pump, Model 2004; Durect Corp, USA) as previously described (Qin et al., 2017). Mice were anaesthetized with isoflurane as previously described, and pumps were implanted subcutaneously in the back in the prone position through a small incision that was closed with sutures (Fu et al., 2013).
The ApoE−/− male mice were randomly divided into four groups: normal saline (NS), Ang II, Ang II + sh-Ctrl, and Ang II + sh-
To quantify rate of apoptosis, an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) cell apoptosis kit was used according to the manufacturer’s instructions (Invitrogen). In brief, 48 h post-transfection, VSMCs were incubated with 10 μM Ang II for an additional 48 h. The total cells were washed twice with PBS buffer (pH 7.4), and re-suspended in the staining buffer provided in the kit. Subsequently, 5 μL Annexin V/FITC and 5 μL PI were mixed with VSMCs. After 10 min of cultivation at room temperature, the mixtures were analysed using the FACScan flow cytometry (BD Biosciences, USA). The percentage of apoptotic cells is indicated by the sum of the numerical values represented in the upper right and lower right quadrants. The cell apoptosis rate is equivalent to the average apoptosis rate from five flow cytometry analyses.
Protein was isolated from VSMCs or aortic tissues using a RIPA lysis buffer kit (Santa Cruz Biotechnology, Inc., USA). The protein content was determined in the supernatants using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., USA). Protein lysates (30 μg/sample) were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore Corp., USA) membranes. The membranes were blocked in 5% fat-free milk overnight at 4°C. Membranes were then probed with the following primary antibodies purchased from Abcam (Cambridge, USA): anti-α-SMA (cat. no. ab5694, 1:1,000), anti-OPN (cat. no. ab8448, 1:1,000), anti-MMP-2 (cat. no. ab92536, 1:1,000), anti-MMP-9 (cat.no. ab38898, 1:1,000), and anti-TIMP-1 (cat. no. ab38978, 1:1,000) and incubated overnight at 4°C. Subsequently, protein bands were detected by incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG, cat. no. ab6789; 1:2,000; Abcam) at room temperature for 1 h. Signals were detected using an enhanced chemiluminescence kit (ECL kit, Wuhan Boster Biotechnology Co., Ltd, China) and exposed to Kodak X-OMAT film (Kodak, Rochester, USA). The band intensity was quantified with the Quantity One software. Each experiment was performed at least three times. GAPDH served as the loading control.
The abdominal aortas of the mice in each group were cut and fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin and cut into serial sections (6 μm thick). Sections were stained with haematoxylin and eosin (HE) and Elastica van Gieson (EVG) as previously described (Watanabe et al., 2014). Immunohistochemistry for MMP-2 and MMP-9 was performed as previously described (Martorell et al., 2016). In brief, a rabbit polyclonal anti-mouse MMP-2 (cat. no. ab37150, 1:100, Abcam) or an anti-mouse MMP-9 (cat. no. ab38898, 1:100, Abcam) antibody was used. Representative images were captured using a fluorescence microscope (Nikon Corporation, Japan).
Apoptotic cells in the aortic tissues were detected by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining, according to the manufacturer’s protocol for the
Cytokine (TNF-α, IL-1β, and IL-6) levels were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol for each ELISA kit (Bender MedSystems, Austria).
Statistical analyses were performed using SPSS statistical software package standard version 16.0 (SPSS, Inc., USA). Statistical differences between two independent groups were determined using Student’s
To investigate the correlation between
Next, we examined relative
To explore the effect of
To assess the potential role of
Four weeks after Ang II infusion, the abdominal aortas of the mice in each group were isolated and cut into serial sections. HE staining revealed modest thinning of the media and marked thickening of the adventitia in the Ang II group compared to the NS group; however, these changes were attenuated by treatment with sh-
Four weeks after Ang II infusion, aortic tissues were isolated from mice and then stored at −80°C for RNA and protein analysis. The data revealed that Ang II infusion significantly upregulated
In the present study, we have shown for the first time that treatment with sh-
AAA is a vascular disease with a high mortality rate. Due to a lack of effective medications to reverse the progression of AAA, surgery is the most commonly recommended treatment for AAA patients. The phenotypic shift and apoptosis of aortic smooth muscle cells (SMCs), degradation of elastin and collagen, accumulation of T lymphocytes, and neovascularization are the best-known features of AAA (Parvizi and Harmsen, 2015). There are many potential mechanisms involved in the pathogenesis of AAA, including inflammation, VSMCs apoptosis, ECM degradation, and oxidative stress (Wang et al., 2017; Zhang et al., 2018). Pro-inflammatory cytokines such as IL-1β, IL-10, IL-6, IL-13, TNF-α, and interferon (IFN)-γ in the diseased aortic wall promote AAA development by modulating MMP secretion (Parvizi and Harmsen, 2015). MMPs are a family of zinc-dependent endopeptidases that include collagenases and elastases. MMPs mainly target and degrade the components of the ECM, such as collagen and elastin (Diaz et al., 2011). It has been shown that the expression of MMPs is increased in the aortic wall (Morris et al., 2014). MMP activation is tightly regulated by the TIMP family of inhibitors, and downregulation of TIMPs in AAA tissue has also been documented (Defawe et al., 2003; Tamarina et al., 1997). Importantly, the increase in MMPs and decrease in TIMPs contribute to ECM disruption and VSMC apoptosis (Li and Maegdefessel, 2017). Apoptosis of VSMCs leads to the massive loss of these contractile cells and, thus, of dilation of the abdominal aortic wall. Meanwhile, the delicate balance of synthetic and contractile phenotype of SMC is tilted to a synthetic phenotype. In these phenotypically shifted SMCs, ECM turnover is also increased, promoting more ECM degradation than ECM production. Together, these results contribute to AAA progression.
To study the mechanism underlying AAA formation, several animal models have been developed in recent years (Sénémaud et al., 2017). Different animal models for AAA may have diverse pathological manifestations. For example, increased aortic dilation and inflammatory response, which are characterized by medial degeneration with accumulation of macrophages, were observed in a CaCl2-induced AAA model (Chiou et al., 2001). Medial degeneration, accumulation of macrophages and distribution of neutrophils were identified as the pathological characteristics of AAA in an elastase-induced model (Pyo et al., 2000). Indeed, one of the most commonly used mouse models of AAA is Ang II perfusion-induced AAA in ApoE−/− mice, which results in many features similar to those of human lesions, including luminal dilatation, leukocyte infiltration, VSMC apoptosis, and ECM deformation (Qin et al., 2017). In this study, we demonstrated that we successfully established the classical murine Ang II-induced AAA model, as evidenced by aortic dilation, marked adventitial thickening, aortic elastin loss, enhanced aortic cell apoptosis, elevated levels of MMP-2 and MMP-9, reduced levels of TIMP-1, and increased levels of pro-inflammatory cytokines.
Some potential therapeutic drugs have been proposed and tested in animal models and clinical trials, such as statins, angiotensin pathway inhibitors, and antiplatelet drugs (Parvizi and Harmsen, 2015). For example, doxycycline, an inhibitor of MMPs, inhibited the development of AAA in animal experiments (Curci et al., 1998). Macrophage chemotaxis protein-induced protein 3 (MCPIP3) may act as an endogenous inhibitor of the inflammatory signalling pathway in endothelial cells and may be a potential therapeutic target in AAA (Zhang et al., 2018). Gingival fibroblasts help prevent the development of experimental AAA and rupture by promoting TIMP-1 production (Giraud et al., 2017). However, studies on the involvement of lncRNAs in AAA remain scarce (Yang et al., 2016; Zhou et al., 2017).
Yang et al. (Yang et al., 2016) utilized microarray analysis and identified 3,688 lncRNAs that were differently expressed between AAA and normal aortic tissues, in which 1,582 lncRNAs were upregulated and 2,106 lncRNAs were downregulated. Among these lncRNAs,
Accumulating evidence has demonstrated that
In summary, we have shown in the present study that sh-
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