2019 Volume 42 Issue 9 Pages 1464-1470
Aortic dissection (AD) diseases are characterized by degeneration of the aortic media. Oxidative stress plays a crucial role in the development of AD. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) deficiency reduces the incidence of aortic dissection induced by angiotensin II, but its mechanism remains to be further elucidated. The expression of Fibulin-5 is decreased in patients with AD, but its upstream mechanism is still unclear. This study was to clarify the relationship between NOX1 and Fibulin-5 in the AD. Results showed that the expressions of NOX1 and Fibulin-5 were increased and decreased in the AD, respectively. Next, by employing gain- and loss-of-function approaches in vitro, NOX1 negatively regulated Fibulin-5 in the vascular smooth muscle cells. Moreover, the blunted activity of NOX1 with VAS2870 could upregulate the expression of Fibulin-5. These findings indicate NOX1 is a negative modulator of Fibulin-5 in the AD.
Aortic dissections (ADs) and aneurysms are the most devastating complications of aortic diseases that afflict numerous individuals, especially in males and the older.1) It refers to a pathological change that the blood in the aortic cavity enters the aortic media from the aortic intimal tear and expands along the long axis of the aorta, resulting in the separation of the true and false aortic cavities.1) The estimated annual incidences of thoracic aortic dissection (TAD) range from 2.9 to 4.3 cases per 100000 individuals per year, and appear to be increasing over time.2,3) Notably, the reported rate is generally lower than the actual incidence because of difficulties in diagnosis.4,5) Without any definitive treatment, the mortality rate could be as high as 50% within the first 48 h for the pivotal cause of death, aortic dissection aneurysm rupture.6) Thus, early identification and proper treatment of aortic dissection are of vital importance.
The key histopathologic feature of AD is the degeneration of the aortic media, a pathological process characterized by smooth muscle cell depletion, fragmentation of elastic fibers and extracellular matrix degradation.7,8) Moreover, multiple studies manifested that these processes are associated with oxidative stress, so there is a mechanistic basis for a potential involvement of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) in the pathogenesis of aortic dissection.9–12) A previous study has shown that NOX1 deletion could reduce the incidence of aortic dissection induced by angiotensin II only through increasing nonlinearly the mRNA and protein levels of the tissue inhibitor of metalloproteinase 1 (TIMP-1), suggesting other mechanisms may be involved in NOX1 regulation of the development of aortic dissection.13) Furthermore, proteinases, such as matrix metalloproteinase, could result in elastin degradation. However, the role of elastogenesis, the de novo synthesis and elastic fiber assembly, in the AD remains to be unknown.14) Fibulin-5, as an extracellular protein, was reported to be associated with the polymerization and assembly of elastin.15,16) Also, previous studies demonstrated decreased expression of Fibulin-5 was observed in patients with TAD and this reduction may facilitate the process of AD via the fragmentation of elastic fibers and a reduction of elastin content.17) However, the study did not elucidate mechanisms about how Fibulin-5 expression was downregulated in the TAD. Therefore, a better understanding of these mechanisms may identify new biomarkers and potential therapeutic targets of aortic dissections and aneurysms.
In the present study, the expressions of NOX1 and Fibulin-5 were increased and decreased in the patients with AD compared with the controls, respectively. Next, by employing gain- and loss-of-function approaches in vitro, NOX1 could negatively regulate the expression of Fibulin-5 in the vascular smooth muscle cells. Besides, the blunted activity of NOX1 with VAS2870 could also increase the expression of Fibulin-5.
Antibodies against NOX1 (molecular weight: 65 kDa, #AP17191c, 1 : 1000 dilution), Fibulin-5 (molecular weight: 50 kDa, #AP18010b, 1 : 1000 dilution), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (molecular weight: 37 kDa, #AP7873a, 1 : 1000 dilution) were purchased from Baiqi Biotechnology (Suzhou, China). Peroxidase-conjugated secondary anti-bodies (1 : 10000 dilution) were purchased from Jackson ImmunoResearch Laboratories (U.S.A.) for visualization. The BCA protein assay kit was purchased from Pierce (U.S.A.). The cell culture reagents and other reagents were purchased from Sigma (U.S.A.).
Patient Enrollment and Tissue CollectionAll of the procedures involving human samples conformed to the principles of the Declaration of Helsinki and were approved by the ethical review committee of Renmin Hospital of Wuhan University. The specimens of the aortic dissection (n = 12) in this study were obtained from the ascending aortic wall undergoing the repair surgery. The clinical characteristics of AD patients were shown in Table 1. The normal control (n = 12) specimens were taken from the donors. The exclusion criteria were Marfan syndrome, Ehlers–Danlos Syndrome, and other known connective tissue disorders. Informed written consent was gained from the patients and the immediate relatives of the donors.
| Characteristics | AD patients |
|---|---|
| Age (y) | 53.6 ± 4.4 |
| Gender (M : F) | 9 : 3 |
| Hypertension | 10 |
| Diabetes mellitus | 2 |
| Smoking history (past or current) | 7 |
| Family history of AD | 1 |
| Maximal aortic diameter (cm) | 5.2 ± 1.2 |
| Ejection fraction (%) | 0.47 ± 0.12 |
| Classification of function capacity of the NYHA | DCM |
| II | 8 |
| III | 3 |
| IV | 1 |
AD: Aortic dissection; NYHA: New York Heart Association (classification).
Specimens were obtained and fixed in 10% formalin before being embedded in paraffin after dehydration. Then, 5-µm-thick sections of the aorta were stained with Verhoeff–van Gieson for the assessments of elastin content in the aortic wall.
Plasmid ConstructionThe full-length Flag-NOX1 plasmids were obtained by cloning the cDNA of NOX1 (Forward Primer: 5′-GGC CGA CAA AUACUACUACUU-3′, Reverse Primer: 5′-GUA GUA GUA UUU GUC GGC CUU-3′) into the CAG-Flag vector. The full-length HA-TSG101 plasmids were obtained by cloning the indicated cDNA of Tsg101 into the pCDNA3.1-HA vector.
Mouse Aorta Smooth Muscle Cells (SMCs) Culture and Immunofluorescence StainingMouse aorta SMCs (hereinbefore and hereinafter referred to as VSMCs) were cultured and seeded at Dulbecco’s modified Eagle’s medium (DMEM)/high glucose supplemented with 10% foetal bovine serum, and 1% penicillin–streptomycin. Three to four days later, the medium was exchanged; subsequently, the media was changed every other day. After 2 to 3 weeks, the plates were 75% to 80% confluent, and the explants were removed. The cells were analyzed before reaching confluence. Immunofluorescence staining was used to evaluate the expression of Fibulin-5 in VSMCs.
Quantitative Real-Time PCRFor real-time PCR, total mRNA from cultured VSMCs was extracted using TRIzol reagent (15596-026, Invitrogen, U.S.A.). Then, the RNA samples were reverse-transcribed into cDNA by using the Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche, Germany). Quantitative real-time PCR was employed by using SYBR Green (04887352001, Roche), and the results were normalized to the corresponding GAPDH gene expression. Primers for amplifying mouse genes were as follows: Mouse GAPDH forward primer, 5′-ACG GAT TTG GTC GTA TTG GG-3′ and reverse primer, 5′-CGC TCC TGG AAG ATG GTG AT-3′; Mouse NOX1 forward primer, 5′-CCC AAG CTT GGA TGG GAA AC-3′ and reverse primer, 5′-CCG GAA TTC AAA TTT TCT TT-3′.
Western BlottingFor Western blotting, total proteins from the VSMCs or the aortic wall tissues were first lysed in RIPA lysis buffer (720 µL of RIPA, 20 µL of phenylmethyl sulphonyl fluoride, 100 µL of complete protease inhibitor cocktail, 100 µL of Phos-stop, 50 µL of NaF, and 10 µL of Na3VO4), and the protein concentrations were measured by BCA protein assay kit (Pierce). Proteins (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen), and transferred to a polyvinylidene difluoride membrane (Millipore, U.S.A.). After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with different primary antibodies at 4°C overnight. After incubation with peroxidase-conjugated secondary antibodies at room temperature for 1 h, the membranes were reacted with ECL reagents prior to visualization using the Bio-Rad (U.S.A.) ChemiDoc™ XRS+ system. The expression levels of specific proteins were normalized to the corresponding GAPDH.
Measurement of Reactive Oxygen Species (ROS) GenerationAs described previously, the fluorescent dye 2′7′-dichlorodihydrofluorescein (H2DCF-DA) was used to measure the intracellular ROS levels.18) Briefly, after the cells were intervened, 20 μM H2DCF-DA dissolved in dimethyl sulfoxide was added. After 30 min, intracellular DCF fluorescence intensity was evaluated, and the DCF fluorescence intensity was converted to a percentage and this represents the ROS level. For fluorescence excitation, the 488 nm band of the argon ion laser of the confocal setup was used. Emission was recorded using an emission band of 525 nm.
Statistical AnalysisStatistical analyses were carried out using the SPSS software for Windows software (version 21.0, IBM Corp., U.S.A.). The results are presented as the mean ± standard error of the mean (S.E.M.). An unpaired Student’s t-test was employed for comparisons between two groups. One-way ANOVA followed by Bonferroni post hoc test (assuming equal variances) or Tamhane post hoc test (without the assumption of equal variances) was performed to calculate differences among more than two groups. A p value <0.05 was considered significant.
Verhoeff–van Gieson staining demonstrated, compared with the control group (Fig. 1A), the elastic fiber morphology and arrangement in the AD group (Fig. 1B) were irregular, broken, and structurally disordered, which suggested that elastin content was reducing in aortic dissection tissue.
n = 12 in A and B.
To investigate the potential role of NOX1 and Fibulin-5 in the initiation and progression of AD, we first determined the expression levels of NOX1 and Fibulin-5 in the aortic wall of AD patients and the controls by Western blot and immunohistochemistry, respectively. The results showed that NOX1 protein expression levels were markedly upregulated in the AD patients when compared with those in the controls, whereas the protein expressions of Fibulin-5 were significantly downregulated (Fig. 2A). Consistently, similar trends in the expression levels of NOX1 and Fibulin-5 were confirmed by immunohistochemistry (Fig. 2B). Collectively, these findings suggest that NOX1 is negatively correlated with Fibulin-5 in the development of AD.
A, Representative Western blots and quantitative analysis of NOX1 and Fibulin-5 protein levels in aorta tissues from the AD patients and the controls (n = 12 per group, * p < 0.05 vs. control, # p < 0.05 vs. control). B, Representative immunohistochemistry staining images of aorta from the AD patients and the controls (n = 12 per group). The data are presented as the mean ± S.E.M.
To explore the regulatory relationship between NOX1 and Fibulin-5, we successfully constructed the plasmids of NOX1 overexpression and transfected them into vascular smooth muscle cells (Fig. 3A). Accordingly, the activity of NOX1 was also enhanced (Fig. 3B). Western blot suggested that while the expression of NOX1 was increased in the pCDNA3.1-NOX1, Fibulin-5 was decreased (Fig. 3C). Then, we labeled Fibulin-5 with green fluorescence in the cellular immunofluorescence assay. Consistently, the fluorescence intensity of Fibulin-5 was also significantly weakened in the NOX1 overexpression group (Fig. 3D).
A and B, Real-time quantitative PCR showing the mRNA levels of NOX1 (A) and detection of ROS (B) in the VSMCs from indicated groups. C, Representative Western blots and quantitative analysis of NOX1 and Fibulin-5 protein levels in the VSMCs from indicated groups. D, Representative immunofluorescence images of VSMCs from indicated groups. n = 3 independent experiments in A–D. * p < 0.05 vs. control or pCDNA3.1 in A–C. # p < 0.05 vs. control or pCDNA3.1 in C.
To further confirm the relationship between NOX1 and Fibulin-5, we employed different small interfering RNAs (siRNAs) to interfere with the expression of NOX1 and screened the group with the lowest expression of NOX1. As shown in Fig. 4A, siRNA2 could minimize the mRNA expression of NOX1. Similarly, the activity of NOX1 was decreased in the siRNA2 group compared with the controls (Fig. 4B). Next, Western blot and immunofluorescence assay both indicated that once the expression of NOX1 was reduced, Fibulin-5 expression would be enhanced (Figs. 4C, D). Altogether, NOX1 is a negative modulator of Fibulin-5 in the AD. To investigate the effect of NADPH oxidase inhibitor (VAS2870) on Fibulin-5 expression, additional experiments were performed. The results showed that the activity of the NOX1 in the VAS2870 group was significantly decreased, and the expression of Fibulin-5 was significantly upregulated when the activity of the NOX1 was blunted (Figs. 4E, F).
A, Real-time quantitative PCR showing the mRNA levels of NOX1 and detection of ROS (B) in the VSMCs from indicated groups. C, Representative Western blots and quantitative analysis of NOX1 and Fibulin-5 protein levels in the VSMCs from indicated groups. D, Representative immunofluorescence images of VSMCs from indicated groups. E, Detection of ROS in the NADPH oxidase inhibitor (VAS2870) group and the controls. F, Representative western blots and quantitative analysis of Fibulin-5 protein levels in the VSMCs from indicated groups. n = 3 independent experiments in A–F. * p < 0.05 vs. control or lipo in A–C, E–F, # p < 0.05 vs. control in C.
Surgical artificial vessels grafts, interventional stenting, and newly emerging hybrid surgery are currently the primary treatments for aortic dissections and aneurysms. However, acute aortic dissection has a mortality rate up to 20–25% within 30 d, even with emergency surgery.6) As the molecular mechanisms of aortic media degeneration are still unclear, there are hitherto no biomarkers to diagnose aortic dissection, and no specific drugs to delay or even reverse the aortic dissection and aneurysm rupture. Therefore, it is imperative to further clarify the mechanisms of aortic dissection in combination with existing researches.
The inflammatory response induced by oxidative stress plays a crucial role in the initiation and development of aortic dissection and aneurysmal diseases, in which the overproduction of ROS and the subsequent aggregation of different metalloproteinases are the threshold process.19,20) Matrix metalloproteinases (MMPs) destroy the vascular structures, including the elastic lamellae and basement membrane, and degrade elastin, which results in vascular dissection.19) Thus, elastin fragmentation is a common characteristic of aortic dissection and aneurysms.21–23) In the present study, our finding indeed confirmed elastin fragmentation and reduced elastin content in aortic dissection tissue, which was in line with other researches.17,24)
NADPH oxidases are the major sources of reactive oxygen species in human and animal vasculature.20) NOX1 deficiency increases angiotensin II-induced TIMP-1 expression, alters the proteolytic balance in the vascular wall, alleviates the degradation of elastin and thereby protects against aortic dissection in mice.13) The treatment with the NOX inhibitor could mitigate oxidative stress in human AAA segments.12) Moreover, although the expression of NOX1 is elevated in the segments of abdominal aortic dissection, this difference does not reach statistical significance due to very low level of expression.12) In the present study, we found that the expression of NOX1 was significantly up-regulated in the aortic wall of thoracic aortic dissection.
Fibulin-5, a member of fibulin family, contains an evolutionally conserved arginine–glycine–aspartic acid (RGD) motif in addition to the similar common domain, calcium-binding epidermal growth factor like domains.16) As an extracellular matrix protein, Fibulin-5 is abundantly expressed in the embryonic vasculature and in elastic fiber-rich tissues in adults, such as aorta.25) Moreover, accumulating evidence suggests that the expression of Fibulin-5 is significantly associated with numerous diseases, such as cancers,26–28) chronic obstructive pulmonary disease29,30) and vascular disease.17,31–34) In the present study, compared with that from controls, the expression of Fibulin-5 from the patients with thoracic aortic dissection was lower, which is consistent with other studies.17)
NOX1 deficiency enhanced the expression of TIMP-1, thereby exerted its ameliorative effect in aortic dissection induced by angiotensin II. Although decreased Fibulin-5 was observed in thoracic aortic dissection, its mechanisms remain to be elusive. In the present study, we found that NOX1 could negatively regulate Fibulin-5 by employing gain- and loss-of-function approaches in vitro. Furthermore, the activity of NOX1 could also affect the expression of Fibulin-5. Therefore, we further clarified the vital role of NOX1 in thoracic aortic dissection. On the one hand, NOX1 represses angiotensin II-induced TIMP-1 expression, alters the protease/antiprotease equilibrium and promotes the degradation of elastin and basement membrane in the vascular wall. On the other hand, NOX1 modulates Fibulin-5 in a negative manner, thereby results in an abnormal assembly of mature elastin in the aortic walls. Moreover, when the expression and activity of NOX1 were enhanced, the production of ROS would be increased, thus the oxidative stress reaction was intensified.35) In the oxidative stress reaction, ROS could inhibit the binding of Fibulin-5 to elastin, and hinder the normal and orderly binding between elastin protein and microfiber scaffolds by hindering the process of cross-linking and deposition of elastin, resulting in loose elastic fiber structure and disordered arrangement, which affected the formation of normal elastic fibers.36) Therefore, we speculated that NOX1 may act on Fibulin-5 via ROS.
In summary, we first observed a negative correlation between NOX1 and Fibulin-5 in the AD. Then, further researches showed that NOX1 could negatively regulate Fibulin-5 in the VSMCs to affect AD.
This work was supported by Grants from the National Natural Science Foundation of China (No. 81700414).
The authors declare no conflict of interest.
