Aortic dissection (AD) is a fatal condition involving the aorta. In recent years, the prevalence of AD has increased worldwide, which led to the high demand for medical resources and social wealth.1 Vascular smooth muscle cells (VSMCs), the main components of aorta media, exhibit two phenotypes, namely, contractile and synthetic.2,3 Contractile VSMCs display weak proliferation and migration abilities, and exhibit a limited ability to synthesize extracellular matrices. By contrast, synthetic VSMCs feature strong proliferation and migration abilities, hypertrophic appearance, and “hill and valley” growth. Synthetic VSMCs show a strong ability to synthesize extracellular matrices (e.g., collagen, elastin, and proteoglycans).4 Most mature and healthy VSMCs are of the contractile phenotype and exhibit a relatively high contractile capability and a low proliferation ability.5 However, different physiological and pathological stimuli may cause the VSMC phenotypic switch, which leads to the proliferation, migration, and synthesis of excessive extracellular matrices.6 These effects result in pathological changes to tissues and organs. The morphological changes of AD primarily occur in aorta media.7 Under normal conditions, contractile VSMCs can maintain the vascular tone and homeostasis of the blood vessel wall. However, the number and proportion of synthetic VSMCs in AD patients are significantly increased, which reduce aortic elasticity and promote rupture.6,8,9 These results suggest that the phenotypic switch is an important factor in the occurrence of AD.6
Polycystin-1 (PC1), a 4302-amino acid (aa) cell-surface protein encoded by the polycystic kidney disease 1 gene (PKD1), is distributed in numerous tissues and involved in cell-matrix interactions and cell signaling.10 This protein contains a short cytoplasmic C terminal (PC1 C-tail, 197 aa), 11 transmembrane receptor-like glycoproteins, and a large N terminal extracellular region.11 Mutations in PC1 are indicators of vascular diseases and therefore of prognostic importance.12 Furthermore, the intracellular C-terminal domain of PC1 comprises specific tyrosine and serine sites for phosphorylation by c-src, focal adhesion kinase, and protein kinase A (PKA), as well as an RRSSR consensus sequence for putative S4166 phosphorylation.13 PC1 has also been implicated in various downstream signaling events; furthermore, it can be cleaved and localized to the nucleus via a nuclear localization signal present within its sequence.14 Overexpression of the C-terminal domain in ventricular myocytes increases [3H]Leucine incorporation by nearly twofold.15 These results suggest that the C-terminal domain of PC1 is an important factor in vascular diseases.
In the present study, we hypothesized that PC1 is an important factor in the VSMC phenotypic switch and in AD occurrence. To test this hypothesis, we determined whether the expression level of PC1 in AD patients is different from that of normal vessel walls and whether PC1 can promote the VSMC phenotypic switch from contractile to synthetic. Then, we explored the mechanism of the PC1 C-terminal domain on several kinases in VSMCs. Furthermore, we confirmed the role of PC1(S4166) on VSMC phenotypic switch.
Materials and Methods
Preparation of aortic tissues
Paraffin sections of aortic tissues were collected from AD patients and normal participants from the First Affiliated Hospital of Anhui Medical University. The study was approved by the Association of Medical Ethics of the hospital. A written informed consent was obtained from all participants.
Human aortic VSMCs were obtained from American Type Culture Collection (Rockville, MD, USA) and were cultured in F12K Kaighn’s modification medium supplemented with 10% fetal bovine serum (FBS) and 1 × Antibiotic-Antimycotic Solution at 37°C, humidified atmosphere containing 5% CO2. The growth medium was replaced every 3-4 day and the cells were seeded onto Petri dishes or multi-well plates at a ratio of 1 to 4 upon 80% confluence, and were starved for 24 h in serum-free medium prior to use.16,17
VSMC proliferation assay and cell cycle analysis
Cell counting kit-8 kits (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China) were used for evaluation VSMCs proliferation. Cell growth was arrested by incubation of the cells in serum-free medium for 24 h prior to be used according to the manufacturer’s instructions. Then, VSMCs were seeded onto a 96-well cell culture plate at a density of 2×103 cells/well for 24 h at 37°C, and treated with different concentrations of salusin-β for 24, 48 or 72 h. Finally, 10 μL of CCK-8 solution was added into each well, and incubated for 2 h at 37°C. The absorbance was conducted at 450 nm using a microplate reader (ELX800, BioTek Instruments, Inc., Vermont, USA). The distribution of various phases of the cell cycle was used to evaluate the VSMCs proliferation with flow cytometry.
Constructs and production of lentivirus
Recombinant lentivirus not expressing PC1 was designed by RNA interference. And recombinant lentivirus expressing GFP was used as a control. The sequence of PC1-shRNA1, sense: 5’-GATCCCGACAAGCAGTCCCTGACCTTCAAGAGAGGTCAGGGACTGCTTGTCGTTTTTTG-3’; The negative control shRNA, sense: 5’-CGCGTCCCCCACCTTTCGGCACTCTCCCTTCAAGAGGGGAGAGTGCCGAAAGGTGTTTTTGGAAAT-3’. Oligo DNA was annealed and subcloned to pLVX-shRNA2 vector, and then the connected compound was transfected with competent cell DH5. The recombinant plasmid was sequenced to verify whether insert sequences were consistent with oligo DNA sequences. A packaging plasmid system pLVX-shRNA2, psPAX2 and pMD2G were co-transfected into 293T cells via Lipofectamine 2000. The transfected cells were incubated for 6 h, and then the complete medium was replaced. The viral particles were subsequently harvested at 48 h after transfection, 0.45-μm cellulose acetate filter was used to remove the debris, under centrifugation at 50,000 rpm (4°C) for 2 h and resolved in formulation buffer. The titer of virions was determined through the transduction of HEK293 cells with serial dilutions of the vector and GFP expression assessment by fluorescent cells counting under fluorescence microscopy after 96 h. Viral stocks were aliquoted and stored until they were used.
Haematoxylin eosin (HE) and Masson’s trichrome staining
The thoracic aorta were fixed in 4% paraformaldehyde in PBS, embedded in paraffin, sectioned into 5-μm thick, and stained with Haematoxylin-Eosin staining or Masson’s trichrome staining according to the instructions. Vascular remodeling was analyzed under light microscopy (BX 45, Olympus Co., Tokyo, Japan).
The thoracic aorta were fixed in 4% paraformaldehyde in PBS, embedded in paraffin, sectioned into 5-μm thick slices, blocked with BSA, the sections were incubated with primary antibody against PC1 (ABT128, Millipore, Billerica, MA, USA) overnight at 4°C and then biotinylated secondary antibody for 60 min at room temperature. Vascular remodeling was analyzed under light microscopy (BX 45, Olympus Corporation, Tokyo, Japan).
RNA extraction and real-time quantitative PCR analysis
The mRNA of PC1, SM22, ACTA2, CNN1, Myc and CyclinD1 were analyzed by real-time quantitative PCR. Total RNA was isolated with Trizol reagent according to the manufacturer’s instructions. RNA concentration and quality were checked by spectrophotometry. cDNA was synthesized from RNA by reverse transcription. Real-time quantitative PCR was performed on an ABI PRISM 7500 sequence detection system. The 25 μL reaction mixture included 12.5 μL 2× SYBR Green PCR Mix, 5 μL diluted cDNA (1:10), and 0.5 μM sense and antisense primers. Amplification was carried out under the following conditions: initial denaturation for 10 min at 95°C, denaturation for 10 s at 95°C, annealing for 30 s at an optimal temperature for each cDNA, and extension for 30 s at 72°C. The mRNA levels of target genes were calculated after normalization to tubulin mRNA.
The content of PC1, SM22, ACTA2, CNN1, p38, ERK, JNK, PI3K and Myc, CyclinD1, and the phosphorylation of p38, ERK, JNK and PI3K were determined by Western blot. Preparation of whole cell lysates and tissue homogenates, and the immunoblotting assay were performed according to previously described procedures.18 The primary antibodies were obtained from the following sources: anti-Polycystin-1 (ABT128; Millipore); anti-SM22 alpha (ab14106; Abcam, Cambridge, UK); anti-alpha smooth muscle Actin (ab5694; Abcam); anti-Calponin (ab46794; Abcam); anti-alpha Tubulin (ab52866; Abcam); anti-p38 (phospho Y182) (ab47363; Abcam); Anti-p38 (ab7952; Abcam); Anti-ERK1 (pT202/pY204) + ERK2 (pT185/pY187) (ab50011; Abcam); Anti-ERK1 + ERK2(ab17942; Abcam); Anti-JNK1+JNK2 (phospho T183 + Y185) antibody (ab4821); Anti-JNK1+JNK2 (ab37228; Abcam); Anti-PI3K p85 (phospho Y607); Anti-PI3K p85 (ab189403; Abcam); anti-myc (ab32072; Abcam) and anti-Cyclin D1 (ab134175; Abcam).
Transwell invasion assay
Culture inserts of 8-μm pore size (Transwell; Costar; Sigma-Aldrich, St. Louis, MO, USA) were coated with Matrigel (BD Biosciences, San Jose, CA, USA) (100 μg/well) and placed into the wells of 24-well culture plates. In the lower chamber, 500 μL of medium containing 10% FBS was added, and 1×105 cells were seeded to the upper chamber. After 36 h of incubation at 37°C with 5% CO2, the number of cells that had migrated through the pores were fixed with 10% formalin and stained with 0.05% crystal violet. Data are the mean ± SD of three independent experiments.
Downregulation of PC1 remodels the aorta of AD patients
HE staining and Masson’s trichrome staining revealed the remodeling of arteries of the AD patients. Furthermore, HE-stained aortic sections showed that the normal aortic wall was composed of an inner layer, a middle layer, and an outer layer. The VSMCs in the middle layer were neatly arranged, and few inflammatory cells were scattered. The middle layer of the aortic wall from each AD patient became thinner and was accompanied by the loss of SMCs and accumulation of the outer layer and inflammatory cells. Masson’s trichrome staining showed that the collagen fibers from AD tissue were thick and disorganized, and their collagen accumulation increased in the aortic media compared with the controls (Figure 1A). These results showed that aorta from an AD patient shows fractures (red arrows) compared with that from a normal person. Moreover, immunohistochemical results showed that the expression level of PC1 was lower in the AD patient than in the controls. Using an antibody against the C-terminal region of PC1, the PC1 C-tail was detected in the nucleus (Figure 1B).
The PC1 mRNA and protein levels in AD patients and normal persons were also detected by Q-PCR and Western blot. PC1 mRNA was significantly lower in the aortic vascular smooth muscle of the AD patients than in the controls (Figure 2A). Similarly, the PC1 protein level was significantly lower in the AD patients than in the controls (Figure 2 B,C).
Genetic inhibition of PC1 promotes the proliferation and migration of VSMCs
The Q-PCR and Western blot were used to detect the efficiency of LV-PC1-kd. As shown in Figure 3 A,B, the PC1 was significantly downregulated by LV-PC1-kd. The proliferation of VSMCs was detected through cell counting kit-8 (CCK8) assay. Results revealed that the proliferation of VSMCs in the group with knock-downed PC1 was enhanced, especially after 72 h (Figure 3C). The mean OD450 values at 24 h were 0.61 and 0.73 in the control and PC1 knockdown groups, respectively (P=0.088). The mean OD450 values increased to 0.73 and 0.99 in the control and PC1 knockdown groups, respectively (P=0.023). Furthermore, the mean value significantly increased to 0.96 and 1.41 in the control and PC1 knockdown groups after 72 h (P=0.008). This results indicates the significant difference between the two groups. PC1 downregulation in the VSMCs reduced the cell ratio in the G0/G1 phase and increased that in the G2/M phase compared with the vehicle treatment in VSMCs (Figure 3D). The percentages of cells in the G0/G1 phase was 60.8 and 46.4 in the control and PC1 knockdown groups, respectively (P=0.010). The percentages of cells in the S phase were 33.1 and 38.8 in the control and PC1 knockdown groups, correspondingly (P=0.168). The percentages of cells in the G2/M phase was 6.02 and 14.8 in the control and PC1 knockdown groups, respectively (P=0.036) (Figure 3E). These data suggests the significant influence of PC1 in cellular proliferation. Moreover, in the transwell assay, the VSMCs with PC1 knockdown showed a strong migration (Figure 3 F,G). These results suggest that PC1 influences the proliferation, cell cycle distribution, and migration of VSMCs.
Genetic inhibition of PC1 promotes the differentiation of VSMCs
SM22, ACTA2 and CNN1 are biomarkers of the VSMCs phenotypic switch from contractile to synthetic; these biomarkers exhibit lower expression levels in the synthetic phenotype than in the contractile phenotype.19,20 In the present study, we determined the expression levels of these three genes to monitor the differentiation of VSMCs. Real-time polymerase chain reaction (PCR) showed that PC1 downregulation decreased the mRNA expression levels of SM22, ACTA2 and CNN1 in VSMCs (Figure 4A). Similarly, Western blot results showed that PC1 knockdown decreased the protein concentrations of SM22, ACTA2 and CNN1 in VSMCs (Figure 4 B,C). Overall, these results suggest that PC1 knockdown promotes VSMC differentiation.
Regulation of ERK phosphorylation in VSMCs
Many signaling pathways regulate the VSMC phenotypic switch.21-23 To determine signalling pathway involved in this process, we identified the phosphorylation levels of p38, JNK and PI3K in VSMCs. PC1 downregulation promoted the phosphorylation of ERK and MEK, but exerted no significant effect on the phosphorylation of p38, JNK and PI3K in VSMCs (Figure 5 A,B). The expression levels of myc and cyclinD1, which can be regulated by ERK were also detected. PC1 knockdown clearly increased the mRNA and protein expression levels of myc and cyclinD1 (Figure 5 C,D). These results indicate that the MEK/ERK/myc signaling pathway plays an important role in the proliferation and differentiation of VSMCs.
ERK phosphorylation in PC1-downregulated VSMCs is inhibited by SCH772984
An ERK inhibitor was used to determine the MEK/ERK/myc signaling pathway involved in the proliferation and differentiation of VSMCs. SCH772984, an inhibitor of ERK1 and ERK2, can inhibit the proliferation of VSMCs. The mean OD450 values of the control, PC1 knockdown, and SCH772984 groups were 0.91, 1.40, and 1.05, respectively, with a significant difference between the PC1 knockdown and SCH772984 groups (P<0.001) (Figure 6A). Flow cytometry results showed that the mean percentage of cells in the G2/M phase of the control, PC1 knockdown, and SCH772984 groups were 10.2, 15.1, and 8.8, respectively, with a significant difference between the PC1 knockdown and SCH772984 groups (P<0.001) (Figure 6 B,C). SCH772984 addition decreased the proliferation of VSMCs with PC1 knockdown. Interestingly, it did not significantly change the protein expression levels of ACTA2, SM22, and cyclinD1 (Figure 6D).
Cytoplasmic tail mutation of PC1 promoted ERK phosphorylation
Considering that PC1 downregulation can promote the proliferation of VSMCs, we hypothesized that altering the expression of wild PC1 affects VSMCs proliferation. Thus, we created a PC1 (pS4166A) mutant to examine the phosphorylation of ERK and the expression of SM22, ACTA2, CNN1, cyclinD1 and myc. Western blot results showed that the protein expression levels of phospho-ERK, ERK, SM22, ACTA2, CNN1, cyclinD1 and myc in the PC1(S4166A) mutant were similar to those in the PC1 knockdown cells (Figure 7 A,B). These results suggest that PC1 C-tail can increase the expression of SM22, ACTA2, and CNN1 which are markers of the contractile phenotype of VSMCs, thereby promoting the phenotypic switch to synthetic phenotype and the proliferation of VSMCs. Furthermore, S4166 is a crucial factor in the function of PC1 C-tail (Figure 7C).
On the basis of these results, we hypothesized that PC1 can regulate the MEK/ERK/myc pathway and induce the VSMC phenotypic switch. However, the mechanism by which PC1 regulates CyclinD1 is still unknown. The PC1 C-tail, especially S4166, is the key factor in the entire process.
In the current study, PC1 was significantly downregulated in the VSMCs of AD patients. PC1 was involved in MEK/ERK/myc pathway and PC1 downregulation promoted VSMC proliferation and phenotypic switch. The PC1 C-tail was also a key factor in the ERK pathway. The effect of overexpressed S4166 mutant in the VSMCs, was similar to that of the downregulated PC1. There results suggest that the PC1 C-tail is the key pathophysiological cause of AD, and is involved in the MEK/ERK/myc signaling pathway. The aorta is composed of three layers, particularly, the tunica adventitia, tunica media, and tunica intima. The tunica media is composed of elastic fibers and VSMCs. The pathology of AD is medial degeneration caused by the VSMC phenotypic switch.24-26 Our findings suggest that the intervention of PC1 can potentially prevent excessive vascular proliferation and medial degeneration, thereby reducing the risk of AD.
PC1 is a transmembrane molecule that plays an essential role in renal tubular morphogenesis. PC1 dysfunction causes human autosomal dominant polycystic kidney disease (ADPKD). Previous studies showed that AD is related to ADPKD, hypertension and atherosclerosis. PC1 downregulation induces the proliferation of human VSMCs. In this regard, CCK8 assay is a convenient colorimetric assay for testing VSMC proliferation. In the current study, we confirmed that PC1 knockdown can stimulate the proliferation of VSMCs in a time-dependent manner. Furthermore, PC1 downregulation increased the ratio of cells in the S and G2/M phases, and promoted VSMC migration. ACTA2 encodes the VSMC-specific α-actin, a component of the contractile complex, SM22 is a 22 kDa lineage-restricted protein of smooth muscle cells (SMC) that is physically associated with cytoskeletal actin filament bundles in contractile VSMCs, CNN1 can bind to actin and calmodulin and can regulate and modulate smooth muscle contraction. Thus, ACTA2, SM22, and CNN1 can reflect VSMC proliferation. The mRNA and protein levels decreased when PC1 knocked down in VSMCs. This effect was also observe in AD patients. These data indicate that PC1 downregulation of PC1 is an important triggering factor of AD formation.
The pathways involved in the pathogenesis of AD includes the TGF-β, Smad3, and angiotensin II signaling pathways and the VSMC pathway.6,27,28 In the current study, we measured the phosphorylation of p38, ERK, JNK, and PI3K and found that ERK phosphorylation significantly increased. The ERK signaling pathway, which involves MEK, myc, and cyclinD, is important in the cell proliferation and migration. By transducing the stimulatory extracellular signals to the nucleus, the MEK/ERK/myc signaling pathway can trigger a series of biological reactions, such as cell proliferation, differentiation, and apoptosis, and plays an important regulatory role in these processes. Recent studies have shown that the MEK/ERK/myc signaling pathway is related to the contractile function of vascular smooth muscle, and contributes to VSMC dysfunction, vasoconstriction, and vascular remodeling.15 PC1 is involved in the regulation of the VSMC phenotypic switch.15 In our previous study, the mutant PC1 was detected in the vessel tissue of patients (unpublished data).
Gene mutations and environmental factors, such as hypertension, can increase the vascular pressure and activate the AD-related pathways.29,30 PC1 can be phosphorylated at S4166 in the C-terminal domain by protein kinase X (PRKX).13 PRKX is a cAMP-dependent CREB kinase that can promote cell migration and proliferation. PRKX shows similar roles to PC1 in VSMCs.31 Mutations in the S4166 of the PC1 C-tail cause ERK phosphorylation, increase the expression levels of cyclinD1 and myc; and decrease ACTA2, SM22, and CNN1. These effects were also observed in VSMCs with PC1 knockdown. These results indicate that the PC1 C-tail plays an important role in the VSMC phenotypic switch.
In conclusion, these findings demonstrate that PC1 regulates AD by modulating the phenotype of VSMCs. PC1 downregulation may induce AD formation, which involves the MEK/ERK/myc signaling pathway. The PC1 C-tail, especially the S4166 site, plays the main role in this process.