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Particularly interesting Cys-His-rich protein is highly expressed in human intracranial aneurysms and resists aneurysmal rupture

Particularly interesting Cys-His-rich protein (PINCH) has several biological functions in cancer development, invasion and metastasis in malignant cells, and the expression of PINCH is upregulated in several cancer types, including breast cancer, gastric adenocarcinoma and rectal cancer. However, the contribution of PINCH to human cerebral aneurysms remains largely unknown. Therefore, the significance of PINCH expression in cerebral aneurysm growth and rupture was examined in the present study. The protein expression levels of alpha-smooth muscle actin, osteopontin (OPN), matrix metalloproteinase (MMP) 9 and PINCH were evaluated using immunohistochemistry and western blot analyses. The results demonstrate that the protein expression levels of OPN, MMP9 and PINCH in the unruptured intracranial aneurysm (UA) and ruptured intracranial aneurysm (RA) groups were markedly higher than those of the control group, whereas OPN and PINCH expression levels were decreased in the RA group compared to those of the UA group. In addition, there was a strong correlation between PINCH and tumor size (r=0.650 and P=0.0026), as well as between PINCH and OPN (r=0.639 and P=0.0033) in the unruptured cerebral aneurysms. However, the correlation between PINCH and tumor size (r=0.450 and P=0.1393) and between PINCH and OPN (r=0.366 and P=0.2426) revealed no obvious difference in the ruptured cerebral aneurysms. In conclusion, PINCH was highly expressed in the UAs, which may be a critical factor for preventing aneurysmal rupture. Moreover, PINCH may facilitate intracranial aneurysm progression, at least partially, through the activation of extracellular signal-regulated kinase signaling and the suppression of c-Jun N-terminal kinase signaling.


Introduction
Intracranial aneurysms (IAs) are relatively common vascular abnormalities of the cerebrum and are the third leading cause of cerebrovascular accidents, accounting for ~3% in the general population (1). Currently, the incidence and operations of IAs have significantly increased, and IA-associated clinical manifestations, such as intracranial hypertension and hemorrhage, are considered to have a diameter of ≥0.6 cm for patients with increasing risk of rupture, which is associated with significant morbidity and mortality (2,3). Notably, subarachnoid hemorrhage remains lethal in up to 65% of cases, and significantly disables 50% of those who survive (4). Complex open surgical repair and endovascular approaches have proven to be a good and potent alternatives to open repair of these aneurysms for older and high-risk patients as well as for aneurysms with optimal morphological suitability (5). However, intervention therapy has the risk of developing a neurological injury (6)(7)(8). Nevertheless, the advances in effective therapy for IAs have been limited due to inherited pathogenic pathways remaining obscure. Therefore, identification of the molecular mechanism for the IAs is required in order to develop an effective treatment.
Particularly interesting Cys-His-rich protein (PINCH), which is composed of 5 LIM domains arrayed in tandem, is expressed in an ubiquitous manner in early embryonic development and adult tissues and has been suggested to be important in processes as diverse as migration, cell adhesion, differentiation, proliferation and survival (9,10). Functional studies have revealed that PINCH loss-of-function leads to embryonic lethality and displays an abnormal epiblast polarity, impaired cavitation, detachment of primitive endoderm (PrE) and severe apoptosis of the PrE (11,12). PINCH-1 promotes B-cell lymphoma (Bcl)-2-dependent survival signalling and inhibits c-Jun N-terminal kinase (JNK)-mediated apoptosis in the PrE (13). Although the function of PINCH remains unknown, increasing numbers and accumulating evidence indicates that PINCH has been correlated with cancer development, invasion and metastasis in malignant cells, including breast cancer (14), pseudomyxoma peritonei (15), gastric adenocarcinoma (16) and rectal cancer (17). For example, PINCH is demonstrated to protect tumor cells from apoptosis by increasing the activity of the pro-survival protein extracellular signal-regulated kinase (ERK)1/2 and Akt (18). In breast cancer cells, Ras suppressor-1 induces apoptosis through the suppression of PINCH-1, and the integrin-linked kinase/PINCH/α-parvin (IPP) complex as a therapeutic target is downregulated in the chelidonine-treated MDA-MB-231 cell line (14,19). To the best of our knowledge there is no literature available regarding the association between PINCH abnormal expression and IAs.
In the present study, an association between PINCH and tumor size was revealed, and PINCH was highly expressed in IAs (including unruptured and ruptured aneurysms), in which it was predominantly localized to the lumen. Furthermore, the present study sought to determine the regulatory events associated with the expression of PINCH in ruptured and unruptured human cerebral aneurysms. Moreover, the levels of PINCH were demonstrated to be significantly higher in unruptured compared to ruptured aneurysms as observed by immunohistochemistry and western blotting. In addition, the rupture of aneurysms was associated with the expression of PINCH in the cerebral aneurysms.

Materials and methods
Patients and specimens. A total of 31 IAs (including 19 unruptured and 12 ruptured cerebral aneurysms) were collected from Fu Xing Hospital Affiliated to Capital Medical University (Beijing, China) between Jan 2008 and June 2014. All the patients recruited in the present study were not subjected to preoperative radiotherapy or chemothera py and were diagnosed with IAs based on histopathological evaluation by digital subtraction angiography (DSA), as previously described (20). The control specimens were 5 intracranial cerebral arteries obtained from patients who underwent surgery. All of the tissue samples collected were immediately stored in liquid nitrogen for western blot analysis or fixed in 10% formalin for immunohistochemical staining. Human samples were obtained with written informed consent from all patients. In addition, the study was approved by the Ethics Committee of the Fu Xing Hospital Affiliated to Capital Medical University (Beijing, China).
Histomorphology and immunohistochemistry. Formalin-fixed and paraffin-embedded tumor tissues were cut into 4 µm sections, which were stained with hematoxylin and eosin (H&E) and visualized under a Leica DM 2500 microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA).
The 5 control artery and 31 aneurysm samples were immunohistochemically evaluated using anti-human alpha-smooth muscle actin (α-SMA; ab5694; 1:200; Abcam, Cambridge, MA, USA), osteopontin (OPN; sc-20788; 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), matrix metalloproteinase (MMP) 9 (ab73734; 1:200; Abcam) and PINCH (sc-136299; 1:50, Santa Cruz Biotechnology, Inc.). Tissues embedded in paraffin were cut into 4-µm sections, mounted on glass slides and stained using indirect immunoperoxidase (P0203; Beyotime Institute of Biotechnology, Haimen, China). The paraffin sections were then baked in an oven at 65˚C for 24 h, then dewaxing to water, and were rinsed with PBS three times (5 min each time). The sections that were washed well were placed in the EDTA buffer for microwave antigen retrieval, boiled, then low heat (100˚C) to boil after an interval of 10 min. Following cooling, the sections were washed with PBS 3 times. They were then placed in 3% hydrogen peroxide solution and incubated at room temperature for 10 min, with the purpose of blocking any endogenous peroxidase. These were then washed with PBS 3 times, and blocked with 5% bovine serum albumin (BSA; ST023; Beyotime Institute of Biotechnology) for 20 min after drying (close charge). Following the removal of the BSA solution, each section was incubated with 50 µl anti-α-SMA, anti-OPN, anti-MMP9 and anti-PINCH primary antibodies overnight at 4˚C, and then washed with PBS 3 times. Following the removal of PBS solution, each slice was incubated with 50-100 µl goat anti-rabbit IgG (A0208) and goat anti-mice IgG (A0216; both 1:5,000; both Beyotime Institute of Biotechnology) secondary antibodies at 4˚C for 50 min, then washed thrice with PBS, and each slice was added to 50-100 µl freshly prepared DAB solution with the help of a microscope in order to observe color. After washing, the sections were counterstained with hematoxylin, rinsed with tap water, dehydrated and mounted. Six sections were randomly selected with no overlapping area (magnification, x200), were observed and photographed. The tissue with brown color (except for the tissue edges) were regarded as α-SMA, OPN, MMP9 and PINCH positive. Image Pro-Plus 6 software (Media Cybernetics, Inc., Rockville, MD, USA) was used for the analysis. The integrated optical density (IOD) was respectively measured for the tumor tissues of immunohistochemical positive staining. The higher the IOD, the higher the expression of the corresponding protein and the lower the expression of the corresponding protein was.
Statistical analysis. All the data were expressed as the mean ± standard deviation. The statistical analyses were performed using the SPSS 13.0 statistical software package (SPSS, Inc., Chicago, IL, USA), and one-way analysis of variance was used to perform comparisons among the different groups. P<0.05 was used to indicate a statistically significant difference.

Results
Clinical characteristics of patients with ruptured and unruptured aneurysms. No significant differences were observed in the mean age (48.8 vs. 43.6 years old), gender distribution (men/women, 9:10 vs. 5:7) and location of aneurysm between patients with unruptured and ruptured cerebral aneurysms (Tables I and II). Furthermore, the mean sizes of unruptured and ruptured aneurysms were 24.84 and 12.67 mm, respectively. However, the tumor size in the unruptured aneurysms group was significantly higher compared to that of the ruptured aneurysms group (P<0.05).
Histological assessment was performed in the normal control (n=5), unruptured (n=19) and ruptured (n=12) cerebral aneurysms. The size of the IAs was distinguished using DSA, and the size of small, large and giant IAs had a diameter <12, 12-25 and >25 mm respectively (Fig. 1A). Moreover, as shown in Tables I and II, the location of IAs was separated by DSA. In the normal control group, H&E staining demonstrated that organized smooth muscle cells and continuous endothelial cells were arranged in the cerebral vessel wall (Fig. 1B). In IAs, there were layers of discontinuous endothelial cells and scattered smooth muscle cells in unruptured aneurysms (Fig. 1B). Fresh or organizing thrombosis lined the luminal, and an extremely thin thrombosis-lined hypocellular wall was observed in the ruptured aneurysms (Fig. 1B).
Expression of α-SMA, OPN, MMP9 and PINCH in IAs. α-SMA positive cells were densely distributed in the control group. However, the levels of α-SMA were significantly decreased in unruptured (P<0.05) and ruptured (P<0.05) cerebral aneurysms compared to those of the control group, and no difference in α-SMA was noted between patients with unruptured and ruptured cerebral aneurysms ( Fig. 2A and B). Moreover, the protein expression levels of OPN, MMP9 and PINCH (all P<0.05) in the UA and RA group were significantly higher than those of the control group ( Fig. 2A and B). In addition, the level of MMP9 (P<0.05) was significantly increased in the RA compared to the UA group. However, OPN and PINCH levels were decreased in the RA group compared to those of the UA group (both P<0.05) ( Fig. 2A and B). In general, these results suggested that OPN and PINCH tend to show a higher expression in the unruptured cerebral aneurysms compared to ruptured cerebral aneurysms.
In order to examine whether there was a correlation between PINCH and tumor size as well as between PINCH and OPN, which was measured in tumor tissues from the same individuals. As shown in Fig. 3A, measurements obtained from the same individuals were strongly correlated between PINCH and tumor size (r=0.650 and P=0.0026) as well as between PINCH and OPN (r=0.639 and P=0.0033) in the unruptured cerebral aneurysms. However, the correlation between PINCH and tumor size (r=0.450, P=0.1393) and between PINCH and OPN (r=0.366 and P=0.2426) revealed no obvious difference in the ruptured cerebral aneurysms (Fig. 3B). Consistent with immunohistochemical methods, the western blot results demonstrated that the protein expression of OPN, MMP9 and PINCH in the UA and RA group was markedly higher than those of the control group (all P<0.05), and OPN and PINCH (both P<0.05) were significantly decreased in the RA group as compared to those of the UA group. However, protein expression of MMP9 was not changed in the RA group compared to the UA group (Fig. 3C). These results indicated that PINCH was highly expressed in the unruptured IAs, which may be a critical factor for preventing aneurysmal rupture.
Diagram depicting the possible regulation mechanism of PINCH in the tumorigenesis of IAs. There are several molecules that have been demonstrated to interact with PINCH signaling. The function of the ternary complex of IPP as a signalling platform is achieved by directly interacting with factors that function as upstream regulators of numerous different signalling pathways (21). The present study summarized the known binding partners of PINCH signaling, which are important in processes as diverse as cell adhesion, migration, proliferation, differentiation, survival and apoptosis (22,23). The results demonstrated a decrease in the pro-apoptotic proteins Bax and caspase3 and an increase in the anti-apoptotic protein Bcl-2 in the RA and UA groups compared to those of the control group (Fig. 4A). Moreover, the JNK signaling pathway was inhibited in unruptured and ruptured cerebral aneurysms, and JNK and p-JNK protein expression were significantly lower in the RA and UA groups compared to the control groups (Fig. 4A). In addition, the steady-state levels of proteins involved in the cell cycle checkpoint were analyzed. The results revealed that cyclin B1, cyclin D1 and CDC2 were upregulated in the RA and UA groups compared to those of the NC group (Fig. 4A). In order to further investigate the potential mechanism that may be involved in the PINCH-associated progression of IAs, the protein expression levels of ERK1/2, p-ERK1/2, p38 and p-p38 in tumor tissues were examined. Western blotting revealed that the protein expression levels of ERK1/2, p-ERK1/2, p38 and p-p38 were markedly reduced in the RA and UA groups compared with the NC group (Fig. 4A). Due to the main role of JNK and ERK signaling in carcinogenesis and maintenance of common cancers, the dysregulated expression of JNK and ERK signaling also affects the expression of its potential downstream targets, which are responsible for a wide range of biological processes, including cell proliferation and differentiation, cell cycle and survival and apoptosis. Thus, the results of the present study indicated that PINCH may facilitate IA progression, at least partially, through the activation of ERK signaling and the suppression of JNK signaling (Fig. 4B).

Discussion
Functional studies reveal that the interaction of PINCH1 with integrin-linked kinase (ILK) is a prerequisite in order to locate both proteins to integrin-mediated adhesion sites and to prevent their proteasome-mediated degradation (24,25). Further studies indicate that PINCH1 regulates the cell-matrix, cell polarity and cell-cell adhesion during the implanting of

A B
mouse embryos (12). Moreover, PINCH1 and ILK function together in controlling cell behavior, including cell shape modulation, motility and survival (12,24). Expression profiles and roles of PINCH in tumor tissues have been previously reported (15,(17)(18)(19). However, the exact role of PINCH signaling in IA progression and rupture is not clear yet. Therefore, the aim of the present study was to investigate the function of PINCH signaling in maintaining the development of IAs and preventing aneurysmal rupture.
In the present study several important observations were made. Initially, the basic expression levels of α-SMA, MMP9, PINCH and OPN in 19 unruptured and 12 ruptured cerebral    aneurysms were studied. Compared to the normal control, the α-SMA, MMP9, PINCH and OPN expression levels were significantly elevated in tumor tissues detected by immunohistochemical staining and western blotting; however, the expression levels of PINCH and OPN were different between unruptured and ruptured aneurysms. Next, the correlation of PINCH with tumor size and OPN was confirmed, and the linear correlation plot of protein expression between PINCH and tumor size, as well as between PINCH and OPN showed a strong positive correlation in the unruptured aneurysms. Furthermore, the possible molecular mechanism of PINCH in the tumorigenesis of IAs was analyzed and it was revealed that PINCH may facilitate IA progression, at least partially, through the activation of ERK signaling and suppression JNK signaling. This could regulate the downstream regulators, which induce cell proliferation, survival and apoptosis (21).
OPN, which is an important immunomodulator, is a matricellular protein that is highly expressed in the aortic wall and was demonstrated to be targeted for cleavage by MMP (26). OPN promotes atherosclerosis through its functions in smooth muscle cell survival, adhesion and migration, and promotes inflammation of carotid plaques in hypertensive patients (27). Furthermore, it mitigates vascular calcification (28) and contributes to the increased amounts of MMP-9 in cardiac and skeletal muscle of mdx mice (29). The present study revealed that the protein levels of MMP9 and OPN were significantly increased in unruptured aneurysms, and the immunohistochemical staining was consistent with the with western blot analysis. It is noteworthy that measurements obtained from the same individuals were strongly correlated between PINCH and OPN (r=0.639 and P=0.0033) in the unruptured cerebral aneurysms. However, the correlation between PINCH and OPN (r=0.366 and P=0.2426) showed no obvious difference in the ruptured cerebral aneurysms. These results suggested that PINCH and OPN offered a mechanism of facilitating IA progression, which may be closely associated with resistance of aneurysmal rupture.
Previous studies suggest that PINCH-1 is likely to have a function in the suppression of apoptosis. For example, depletion of PINCH-1 from human HeLa cervical carcinoma cells promotes apoptosis, and an increase of apoptotic endodermal cells, mouse embryos and embryonic neural crest cells are observed with PINCH loss-of-function (10,11,18). A prior study suggested that PINCH-1 regulates the antiand pro-apoptotic pathways and contributes to apoptosis resistance in all types of cancer cells (18). The apoptosis signaling pathway is activated by the loss of PINCH-1, and the level of Bim is significantly increased in response to loss of PINCH-1 in several types of PINCH-1-dependent cancer cells. However, Bim depletion completely blocks the increase of apoptosis induced by the loss of PINCH-1 (18). Moreover, activation of the phosphorylation of the Src family kinase and ERK1/2 is promoted by PINCH-1 (18). In the PrE, PINCH-1 is a pro-survival factor, which prevents apoptosis of PrE cells by modulating two independent signalling pathways; PINCH-1 inhibits JNK-mediated apoptosis by stabilizing the PINCH-1 binding protein RSU-1 and promotes Bcl-2-dependent pro-survival signalling downstream of integrins (13). In the present study, a correlation was identified between the activation of ERK signaling and the suppression of JNK signaling with the expression of PINCH in IAs. The downstream regulators, including anti-apoptotic (Bcl-2), pro-apoptotic proteins (Bax and caspase3) and cell cycle proteins (cyclin B1, cyclin D1 and CDC2) were involved in PINCH-mediated tumorigenesis and progression in IAs.
To the best of our knowledge, this is the first study to report that PINCH is highly expressed in IAs and resists aneurysmal rupture. The study also demonstrated that PINCH may facilitate IA progression, at least partially, through the activation of ERK and suppression of JNK signaling, which could regulate the downstream regulators including anti-and pro-apoptotic protein and cell cycle proteins.