REV7 is involved in outcomes of platinum-based chemotherapy in pancreatic cancer by controlling the DNA damage response.

REV7 is a multifunctional protein implicated in various biological processes, including DNA damage response. REV7 expression in human cancer cells affects their sensitivity to DNA-damaging agents. In the present study, we investigated the significance of REV7 in pancreatic ductal adenocarcinoma (PDAC). REV7 expression was immunohistochemically examined in 92 resected PDAC specimens and 60 endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) specimens of unresectable PDAC treated with platinum-based chemotherapy, and its association with clinicopathologic features was analyzed. Although REV7 expression was not significantly associated with the progression of primary tumors (T-factor and Stage) in either resected or unresectable PDAC, decreased levels of REV7 expression in EUS-FNAB specimens of unresectable PDAC were significantly associated with better outcomes of platinum-based chemotherapy and a favorable prognosis. REV7-deficient PDAC cell lines showed suppressed cell growth and enhanced sensitivity to cisplatin in vitro. Tumor-bearing mice generated using REV7-deficient PDAC cell lines also showed enhanced sensitivity to cisplatin in vivo. RNA sequencing analysis using WT and REV7-deficient PDAC cell lines revealed that REV7 inactivation promoted the downregulation of genes involved in the DNA repair and the upregulation of genes involved in apoptosis. Our results indicate that decreased expression of REV7 is associated with better outcomes of platinum-based chemotherapy in PDAC by suppressing the DNA damage response. It is also suggested that REV7 is a useful biomarker for predicting the outcome of platinum-based chemotherapy and the prognosis of unresectable PDAC and is a potential target for PDAC treatment.

Identification of the promoter region regulating the transcription of the REV7 gene.

REV7 is involved in various biological processes including DNA repair and mutagenesis, cell cycle regulation, gene transcription, and carcinogenesis. REV7 is highly expressed in adult testicular germ cells as well as several malignant tumors. REV7 expression levels are associated with prognosis in several human cancers, however, the mechanism of REV7 transcriptional regulation has not been elucidated. In this study, we characterized the promoter region of the REV7 gene. A luciferase reporter assay using the human germ cell tumor cell line NEC8 was utilized to examine the upstream genomic region of REV7 for transcriptional activity, and two transcriptional activation regions were identified. We determined a small genomic region important for transcriptional activation using site-directed mutagenesis; this region is shared by several putative binding motifs for transcription factors, including the cAMP-responsive element modulator (CREM), cAMP-response element binding protein (CREB), and B-lymphocyte-induced maturation protein-1 (BLIMP-1). Exogenous CREM and CREB expression had no effect on the transcriptional activity in NEC8 cells or the human embryonic kidney cell line HEK293T. In contrast, exogenous BLIMP-1 expression increased luciferase reporter activity in HEK293T cells but unexpectedly decreased activity in NEC8 cells. Chromatin immunoprecipitation analysis demonstrated that BLIMP-1 binds to the genomic region near the binding motif in the REV7 promoter. Additionally, BLIMP-1 overexpression promoted endogenous REV7 expression in HEK293T cells. These findings suggest that BLIMP-1 may be a putative transcriptional regulator of REV7 in mammalian cells.

Upregulation of REV7 correlates with progression of malignant melanoma.

REV7 is a multifunctional protein implicated in DNA damage tolerance, cell cycle control, and gene expression, and is involved in the carcinogenesis of various human tumors. It has been reported that REV7 expression is associated with ultraviolet-induced mutagenesis; however, the role of REV7 expression in skin cancers, including malignant melanomas, remains unclear. In the present study, we investigated the clinical and biological significance of REV7 in malignant melanoma. Levels of REV7 expression in human skin cancers were evaluated immunohistochemically. Positive expression of REV7 was frequently observed in malignant melanomas, as well as in squamous cell carcinomas and basal cell carcinomas. Enhanced immunoreactivity to REV7 was closely linked with cell proliferation assessed by Ki-67 labeling indexes in the three skin cancers, and was related with tumor thickness in malignant melanomas. REV7 depletion in malignant melanoma cells MEWO and G361 suppressed cell proliferation, migration, and invasion abilities. REV7 depletion also affected the expression of intracellular signaling molecules AKT and ERK in MEWO cells, resulting in downregulation of ERK signal activation. In addition, REV7 depletion facilitated sensitivity to cisplatin, but not to dacarbazine, in MEWO cells. Our results suggest that REV7 expression correlates with disease progression of malignant melanoma.

Increased expression of REV7 in small cell lung carcinomas and its association with tumor cell survival and proliferation.

REV7 is involved in multiple biological processes including DNA damage tolerance, cell cycle regulation and gene expression, and is an accessory subunit of the mutation-prone DNA polymerase ζ. It has been reported that REV7 expression is associated with poor prognosis in several human cancers. The aim of this study is to investigate the significance of REV7 in lung carcinogenesis. Immunohistochemical analyses of surgically resected lung cancer specimens revealed that REV7 shows an increased expression in small cell lung carcinomas (SCLCs) when compared with other histological types of lung carcinoma. Association between REV7 expression levels and clinicopathological factors was investigated using SCLC cases with or without surgical resection. Our analyses revealed that high REV7 expression significantly correlated with tumor cell proliferation, assessed by Ki-67 labeling indices, and was negatively associated with distant metastasis and extensive-stage disease. No significant association was detected between REV7 expression and other factors, including prognosis or response to chemoradiotherapy in SCLC. Increase in REV7 expression in SCLC was confirmed using SCLC cell lines. In addition, siRNA-mediated depletion of REV7 activated the apoptotic pathway and suppressed cell growth in SCLC cells. These results suggest that REV7 plays an important role in tumor cell survival and proliferation in SCLC.

CD109 regulates in vivo tumor invasion in lung adenocarcinoma through TGF-ß signaling.

Stromal invasion is considered an important prognostic factor in patients with lung adenocarcinoma. The mechanisms underlying the formation of tumor stroma and stromal invasion have been studied in the lung; however, they are still unclear. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein highly expressed in several types of human malignant tumors including lung cancers. In this study, we investigated the in vivo functions of CD109 protein in malignant lung tumors. Initially, we identified an association between higher expression of CD109 protein in human lung adenocarcinoma and a significantly worse prognosis, according to immunohistochemical analysis. We also showed that CD109 deficiency significantly reduced the area of stromal invasive lesions in a genetically engineered CD109-deficient lung adenocarcinoma mouse model, which correlated with the results observed in human lung adenocarcinoma. Furthermore, we identified latent TGF-ß binding protein-1 (LTBP1) as a CD109-interacting protein using mass spectrometry and confirmed their interaction by co-immunoprecipitation. Importantly, increased CD109 expression enhanced stromal TGF-ß activation in the presence of LTBP1. Therefore, these data suggest the significance of the regulation of TGF-ß signaling through CD109 and LTBP1 interaction in tumor stroma and also reveal the importance of CD109 expression levels in promoting lung cancer cell proliferation, migration, and invasion, and thus predicting the outcome of patients suffering from lung adenocarcinoma. Therefore, CD109 protein could be a potential therapeutic target for this disease.

Expression of CA2 and CA9 carbonic anhydrases in ulcerative colitis and ulcerative colitis-associated colorectal cancer.

Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic mucosa and submucosa with repeating relapse and remission, but the pathogenesis is unknown. Patients with long-standing UC are at high risk of neoplasm development. The aim of the present study was to identify molecules whose expression is associated with UC and UC-associated colorectal cancer (UCCA). Biopsy specimens from UC and normal colonic mucosae were analyzed using a proteomics approach, in which carbonic anhydrase 2 (CA2) was identified as a molecule downregulated in UC mucosae. Immunohistochemically, CA2 expression was detected in normal and diverticulitis mucosal epithelia, and its expression decreased as UC activity increased. CA2 expression was almost undetectable in UCCA. We also analyzed the expression of another carbonic anhydrase, CA9, and its upstream molecule, hypoxia-inducible factor-1α (HIF-1α), both of which are induced under hypoxic conditions. It was revealed that CA9 expression was relatively low in normal, diverticulitis and UC mucosae, and was upregulated in UCCA. HIF-1α expression was consistently low in all tissue types examined. In conclusion, these results suggest that CA2 and CA9 may be possible indicators of UC activity and UCCA development, respectively.

CD109 deficiency induces osteopenia with an osteoporosis-like phenotype in vivo.

Osteoporosis is a global public health problem that is increasing along with an aging population. A major determinant of osteoporosis is high bone turnover, which results from osteoclast activation. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, a deficiency that leads to a psoriasis-like skin inflammation in mice. Although the expression of CD109 has been reported in mouse pre-osteoclast cells, its function in osteoclasts in vivo remains largely unknown. To investigate the physiological role of CD109 in bone metabolism, we analyzed bones from wild-type and CD109-deficient adult mice. Micro-computed tomography analysis of the femur (thigh bone) showed that bone volume was lower in CD109-deficient mice than in wild-type mice. Bone histomorphometric analysis showed not only a reduction in bone volume but also an increase in bone turnover in CD109-deficient mice as compared with wild-type mice. Additionally, we measured serum levels of several markers of bone turnover and found a significant increase in the N-terminal telopeptide of type I collagen, a bone resorption marker, as well as alkaline phosphatase, a bone formation marker, in CD109-deficient mice. These results indicate that CD109 deficiency induces a high-turnover, osteoporosis-like phenotype, which suggests that CD109 plays a role in bone metabolism in vivo.

Cytoskeleton-Associated Protein 4 Is a Novel Serodiagnostic Marker for Lung Cancer.

Our aim was to develop a serodiagnostic marker for lung cancer. Monoclonal antibodies were generated, and one antibody designated as KU-Lu-1, recognizing cytoskeleton-associated protein 4 (CKAP4), was studied further. To evaluate the utility of KU-Lu-1 antibody as a serodiagnostic marker for lung cancer, reverse-phase protein array analysis was performed with sera of 271 lung cancer patients and 100 healthy controls. CKAP4 was detected in lung cancer cells and tissues, and its secretion into the culture supernatant was also confirmed. The serum CKAP4 levels of lung cancer patients were significantly higher than those of healthy controls (P < 0.0001), and the area under the curve of receiver-operating characteristic curve analysis was 0.890, with 81.1% sensitivity and 86.0% specificity. Furthermore, the serum CKAP4 levels were also higher in patients with stage I adenocarcinoma or squamous cell carcinoma than in healthy controls (P < 0.0001). Serum CKAP4 levels may differentiate lung cancer patients from healthy controls, and they may be detected early even in stage I non-small cell lung cancer. Serum CKAP4 levels were also significantly higher in lung cancer patients than in healthy controls in the validation set (P < 0.0001). The present results provide evidence that CKAP4 may be a novel early serodiagnostic marker for lung cancer.

CD109: a multifunctional GPI-anchored protein with key roles in tumor progression and physiological homeostasis.

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein and a member of the α2 -macroglobulin/C3,C4,C5 family of thioester-containing proteins first identified as being expressed on blood cells, including activated T cells and platelets, and a subset of CD34 + bone marrow cells containing megakaryocyte progenitors. Although CD109 carries the biallelic platelet-specific alloantigen Gov, the physiological functions or roles of CD109 in human disease remain largely unknown. It was recently demonstrated that CD109 is expressed in many malignant tumors, including various squamous cell carcinomas and adenocarcinomas, and plays a role as a multifunctional coreceptor. CD109 reportedly associates with transforming growth factor (TGF)-ß receptors and negatively regulates TGF-ß signaling in keratinocytes. Additionally, CD109 is potentially related to signal transducer and activator of transcription-3 signaling and aberrant cell proliferation. In this review, we describe recent evidence of CD109-specific significance in malignant tumors shown in mouse models and human tissues. Furthermore, we discuss the physiological functions of CD109 in vitro and in vivo, including results of phenotype analyses of CD109-deficient mice exhibiting epidermal hyperplasia and osteopenia.

Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin.

In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

CD109 and squamous cell carcinoma.

Squamous cell carcinoma (SCC) is well-known for its high rate of metastasis with poor prognosis. CD109 is a glycosylphosphatidylinositol-anchored cell-surface glycoprotein. Recently, CD109 emerges as a potential biomarker and a therapeutic target for SCCs. Accumulating studies have reported that CD109 is highly expressed in human SCCs of multiple organs, and may contribute to the progression of SCCs. In this review, we summarized the findings on expression pattern of CD109 in SCCs, and discussed the molecular mechanisms underlying the roles of CD109 in pathogenesis of SCCs.

Significance of perivascular tumour cells defined by CD109 expression in progression of glioma.

In the progression of glioma, tumour cells often exploit the perivascular microenvironment to promote their survival and resistance to conventional therapies. Some of these cells are considered to be brain tumour stem cells (BTSCs); however, the molecular nature of perivascular tumour cells has not been specifically clarified because of the complexity of glioma. Here, we identified CD109, a glycosylphosphatidylinositol-anchored protein and regulator of multiple signalling pathways, as a critical regulator of the progression of lower-grade glioma (World Health Organization grade II/III) by clinicopathological and whole-genome sequencing analysis of tissues from human glioma. The importance of CD109-positive perivascular tumour cells was confirmed not only in human lower-grade glioma tissues but also in a mouse model that recapitulated human glioma. Intriguingly, BTSCs isolated from mouse glioma expressed high levels of CD109. CD109-positive BTSCs exerted a proliferative effect on differentiated glioma cells treated with temozolomide. These data reveal the significance of tumour cells that populate perivascular regions during glioma progression, and indicate that CD109 is a potential therapeutic target for the disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The molecular chaperone Hsp90 regulates accumulation of DNA polymerase eta at replication stalling sites in UV-irradiated cells.

DNA polymerase eta (Pol eta) is a member of the mammalian Y family polymerases and performs error-free translesion synthesis across UV-damaged DNA. For this function, Pol eta accumulates in nuclear foci at replication stalling sites via its interaction with monoubiquitinated PCNA. However, little is known about the posttranslational control mechanisms of Pol eta, which regulate its accumulation in replication foci. Here, we report that the molecular chaperone Hsp90 promotes UV irradiation-induced nuclear focus formation of Pol eta through control of its stability and binding to monoubiquitinated PCNA. Our data indicate that Hsp90 facilitates the folding of Pol eta into an active form in which PCNA- and ubiquitin-binding regions are functional. Furthermore, Hsp90 inhibition potentiates UV-induced cytotoxicity and mutagenesis in a Pol eta-dependent manner. Our studies identify Hsp90 as an essential regulator of Pol eta-mediated translesion synthesis.

Basigin expression as a prognostic indicator in stage I pulmonary adenocarcinoma.

We established the KU-Lu-8 monoclonal antibody (MoAb) using a lung cancer cell line as an antigen and a random immunization method. The KU-Lu-8 MoAb recognizes basigin (BSG), which is a transmembrane-type glycoprotein that is strongly expressed on the cell membranes of lung cancer cells. This study aimed to clarify the relationships between BSG expression and clinicopathological parameters and determine the prognostic significance of BSG expression in pulmonary adenocarcinoma (AC) patients. To evaluate the significance of BSG expression in lung cancer, we immunohistochemically analyzed 113 surgically resected pulmonary adenocarcinomas, and the associations between BSG expression and various clinicopathological parameters were evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to investigate the effects of BSG expression on survival. Clinicopathologically, BSG expression was significantly associated with tumor differentiation, vascular invasion, lymphatic invasion, and a poor prognosis. In particular, BSG expression was significantly correlated with poorer survival in patients with stage I AC. The high BSG expression group (compared with the low BSG expression group) exhibited adjusted hazard ratios for mortality of 4.694. BSG expression is indicative of a poor prognosis in AC patients, particularly in those with stage I disease.

Impact of PD-L1 Expression in Patients with Surgically Resected Non-Small-Cell Lung Cancer.

BACKGROUND: Immunotherapy can become a crucial therapeutic option to improve the prognosis of patients with non-small-cell lung cancer (NSCLC). Here, we evaluated the impact of programmed cell death ligand-1 (PD-L1) expression in surgically resected NSCLCs. METHODS: We estimated PD-L1 expression in 229 consecutive NSCLC specimens using rabbit polyclonal antibodies to human PD-L1 in a SP263 immunohistochemical assay and evaluated PD-L1 expression for potential associations with clinicopathological parameters and survival time. RESULTS: PD-L1 expression was significantly higher in tumors from men or current smokers. Squamous cell carcinoma histology was independently associated with high PD-L1 expression according to multivariate analysis (p = 0.015). The 5-year survival rate of patients was 70%, and the difference in the 5-year survival rate according to PD-L1 expression was not statistically significant (high expression group [67%] vs. low expression group [68%]); however, the squamous cell carcinoma group exhibited significantly lower 5-year survival rates as compared to the non-squamous cell carcinoma group (53 and 71%, respectively; p = 0.026). CONCLUSION: Here, we revealed high PD-L1 expression and poor prognosis observed in patients with surgically resected squamous NSCLC as compared with non-squamous NSCLC. Our results support the identification of patient subsets that most likely respond to anti-PD-1 therapy as the first step in precision medicine.

CD109 is a component of exosome secreted from cultured cells.

Exosomes are 50-100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-ß signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome.

CD109 attenuates TGF-ß1 signaling and enhances EGF signaling in SK-MG-1 human glioblastoma cells.

CD109 is a glycosylphosphatidylinositol-anchored cell surface protein that is frequently detected in squamous cell carcinomas. CD109 is a negative regulator of TGF-ß1 signaling in human keratinocytes, and the N-terminal fragment of CD109 secreted from cells after cleavage by the furin protease is important for modulating TGF-ß1 signaling. Previously, we found that CD109 is expressed in human glioblastoma cells; however, the role of CD109 in glioblastoma cells is not established. Here, we describe the effects of CD109 in human glioblastoma cell lines. Three glioblastoma cell lines, SK-MG-1, U251MG and MG178, were tested and CD109 overexpression attenuated TGF-ß1 signaling and enhanced EGF signaling in SK-MG-1, but not in U251MG or MG178. The N-terminal CD109 fragment in SK-MG-1 was hyperglycosylated compared with that in MG178 or U251MG. The conditioned medium of CD109-overexpressing SK-MG-1, containing the secreted N-terminal CD109, had a negative effect on TGF-ß1 signaling in wild-type SK-MG-1 and MG178, whereas it did not show any effect on EGF signaling. In addition, cell surface CD109 interacts with EGF receptor in SK-MG-1 overexpressing CD109, and exhibited enhanced cell migration and invasion. These findings suggest that CD109 attenuates TGF-ß1 signaling and enhances EGF signaling in SK-MG-1 cells and that the membrane-anchored CD109 may play major roles in the EGF signaling pathway.

The REV7 subunit of DNA polymerase ζ is essential for primordial germ cell maintenance in the mouse.

REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene expression, and carcinogenesis. In vitro studies show that REV7 interacts with several proteins and regulates their function. It has been reported that human REV7 is highly expressed in the adult testis by Northern blot analysis. However, the significance of REV7 in mammalian development has not been elucidated. Here, we present analyses of REV7-deficient (Rev7(-/-)) mice to clarify the significance of Rev7 in mouse development. In WT mice (Rev7(+/+)), Rev7 expression was ubiquitously observed in the embryo and confined to germ cells in the testes after birth. Rev7(-/-) mice exhibited growth retardation and a partial embryonic lethal phenotype. Mice that survived to adulthood were infertile in both sexes and showed germ cell aplasia in the testes and ovaries. Analyses of Rev7(-/-) embryos revealed that primordial germ cells (PGCs) were present at embryonic day 8.5 (E8.5). However, progressive loss of PGCs was observed during migration, and PGCs were absent in the genital ridges at E13.5. An increase of apoptotic cells was detected not only among PGCs but also in the forebrain of the Rev7(-/-) embryo, whereas cell proliferation was unaffected. Moreover, DNA damage accumulation and increased levels of histone methylation were detected in Rev7(-/-) embryos, and expression of Oct4 and Nanog was deregulated by REV7 deficiency at E8.5. These findings indicate that Rev7 is essential for PGC maintenance by prevention of apoptotic cell death in the mouse.

Suppression of REV7 enhances cisplatin sensitivity in ovarian clear cell carcinoma cells.

Human REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene transcription, and carcinogenesis. In this study, we evaluated the expression of REV7 in epithelial ovarian cancer (EOC) and analyzed the association between its expression and chemosensitivity in ovarian clear cell carcinoma (CCC) cells. Expression of REV7 in human EOC tissues was assessed by immunohistochemical staining. Expression was detected in the majority of EOCs (92.0%) with especially high levels of expression frequently observed in CCCs (73.5%) compared with that of non-CCCs (53.4%). Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stage II-IV) EOC as assessed using Kaplan-Meier curves and log-rank tests. The effects of REV7 knockdown on cell proliferation and chemosensitivity in CCC cells were also analyzed in vitro and in vivo. Knockdown of REV7 in CCC cells decreased cell proliferation without affecting cell cycle distribution. Additionally, the number of apoptotic cells and DNA damaged cells were increased after cisplatin treatment. In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group. These findings indicate that depletion of REV7 enhances sensitivity to cisplatin treatment in CCC, suggesting that REV7 is a candidate molecular target in CCC management.

Role of Girdin in intimal hyperplasia in vein grafts and efficacy of atelocollagen-mediated application of small interfering RNA for vein graft failure.

OBJECTIVE: Intimal hyperplasia is a major obstacle to patency in grafted veins. Although migration and proliferation of vascular smooth muscle cells (SMCs) pivotally affect the vascular remodeling process, no therapy has been established to prevent intimal hyperplasia of vein grafts. We previously reported that the actin-binding protein Girdin crucially affects arterial remodeling. In this study, we investigated the role of Girdin in venous SMCs and evaluated a therapeutic strategy for vein graft failure in vivo using small interfering RNA (siRNA) that targets Girdin. METHODS: We investigated the relationship between Girdin expression and intimal hyperplasia using a rabbit vein graft model. Vein grafts under low-flow conditions were performed in Japanese White rabbits. For in vitro analyses, we isolated primary venous SMCs from vein graft neointima. siRNA that targets Girdin was mixed with atelocollagen, which stabilizes and releases nucleic acid reagents slowly and is applied perivascularly to the vein grafts at operation. Intimal hyperplasia was evaluated 4 weeks later. RESULTS: In the rabbit model, increased Girdin expression was seen in the neointima after the grafting operation. Using primary venous SMCs, we showed that Girdin is required for rearrangement of the actin cytoskeleton in venous SMCs and that siRNA-mediated Girdin knockdown significantly reduced venous SMC migration and proliferation. Girdin knockdown via perivascular application of siRNA using atelocollagen markedly reduced intimal thickening after the grafting operation. CONCLUSIONS: Depletion of Girdin attenuated venous SMCs migration and proliferation in vitro and intimal hyperplasia in vein grafts in vivo. Our findings suggest that Girdin affects migration and proliferation of vascular SMCs in vein grafts and that controlled release of Girdin siRNA using atelocollagen could be a novel therapeutic strategy for vein graft failure.