POPDC1 also known as BVES, is a highly conserved transmembrane protein, important for striated muscle function and homeostasis. Pathogenic variants in the POPDC1 gene are associated with limb-girdle muscular dystrophy type 25 (LGMDR25). In the present study, we performed trio-whole exome sequencing (WES) followed by Sanger sequencing on a single family having LGMD clinical features. Protein modeling of all POPDC1 missense variants (POPDC1Pro134Leu , POPDC1Ile193Ser , and POPDC1Ser201Phe ) associated with LGMDR25 were performed using Molecular Dynamics (MD) simulation. We identified a homozygous missense variant (c.401C>T; p.Pro134Leu) in the POPDC1 gene. Altered 3D structure, disruptive fluctuation, less compactness, and instability were observed in all the three variants of POPDC1 protein models. In comparison, POPDC1Ser201Phe protein dynamics were more unstable than other variants. Functional study of newly identified variant would add key answers to underlying mechanisms of the disease.

Tumor cells detachment from primary lesions is an early event for hepatocellular carcinoma (HCC) metastasis, in which cell adhesion molecules play an important role. The role of mechanical crowding has attracted increasing attention. Previous studies have found that overcrowding can induce live cells extrusion to maintain epithelial cell homeostasis, and normally, live extruded cells eventually die through a process termed anoikis, suggesting the potential of tumor cells resistant to anoikis might initiate metastasis from primary tumors by cell extrusion. We have demonstrated transmembrane adhesion molecule blood vessel epicardial substance (BVES) suppression as an early event in HCC metastasis. However, whether its suppression is involved in HCC cell extrusion, especially in HCC metastasis, remains unknown. This study aims to investigate the role of BVES in tumor cells extrusion in HCC metastasis, as well as the underlying mechanisms.
Cells extrusion was observed by silicone chamber, petri dish inversion, and three-dimensional cell culture model. Polymerase chain reaction, western blotting, immunohistochemistry, immunofluorescence, co-immunoprecipitation, and RhoA activity assays were used to explore the underlying mechanisms of cell extrusion regulated by BVES. An orthotopic xenograft model was established to investigate the effects of BVES and cell extrusion in HCC metastasis in vivo.
Tumor cell extrusion was observed in HCC cells and tissues. BVES expression was decreased both in HCC and extruded tumor cells. BVES overexpression led to the decrease in HCC cells extrusion in vitro and in vivo. Moreover, our data showed that BVES co-localized with ZO-1 and GEFT, regulating ZO-1 expression and localization, and GEFT distribution, thus modulating RhoA activity.
The present study revealed that BVES downregulation in HCC enhanced tumor cells extrusion, thus promoting HCC metastasis, which contributed to a more comprehensive understanding of tumor metastasis, and provided clues for developing novel HCC therapy strategies. Video abstract.

OBJECTIVE: Vascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear.
METHODS: We used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development.
RESULTS: Here, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation.
CONCLUSIONS: Bves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

Limb girdle muscular dystrophy type R25 (LGMDR25) is a rare genetic disorder due to loss-of-function mutations in BVES, characterized by progressive proximal lower limb weakness and atrioventricular block. Here we report a young Chinese man with LGMDR25 who presented with asymmetrical lower limb weakness, myalgia, palpitations and dyspnea on exertion. Muscle imaging demonstrated fatty infiltration of the long head of biceps femoris, adductor magnus, gastrocnemius and soleus, and myoedema of semitendinosus and quadriceps, sparing rectus femoris. ECG showed only mild sinus tachycardia but pulmonary function test suggested prominent respiratory muscle weakness. Our report expands the phenotypical spectrum and indicates the importance of monitoring respiratory function in LGMDR25 patients.

BACKGROUND: Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions.
RESULTS: In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation.
CONCLUSIONS: Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

The underexpression of the long noncoding RNA blood vessel epicardial substance antisense RNA 1 (BVES-AS1) has been shown in colon adenocarcinoma (COAD) patients. However, its role in COAD remains to be explored. This study aimed to investigate the function and potential mechanism of BVES-AS1 in COAD. Colony formation, Cell Counting Kit-8, JC-1 mitochondrial membrane potential assay, wound healing, transwell, and western blot analyses were used to measure cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) in COAD cells. RNA pull-down, luciferase reporter, and RNA binding protein immunoprecipitation assays were used to detect the interaction of BVES-AS1 and downstream genes. BVES-AS1 was expressed at low levels in COAD cells. Overexpressed BVES-AS1 inhibited COAD cell proliferation, migration, invasion, and EMT while elevating cell apoptosis. Mechanistically, BVES-AS1 functioned as a competing endogenous RNA sponging miR-522-3p to regulate the expression of nearby gene blood vessel epicardial substance (BVES). Besides this, BVES-AS1 recruited TATA-box binding protein associated factor 15 (TAF15) to promote BVES messenger RNA stability. Taken together, our study confirmed that BVES-AS1 inhibited COAD progression via interacting with miR-522-3p and TAF15 to regulate BVES expression, which might offer a perspective for COAD treatment.

Background: Tetralogy of Fallot (TOF) accounts for ∼10% of congenital heart disease cases. The blood vessel epicardial substance (BVES) gene has been reported to play a role in the function of adult hearts. However, whether allelic variants in BVES contribute to the risk of TOF and its possible mechanism remains unknown. Methods: The open reading frame of the BVES gene was sequenced using samples from 146 TOF patients and 100 unrelated healthy controls. qRT-PCR and western blot assays were used to confirm the expression of mutated BVES variants in the TOF samples. The online software Polyphen2 and SIFT were used to predict the deleterious effects of the observed allelic variants. The effects of these allelic variants on the transcriptional activities of genes were examined using dual-fluorescence reporter assays. Results: We genotyped four single nucleotide polymorphisms (SNPs) in the BVES gene from each of the 146 TOF patients. Among them, the minor allelic frequencies of c.385C>T (p.R129W) were 0.035% in TOF, but ∼0.025% in 100 controls and the Chinese Millionome Database. This allelic variant was predicted to be a potentially harmful alteration by the Polyphen2 and SIFT softwares. qRT-PCR and western blot analyses indicated that the expression of BVES in the six right ventricular outflow tract samples with the c.385C>T allelic variant was significantly downregulated. A dual-fluorescence reporter system showed that the c.385C>T allelic variant significantly decreased the transcriptional activity of the BVES gene and also decreased transcription from the GATA4 and NKX2.5 promoters. Conclusions: c.385C>T (p.R129W) is a functional SNP of the BVES gene that reduces the transcriptional activity of BVES in vitro and in vivo in TOF tissues. This subsequently affects the transcriptional activities of GATA4 and NKX2.5 related to TOF. These findings suggest that c.385C>T may be associated with the risk of TOF in the Han Chinese population.

The axon guidance cues netrin-1 has been reported to be associated with cancer progression in various types of human cancers. However, the underlying molecular mechanism of netrin-1-mediated metastasis remains obscure. In this study, we found that overexpression of netrin-1 promoted HCC cell migration and invasion as determined by transwell assay and 3D cell culture assay. However, netrin-1 knockdown inhibited these processes. Further investigation indicated that netrin-1 decreased the expression of Blood Vessel Epicardial Substance (BVES), which was down-regulated in HCC. Interestingly, LY294002, a special inhibitor to PI3K/AKT signaling which was determined as a downstream pathway of netrin-1, restored the reduction in BVES caused by netrin-1. In addition, BVES exhibited an opposite effect on HCC cell metastasis to that of netrin-1. Importantly, up-regulating BVES expression significantly attenuated netrin-1-enhanced migration and invasion, whereas silencing BVES expression rescued the metastatic phenotype in netrin-1 knockdown cells. Moreover, netrin-1 expression was negatively correlated with BVES in HCC tissues and cell lines with different metastatic potential. Taken together, these results reveal that netrin-1 promotes HCC cell metastasis by regulating BVES expression via AKT activation.