Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone.

Adult neural stem cells (NSCs) reside in specialized niches, which hold a balanced number of NSCs, their progeny, and other cells. How niche capacity is regulated to contain a specific number of NSCs remains unclear. Here, we show that ependyma-derived matricellular protein CCN1 (cellular communication network factor 1) negatively regulates niche capacity and NSC number in the adult ventricular-subventricular zone (V-SVZ). Adult ependyma-specific deletion of Ccn1 transiently enhanced NSC proliferation and reduced neuronal differentiation in mice, increasing the numbers of NSCs and NSC units. Although proliferation of NSCs and neurogenesis seen in Ccn1 knockout mice eventually returned to normal, the expanded NSC pool was maintained in the V-SVZ until old age. Inhibition of EGFR signaling prevented expansion of the NSC population observed in CCN1 deficient mice. Thus, ependyma-derived CCN1 restricts NSC expansion in the adult brain to maintain the proper niche capacity of the V-SVZ.

Cellular communication network factor 1 promotes retinal leakage in diabetic retinopathy via inducing neutrophil stasis and neutrophil extracellular traps extrusion.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of blindness and is characterized by dysfunction of the retinal microvasculature. Neutrophil stasis, resulting in retinal inflammation and the occlusion of retinal microvessels, is a key mechanism driving DR. These plugging neutrophils subsequently release neutrophil extracellular traps (NETs), which further disrupts the retinal vasculature. Nevertheless, the primary catalyst for NETs extrusion in the retinal microenvironment under diabetic conditions remains unidentified. In recent studies, cellular communication network factor 1 (CCN1) has emerged as a central molecule modulating inflammation in pathological settings. Additionally, our previous research has shed light on the pathogenic role of CCN1 in maintaining endothelial integrity. However, the precise role of CCN1 in microvascular occlusion and its potential interaction with neutrophils in diabetic retinopathy have not yet been investigated. METHODS: We first examined the circulating level of CCN1 and NETs in our study cohort and analyzed related clinical parameters. To further evaluate the effects of CCN1 in vivo, we used recombinant CCN1 protein and CCN1 overexpression for gain-of-function, and CCN1 knockdown for loss-of-function by intravitreal injection in diabetic mice. The underlying mechanisms were further validated on human and mouse primary neutrophils and dHL60 cells. RESULTS: We detected increases in CCN1 and neutrophil elastase in the plasma of DR patients and the retinas of diabetic mice. CCN1 gain-of-function in the retina resulted in neutrophil stasis, NETs extrusion, capillary degeneration, and retinal leakage. Pre-treatment with DNase I to reduce NETs effectively eliminated CCN1-induced retinal leakage. Notably, both CCN1 knockdown and DNase I treatment rescued the retinal leakage in the context of diabetes. In vitro, CCN1 promoted adherence, migration, and NETs extrusion of neutrophils. CONCLUSION: In this study, we uncover that CCN1 contributed to retinal inflammation, vessel occlusion and leakage by recruiting neutrophils and triggering NETs extrusion under diabetic conditions. Notably, manipulating CCN1 was able to hold therapeutic promise for the treatment of diabetic retinopathy.

Global Proteomics Analysis of Lysophosphatidic Acid Signaling in PC-3 Human Prostate Cancer Cells: Role of CCN1.

Cysteine-rich angiogenic factor 61 (CCN1/Cyr61) is a matricellular protein that is induced and secreted in response to growth factors. Our previous work showed that 18:1-lysophosphatidic acid (LPA), which activates the G protein-coupled receptor LPAR1, induces CCN1 between 2-4 h in PC-3 human prostate cancer cells in a manner than enhances cell-substrate adhesion. While the time course of induction suggests that CCN1 contributes to intermediate events in LPA action, the roles of CCN1 in LPA-mediated signal transduction have not been fully elucidated. This study utilized a comprehensive global proteomics approach to identify proteins up- or down-regulated in response to treatment of PC-3 cells with LPA for three hours, during the time of peak CCN1 levels. In addition, the effects of siRNA-mediated CCN1 knockdown on LPA responses were analyzed. The results show that, in addition to CCN1, LPA increased the levels of multiple proteins. Proteins up-regulated by LPA included metastasis-associated in colon cancer protein 1 (MACC1) and thrombospondin-1 (TSP1/THBS1); both MACC1 and TSP1 regulated cancer cell adhesion and motility. LPA down-regulated thioredoxin interacting protein (TXNIP). CCN1 knockdown suppressed the LPA-induced up-regulation of 30 proteins; these included MACC1 and TSP1, as confirmed by immunoblotting. Gene ontology and STRING analyses revealed multiple pathways impacted by LPA and CCN1. These results indicate that CCN1 contributes to LPA signaling cascades that occur during the intermediate phase after the initial stimulus. The study provides a rationale for the development of interventions to disrupt the LPA-CCN1 axis.

Increased CYR61 expression activates CCND1/c-Myc pathway to promote nasal epithelial cells proliferation in chronic rhinosinusitis with nasal polyps.

PURPOSE: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic sinonasal inflammatory disease characterized histologically by hyperplastic nasal epithelium and epithelial cells proliferation. Cysteine-rich angiogenic inducer 61 (CYR61) acts as a positive regulator of cell cycle process. Cyclin D1 (CCND1) and c-Myc play key roles in the processes of cell cycle and cell growth. The purpose of our research was to explore the expression and roles of CYR61, CCND1 and c-Myc in CRSwNP. METHODS: FeaturePlot and vlnPlot functions embedded in the seurat package (version 4.1.1) of R software (version 4.2.0) were applied to explore the cellular distribution of CYR61, CCND1 and c-Myc in the single-cell RNA sequencing (scRNA-seq) dataset of nasal tissue samples. CYR61, CCND1 and c-Myc immunolabeling and mRNA levels in nasal tissue samples were assessed by immunohistochemistry and real-time PCR. Co-localization of CYR61, CCND1 and c-Myc with basal epithelial cell marker P63 was assayed using double-label immunofluorescence staining. Furthermore, we collected and cultured human nasal epithelial cells (HNEC) to assess the regulation and role of CYR61 in vitro study. RESULTS: CYR61, CCND1 and c-Myc were primarily expressed by nasal epithelial cells. Significant upregulation of CYR61, CCND1 and c-Myc positive cells and increased levels of CYR61, CCND1 and c-Myc mRNA were found in nasal polyps in comparison to control samples. Of note, CYR61 mRNA and protein levels were altered by SEB, LPS, IFN-γ, IL-13, IL-17A and TGF-ß1 in HNEC. In addition, CYR61 intervention could increase CCND1 and c-Myc mRNA and protein levels to promote HNEC proliferation, and siRNA against ITGA2 (si-ITGA2) could reverse CYR61 induced upregulation of CCND1 and c-Myc mRNA and protein levels in HNEC and cell proliferation of HNEC. CONCLUSIONS: CYR61, CCND1 and c-Myc were primarily expressed by epithelial cells in nasal mucosa. CYR61, CCND1 and c-Myc expression levels were increased in CRSwNP compared with controls. CYR61 could interact with ITGA2 to enhance HNEC proliferation via upregulating CCND1 and c-Myc levels in the HNEC, leading to hyperplastic nasal epithelium in CRSwNP.

CircUSP48 promotes malignant behavior by regulating CYR61 via miR-365 in osteosarcoma.

Even though circular RNAs (circRNAs), a class of non-coding endogenous RNA, play a crucial role in the progression of osteosarcoma (OS), the specific function of hsa_circ_0000028 (circUSP48) remains unclear. This study aims to elucidate the mechanism by which circUSP48 regulates OS. We employed qRT-PCR and western blot techniques to quantify circDOCK1, miR-186, and DNMT3A levels. Cell proliferation was assessed using the cell counting kit-8 (CCK-8), 5-Ethynyl-20-deoxyuridine (EdU) assay, and colony formation assay. Cell migration and invasion were evaluated through Transwell and cell scratch assays. Furthermore, we performed dual-luciferase reporter, RIP, and RNA pull-down assays to investigate the association between circUSP48, miR-365, and CYR61. In addition, an in vivo xenograft model was utilized to assess the functional role of circUSP48. High levels of circUSP48 and CYR61 were observed in OS tissues and cells, while miR-365 levels were low. Knockdown of circUSP48 suppressed the multiplication, motility, and invasion of OS cells, thereby reducing carcinoma growth. Moreover, inhibition of miR-365 reversed the OS cell-suppressive effect caused by circUSP48 knockdown through direct interaction with circUSP48. Additionally, circUSP48 upregulated the expression of CYR61 by sponging miR-365. The findings suggest that circUSP48 promotes malignant behavior in OS by regulating the expression of CYR61 through miR-365, making it a potential therapeutic target for OS.

CCN1 secreted by human adipose-derived stem cells enhances wound healing and promotes angiogenesis through activating the AKT signalling pathway.

The study aimed to explore the role of cellular communication network factor 1 (CCN1) an extracellular matrix protein in hADSC-treated wound healing. Immunofluorescence and enzyme-linked immunosorbent assays (ELISA) were used to demonstrate the secretion of CCN1 by hADSCs, isolated from human fat tissue. We investigated the role of CCN1 in wound healing by knockdown of CCN1 expression in hADSCs using CCN1 siRNA. Conditioned medium of hADSCs or hADSCs with CCN1 knocked down (hADSC-CMCCN1↓ ) was collected. After treatment with plain DMEM/F12, hADSC-CM, hADSC-CMCCN1↓ , or recombinant human CCN1 (rhCCN1), the wound healing abilities of human umbilical vascular endothelial cells (HUVECs) were assayed, and the AKT, also known as protein kinase B (PKB), signalling pathway was detected using western blotting. Next, we created full-thickness skin wounds on the backs of the mice and different treatments were applied to the wound surface. Wound size was measured using a digital camera on days 0-10, and evaluated. H&E and immunohistochemical staining were performed, and laser Doppler perfusion imaging was used to evaluate blood perfusion. The wound model and wound-healing assay showed that the hADSCs-CM and rhCCN1 groups had enhanced wound healing compared to the hADSCs-CMCCN1↓ group. Further, CCN1 and hADSCs-CM promoted the proliferation and migration of HUVECs through the AKT signalling pathway. We concluded that CCN1 secreted by hADSCs enhances wound healing and promotes angiogenesis by activating the AKT signalling pathway. CCN1 plays a vital role in the regulation of hADSCs-CM during wound healing.

Endothelial cyclin I reduces vulnerability to angiotensin II-induced vascular remodeling and abdominal aortic aneurysm risk.

BACKGROUND: Retinoblastoma protein (Rb) supports vasoprotective E2F Transcription Factor 1 (E2f1)/Dihydrofolate Reductase (Dhfr) pathway activity in endothelial cells. Cyclin I (Ccni) promotes Cyclin-Dependent Kinase-5 (Cdk5)-mediated Rb phosphorylation. Therefore, we hypothesized that endothelial Ccni may regulate cardiovascular homeostasis, vessel remodeling, and abdominal aortic aneurysm (AAA) formation. METHODS: Aortic CCNI mRNA expression was analyzed in the Gene Expression Omnibus (GEO) GSE57691 cohort consisting of AAA patients (n = 39) and healthy controls (n = 10). We employed wild-type (WT) mice and endothelial Ccni knockout (Ccnifl/flTie2-Cre) mice to conduct in vivo and ex vivo experimentation using an Angiotensin (Ang) II hypertension model and a CaCl2 AAA model. Mice were assessed for Rb/E2f1/Dhfr signaling, biopterin (i.e., biopterin [B], dihydrobiopterin [BH2], and tetrahydrobiopterin [BH4]) production, cardiovascular homeostasis, vessel remodeling, and AAA formation. RESULTS: Aortic CCNI mRNA expression was downregulated in AAA patients. Both Ang II- and CaCl2-induced WT mice showed aortic Ccni upregulation coupled with vasculoprotective upregulation of Rb/E2f1/Dhfr signaling and biopterins. Endothelial Ccni knockout downregulated medial Rb/E2f1/Dhfr signaling and biopterins in Ang II-induced hypertensive mice, which exacerbated eNos uncoupling and H2O2 production. Endothelial Ccni knockout impaired in vivo hemodynamic responses and endothelium-dependent vasodilatation in ex vivo mesenteric arteries in response to Ang II. Endothelial Ccni knockout exacerbated mesenteric artery remodeling and AAA risk in response to Ang II and CaCl2. CONCLUSIONS: Endothelial Ccni acts as a critical negative regulator of eNos uncoupling-mediated ROS generation and thereby reduces vulnerability to hypertension-induced vascular remodeling and AAA development in mice.

The Role of CCN1 in Esophageal Adenocarcinoma: What We Have Learned From the Lab.

Background: Esophageal cancer is one of the most common and deadliest cancers in the world, particularly esophageal adenocarcinoma. There has never been a special drug to treat it.Purpose: This article summarizes the work that we have done in our laboratory about the role of CCN1 in esophageal cancer and gives a new perspective of CCN1 biology.Research Design: This is a review article. Study Sample: The work was done using validated cell lines and fixed human tissue slides.Data Collection and Analysis: This is a review article, therefore, no data collection or analysis was involved.Results: CCN1 is a matricellular protein supporting adhesion, migration, and survival in normal cells, but in the esophageal cancer cells, it induces TRAIL-mediated apoptosis. CCN1 promotes TRAIL and its death receptor expression but downregulates the decoy receptors and survivin in a p53-dependant manner. It was thought that CCN1 relies on TNF to induce apoptosis, but our study found that these two molecules antagonize each other. CCN1 promotes TNFR1 cleavage and uses the soluble product to block TNF signaling, while TNF upregulates PGLYRP1 to overcome this obstacle because PGLYRP1 is a secreted protein that competes with TNF for TNFR1 binding. As a result, when CCN1 and TNF are present together in the vicinity of esophageal tumors, they cancel each other out.Conclusions: Based on our laboratory study, CCN1 has much potential to be a candidate for the treatment of esophageal cancer.

Effects of umbilical cord mesenchymal stem cells on expression of CYR61, FSH, and AMH in mice with premature ovarian failure.

This study aimed to investigate the effects of umbilical cord mesenchymal stem cells on the expression of CYR61, FSH and AMH in mice with premature ovarian failure. For this purpose, thirty SPF female SD mice were selected as the research object, 10 of which were control group, namely group α, and 20 mice with premature ovarian failure model were established by cyclophosphamide. The mice were divided into the model group, namely the ß group and the umbilical cord mesenchymal stem cell transplantation group (γ group), with 10 mice in each group. ELSA method was used to determine the levels of follicle-stimulating hormone (FSH), Luteinizing hormone (LH), estradiol (Estradiol) in serum. The changes of E2, Antimullerian hormone (AMH) and cysteine-rich protein 61 in ovarian tissues were determined by the protein imprinting method. Connective tissue growth factor (CTGF) and caspase-3 protein expression. Results showed that in fertility rate, γ group > α group > ß group, the difference was statistically significant (P<0.05), in litter size, α group > γ group > ß group, the difference was statistically significant (P<0.05). The levels of serum E2 and AMH in α group > γ group > ß group, and the levels of serum FSH and LH in ß group > γ group > α group were statistically significant (P<0.05). The growth follicles were α group > γ group > ß group, and the atresia follicles were ß group > γ group > α group, and there was a statistical difference among all groups (P<0.05). There was no difference in luteal number among the three groups (P>0.05). In terms of CYR61 and CTGF protein expression, α group > γ group > ß group, and in terms of caspase-3, ß group > γ group > α group had statistical significance (P<0.05). It is concluded that intervention with umbilical cord mesenchymal stem cells can significantly improve the expression levels of CYR61 and AMH, reduce the level of FSH, promote cell survival, improve the reproductive quality of mice, and restore the physiological function of the ovary. It is feasible to treat premature ovarian failure with umbilical cord mesenchymal stem cells.

Involvement of CCN1 Protein and TLR2/4 Signaling Pathways in Intestinal Epithelial Cells Response to Listeria monocytogenes.

CCN1 is well studied in terms of its functions in injury repair, cell adhesion survival and apoptosis, bacterial clearance and mediation of inflammation-related pathways, such as the TLR2/4 pathways. However, the role of CCN1 protein and its interaction with TLR2/4 pathways in intestinal epithelial cells was not elucidated after Listeria monocytogenes infection. The results of this study confirm that L. monocytogenes infection induced intestinal inflammation and increased the protein expression of CCN1, TLR2, TLR4 and p38, which followed a similar tendency in the expression of genes related to the TLR2/4 pathways. In addition, organoids infected by L. monocytogenes showed a significant increase in the expression of CCN1 and the activation of TLR2/4 pathways. Furthermore, pre-treatment with CCN1 protein to organoids infected by L. monocytogenes could increase the related genes of TLR2/4 pathways and up-regulate the expression of TNF, and increase the count of pathogens in organoids, which indicates that the interaction between the CCN1 protein and TLR2/4 signaling pathways in intestinal epithelial cells occurred after L. monocytogenes infection. This study will provide a novel insight of the role of CCN1 protein after L. monocytogenes infection in the intestine.

NR4A1 knockdown confers hepatoprotection against ischaemia-reperfusion injury by suppressing TGFß1 via inhibition of CYR61/NF-κB in mouse hepatocytes.

Nuclear receptor subfamily 4, group A, member 1 (NR4A1) can aggravate ischaemia-reperfusion (I/R) injury in the heart, kidney and brain. Thus, the present study aimed to unravel the role of NR4A1 on hepatic I/R injury. For this purpose, the mouse hepatic I/R model and H/R-exposed mouse hepatocytes model were established to stimulate the hepatic and hepatocellular damage. Then, the levels of ALT and AST as well as TNF-α and IL-1ß expression were measured in the mouse serum and supernatant of hepatocyte s, respectively. Thereafter, we quantified the levels of NR4A1, CYR61, NF-kB p65 and TGFß1 under pathological conditions, and their interactions were analysed using ChIP and dual-luciferase reporter gene assays. The in vivo and in vitro effects of NR4A1, CYR61, NF-kB p65 and TGFß1 on I/R-induced hepatic and H/R-induced hepatocellular damage were evaluated using gain- and loss-of-function approaches. NR4A1 was up-regulated in the hepatic tissues of I/R-operated mice and in H/R-treated hepatocytes. Silencing NR4A1 relieved the I/R-induced hepatic injury, as supported by suppression of ALT and AST as well as TNF-α and IL-1ß. Meanwhile, NR4A1 knockdown attenuated the H/R-induced hepatocellular damage by inhibiting the apoptosis of hepatocyte s. Moreover, we also found that NR4A1 up-regulated the expression of CYR61 which resulted in the activation of the NF-κB signalling pathway, thereby enhancing the transcription of TGFß1, which was validated to be the mechanism underlying the contributory role of NR4A1 in hepatic I/R injury. Taken together, NR4A1 silencing reduced the expression of CYR61/NF-κB/TGFß1, thereby relieving the hepatic I/R injury.

MiR-181b suppresses angiogenesis by directly targeting cellular communication network factor 1.

Angiogenesis is essential for various physiological and pathological processes. Previous studies have shown that miRNAs play an important role in blood vessel development and angiogenesis. Recent studies have suggested that miR-181b might be involved in the regulation of angiogenesis in tumors. However, whether miR-181b plays a role in angiogenesis in nontumor diseases is unclear. We found that miR-181b expression was downregulated in hypoxia-stimulated primary human umbilical vein endothelial cells (HUVECs) and a mouse hindlimb ischemia (HLI) model. Gain- and loss-of-function studies showed that a miR-181b mimic inhibited HUVEC migration and tube formation in vitro, and a miR-181b inhibitor had the opposite effects. In vivo, agomir-181b suppressed perfusion recovery in the HLI model and capillary density in a Matrigel plug assay, while perfusion recovery and capillary density were increased by injection of antagomir-181b. Mechanistically, we showed with a reporter assay that cellular communication network factor 1 (CCN1) was a direct target of miR-181b. Moreover, miR-181b suppressed angiogenesis at least in part by targeting CCN1 to inhibit the AMPK signaling pathway. Our research suggests that miR-181b suppresses angiogenesis by directly targeting CCN1, which provides new clues for pro-angiogenic treatment strategies.

ZNF213 negatively controls triple negative breast cancer progression via Hippo/YAP signaling.

Breast cancer is one of the most commonly diagnosed malignancies worldwide, while the triple negative breast cancer (TNBC) is the most aggressive and virulent subtype in breast cancers. Compared with luminal type breast cancers, which could be well controlled by endocrine treatment, TNBC is worse in prognosis and lack of effective targeted therapy. Thus, it would be interesting and meaningful to identify novel therapeutic targets for TNBC treatments. Recent genomic data showed the activation of Hippo/YAP signaling in TNBC, indicating its critical roles in TNBC carcinogenesis and cancer progression. Hippo/YAP signaling could subject to several kinds of protein modifications, including ubiquitination and phosphorylation. Quite a few studies have demonstrated these modifications, which controlled YAP protein stability and turnover, played critical role in Hippo signaling activation In our current study, we identified ZNF213 as a negative modifier for Hippo/YAP axis. ZNF213 depletion promoted TNBC cell migration and invasion, which could be rescued by further YAP silencing. ZNF213 knocking down facilitated YAP protein stability and Hippo target gene expression, including CTGF and CYR61. Further mechanism studies demonstrated that ZNF213 associated with YAP and facilitated YAP K48-linked poly-ubiquitination at several YAP lysine sites (K252, K254, K321 and K497). Besides, the clinical data showed that ZNF213 negatively correlated with YAP protein level and Hippo target gene expression in TNBC samples. ZNF213 expression correlated with good prognosis in TNBC patients. Our data provided novel insights in YAP proteolytic regulation and TNBC progression.

Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis.

BACKGROUND AND AIMS: Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis. APPROACH AND RESULTS: Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor ß2) and inflammation (tumor necrosis factor, interleukin 1ß). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl4 injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis. CONCLUSIONS: Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis.

Sutural fibroblasts exhibit the function of vascular endothelial cells upon mechanical strain.

Midfacial hypoplasia is a type of facial dysplasia. The technique of trans-sutural distraction osteogenesis promotes midface growth so as to ameliorate this symptom. In the process of distraction osteogenesis, the fiber matrix in the suture acts as a mechanical sensor. Compared with osteogenesis, the formation of collagen fibers by fibroblasts is significant in the early stage of sutural distraction. However the transformation of fibroblasts during sutural bone formation induced by tensile force is poorly characterized. Here, we used single-cell RNA sequencing to define the cell classification of the zygomatic maxillary suture and the changes of cell clusters in the suture before and after seven-day distraction. We identified twenty-nine cell subsets spanning monocyte/macrophages, neutrophils, red blood cells, B cells and fibroblasts. Compared with the control group, Monocle analysis revealed the emergence of a unique fibroblast subset (Cdh5+, Col4a1+, Fat1-, and Acta2-) (cluster 27) that expressed vascular endothelial cell genes within the distracted zygomatic maxillary suture. We constructed the differentiation trajectories of the fibroblast population (cluster 23, 27) in the suture before and after distraction. In addition, we clarified that a subset of fibroblasts (cluster 27) lost expression of Fat1, an upregulator of the Hippo pathway, and upregulated Cyr61, a downstream gene of the Hippo pathway, during the distraction process. Further enrichment analysis suggests that cells of the new subset (cluster 27) are undergoing conversion of their identity into a vascular endothelial cell-like state in response to mechanical stimulation, associated with upregulation of angiogenesis genes along the single-cell trajectory. Further immunofluorescence staining confirmed this phenomenon. A combined general transcriptome RNA sequencing data analysis demonstrated that the fibroblasts expressed a number of extracellular matrix-related genes under mechanical strain. These data together provide a new view of the role of fibroblasts in tension-induced sutural angiogenesis via interaction with the Hippo pathway.

Cumulus cells of euploid versus whole chromosome 21 aneuploid embryos reveal differentially expressed genes.

RESEARCH QUESTION: Can cumulus cells be used as a non-invasive target for the study of determinants of preimplantation embryo quality? DESIGN: Cumulus cells were collected from monosomy 21, trisomy 21 and euploid embryos and subjected to RNA sequencing analysis and real-time polymerase chain reaction assays. The differential gene expression was analysed for different comparisons. RESULTS: A total of 3122 genes in monosomy 21 cumulus cells and 19 genes in trisomy 21 cumulus cells were differentially expressed compared with euploid cumulus cells. Thirteen of these genes were differentially expressed in both monosomy and trisomy 21, compared with euploid, including disheveled segment polarity protein 2 (DVL2), cellular communication network factor 1 (CCN1/CYR61) and serum response factor (SRF), which have been previously implicated in embryo developmental competence. In addition, ingenuity pathway analysis revealed cell-cell contact function to be affected in both monosomy and trisomy 21 cumulus cells. CONCLUSIONS: These findings support the use of cumulus cell gene expression analysis for the development of biomarkers evaluating oocyte quality for patients undergoing fertility preservation of oocytes.

Simvastatin Inhibits CYR61 Expression in Orbital Fibroblasts in Graves' Ophthalmopathy through the Regulation of FoxO3a Signaling.

Graves' ophthalmopathy (GO), which is characterized by orbital tissue inflammation, expansion, and fibrosis, is the ocular manifestation in 25% to 50% of patients with Graves' disease. As the pathology of GO is driven by autoimmune inflammation, many proinflammatory cytokines/chemokines, including TNF-α, IL-1ß, IL-6, and CCL20, are crucial in the pathogenesis of GO to activate the orbital fibroblasts. Cysteine-rich protein 61 (CYR61), which is known to regulate cell proliferation, adhesion, and migration, plays a proinflammatory role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis. CYR61 was considered a potential biomarker of GO in recent studies. Statins, which are cholesterol-lowering drugs, were found to reduce the risk of GO, probably through their anti-inflammatory and immunomodulatory effects. In this study, we established a link between CYR61 and statins in the pathogenesis and potential treatment for GO. Firstly, our data showed the overexpression of CYR61 in the orbital tissue (n = 4) and serum specimens (n = 6) obtained from the patients with inactive GO. CYR61 could induce the production of IL-6 and CCL20 in cultured GO orbital fibroblasts. The expression of CYR61 in cultured GO orbital fibroblasts was upregulated via TNF-α stimulation. Secondly, we pretreated cultured GO orbital fibroblasts using simvastatin, a statin, followed by TNF-α stimulation. The data revealed that simvastatin could inhibit TNF-α-induced CYR61 expression by modulating the activity of transcription factor FoxO3a. Our results provided insights into some cellular mechanisms that may explain the possible protective effects of simvastatin against the development of GO.

The CCN1 (CYR61) protein promotes skin growth by enhancing epithelial-mesenchymal transition during skin expansion.

The skin expansion technique is widely used to induce skin growth for large-scale skin deformity reconstruction. However, the capacity for skin expansion is limited and searching for ways to improve the expansion efficiency is a challenge. In this study, we aimed to explore the possible mechanism of skin expansion and to find a potential therapeutic target on promoting skin growth. We conducted weighted gene coexpression network analysis (WGCNA) of microarray data generated from rat skin expansion and found CCN1 (CYR61) to be the central hub gene related to epithelial-mesenchymal transition (EMT). CCN1 up-regulation was confirmed in human and rat expanded skin and also in mechanically stretched rat keratinocytes, together with acquired mesenchymal phenotype. After CCN1 stimulation on keratinocytes, cell proliferation was promoted and partial EMT was induced by activating ß-catenin pathway. Treatment of CCN1 protein could significantly increase the flap thickness, improve the blood supply and restore the structure in a rat model of skin expansion, whereas inhibition of CCN1 through shRNA interference could dramatically reduce the efficiency of skin expansion. Our findings demonstrate that CCN1 plays a crucial role in skin expansion and that CCN1 may serve as a potential therapeutic target to promote skin growth and improve the efficiency of skin expansion.

Shear-Induced CCN1 Promotes Atheroprone Endothelial Phenotypes and Atherosclerosis.

BACKGROUND: Atherosclerosis occurs preferentially at the blood vessels encountering blood flow turbulence. The matricellular protein CCN1 is induced in endothelial cells by disturbed flow, and is expressed in advanced atherosclerotic lesions in patients and in the Apoe-/- mouse model. The role of CCN1 in atherosclerosis remains undefined. METHODS: To assess the function of CCN1 in vivo, knock-in mice carrying the integrin α6ß1-binding-defective mutant allele Ccn1-dm on the Apoe-/- background were tested in an atherosclerosis model generated by carotid artery ligation. Additionally, CCN1-regulated functional phenotypes of human umbilical vein endothelial cells, or primary mouse aortic endothelial cells isolated from wild-type and Ccn1 dm/dm mice, were investigated in the in vitro shear stress experiments under unidirectional laminar shear stress (12 dyn/cm2) versus oscillatory shear stress (±5 dyn/cm2) conditions. RESULTS: We found that Ccn1 expression was upregulated in the arterial endothelium 3 days after ligation before any detectable structural changes, and intensified with the progression of atherosclerotic lesions. Compared with Apoe-/- controls, Ccn1 dm/dm/ Apoe-/- mice were remarkably resistant to ligation-induced plaque formation (n=6). These mice exhibited lower oxidative stress, expression of endothelin-1 and monocyte chemoattractant protein-1, and monocyte homing. CCN1/α6ß1 critically mediated flow-induced activation of the pleiotropic transcription factor nuclear factor-κB and therefore the induction of atheroprone gene expression in the mouse arterial endothelium after ligation (n=6), or in cultured human umbilical vein endothelial cells or primary mouse aortic endothelial cells exposed to oscillatory shear stress (n=3 in triplicate). Interestingly, the activation of nuclear factor-κB by CCN1/α6ß1 signaling prompted more production of CCN1 and α6ß1. Blocking CCN1-α6ß1 binding by the Ccn1-dm mutation or by T1 peptide (derived from an α6ß1-binding sequence of CCN1) disrupted the positive-feedback regulation between CCN1/α6ß1 and nuclear factor-κB, and prevented flow-induced atheroprone phenotypic alterations in endothelial cells or atherosclerosis in mice. CONCLUSIONS: These data demonstrate a causative role of CCN1 in atherosclerosis via modulating endothelial phenotypes. CCN1 binds to its receptor integrin α6ß1 to activate nuclear factor-κB, thereby instigating a vicious circle to persistently promote atherogenesis. T1, a peptide antagonist selectively targeting CCN1-α6ß1, can be further optimized for developing T1-mimetics to treat atherosclerosis.