Changes and related factors of blood CCN1 levels in diabetic patients.

Objective: To study the differences in blood cellular communication network factor 1 (CCN1) levels between patients with diabetes mellitus (DM) and healthy individuals and to explore the relationship between CCN1 and diabetic retinopathy (DR). Methods: Plasma CCN1 levels were detected using ELISA in 50 healthy controls, 74 patients with diabetes without diabetic retinopathy (DM group), and 69 patients with diabetic retinopathy (DR group). Correlations between CCN1 levels and age, body mass index, mean arterial pressure, hemoglobin A1c, and other factors were analyzed. The relationship between CCN1 expression and DR was explored using logistic regression after adjusting for confounding factors. Blood mRNA sequencing analysis was performed for all subjects, and the molecular changes that may be related to CCN1 were explored. The retinal vasculature of streptozotocin-induced diabetic rats was examined using fundus fluorescein angiography; in addition, retinal protein expression was examined using western blotting. Results: Plasma CCN1 levels in patients with DR were significantly higher than in the control and DM groups; however, no significant differences were observed between healthy controls and patients with DM. CCN1 levels negatively correlated with body mass index and positively correlated with the duration of diabetes and urea levels. It was observed that high (OR 4.72, 95% CI: 1.10-20.25) and very high (OR 8.54, 95% CI: 2.00-36.51) levels of CCN1 were risk factors for DR. Blood mRNA sequencing analysis revealed that CCN1-related pathways were significantly altered in the DR group. The expression of hypoxia-, oxidative stress-, and dephosphorylation-related proteins were elevated, while that of tight junction proteins were reduced in the retinas of diabetic rats. Conclusion: Blood CCN1 levels are significantly elevated in patients with DR. High and very high levels of plasma CCN1 are risk factors for DR. Blood CCN1 level may be a potential biomarker for diagnosis of DR. The effects of CCN1 on DR may be related to hypoxia, oxidative stress, and dephosphorylation.

Integrated genomic and proteomic analyses identify stimulus-dependent molecular changes associated with distinct modes of skeletal muscle atrophy.

Skeletal muscle atrophy is a debilitating condition that occurs with aging and disease, but the underlying mechanisms are incompletely understood. Previous work determined that common transcriptional changes occur in muscle during atrophy induced by different stimuli. However, whether this holds true at the proteome level remains largely unexplored. Here, we find that, contrary to this earlier model, distinct atrophic stimuli (corticosteroids, cancer cachexia, and aging) induce largely different mRNA and protein changes during muscle atrophy in mice. Moreover, there is widespread transcriptome-proteome disconnect. Consequently, atrophy markers (atrogenes) identified in earlier microarray-based studies do not emerge from proteomics as generally induced by atrophy. Rather, we identify proteins that are distinctly modulated by different types of atrophy (herein defined as "atroproteins") such as the myokine CCN1/Cyr61, which regulates myofiber type switching during sarcopenia. Altogether, these integrated analyses indicate that different catabolic stimuli induce muscle atrophy via largely distinct mechanisms.

Expression of ACE2 and a viral virulence-regulating factor CCN family member 1 in human iPSC-derived neural cells: implications for COVID-19-related CNS disorders.

It has been reported that coronavirus disease 2019 (COVID-19) causes not only pneumonia but also systemic inflammations including central nervous system (CNS) disorders. However, little is known about the mechanism that triggers the COVID-19-associated CNS disorders, due to the lack of appropriate experimental systems. Our present study showed that angiotensin-converting enzyme-2 (ACE2), a cellular receptor for SARS-CoV-2, is expressed in human induced pluripotent stem cell (iPSC)-derived neural stem/progenitor cells (hiPSC-NS/PCs) and young neurons. Furthermore, together with database analysis, we found that a viral virulent factor CCN family member 1 (CCN1), which is known to be induced by SARS-CoV-2 infection, is expressed in these cells at basal levels. Considering the role of CCN1 which is known to be involved in viral toxicity and inflammation, hiPSC-NS/PCs could provide an excellent model for COVID-19-associated CNS disorders from the aspect of SARS-CoV-2 infection-ACE2-CCN1 axis. In addition, we identified compounds that reduce CCN1 expression. Collectively, our study using hiPSC-NS/PCs may aid in the development of a therapeutic target for COVID-19-related CNS disorders.

CCN1/Cyr61 Is Required in Osteoblasts for Responsiveness to the Anabolic Activity of PTH.

CCN1/Cyr61 is a dynamically expressed matricellular protein that serves regulatory functions in multiple tissues. Previous studies from our laboratory demonstrated that CCN1 regulates bone maintenance. Using an osteoblast and osteocyte conditional knockout mouse model (Ccn1OCN ), we found a significant decrease in trabecular and cortical bone mass in vivo, in part through suppression of Wnt signaling since the expression of the Wnt antagonist sclerostin (SOST) is increased in osteoblasts lacking CCN1. It has been established that parathyroid hormone (PTH) signaling also suppresses SOST expression in bone. We therefore investigated the interaction between CCN1 and PTH-mediated responses in this study. We find that loss of Ccn1 in osteoblasts leads to impaired responsiveness to anabolic intermittent PTH treatment in Ccn1OCN mice in vivo and in osteoblasts from these mice in vitro. Analysis of Ccn1OCN mice demonstrated a significant decrease in parathyroid hormone receptor-1 (PTH1R) expression in osteoblasts in vivo and in vitro. We investigated the regulatory role of a non-canonical integrin-binding domain of CCN1 because several studies indicate that specific integrins are critical to mechanotransduction, a PTH-dependent response, in bone. These data suggest that CCN1 regulates the expression of PTH1R through interaction with the αvß3 and/or αvß5 integrin complexes. Osteoblasts that express a mutant form of CCN1 that cannot interact with αvß3/ß5 integrin demonstrate a significant decrease in mRNA and protein expression of both PTH1R and αv integrin. Overall, these data suggest that the αvß3/ß5-binding domain of CCN1 is required to endow PTH signaling with anabolic activity in bone cells. © 2020 American Society for Bone and Mineral Research (ASBMR).

Caught between a "Rho" and a hard place: are CCN1/CYR61 and CCN2/CTGF the arbiters of microvascular stiffness?

The extracellular matrix (ECM) is a deformable dynamic structure that dictates the behavior, function and integrity of blood vessels. The composition, density, chemistry and architecture of major globular and fibrillar proteins of the matrisome regulate the mechanical properties of the vasculature (i.e., stiffness/compliance). ECM proteins are linked via integrins to a protein adhesome directly connected to the actin cytoskeleton and various downstream signaling pathways that enable the cells to respond to external stimuli in a coordinated manner and maintain optimal tissue stiffness. However, cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, ischemia and aging compromise the mechanical balance of the vascular wall. Stiffening of large blood vessels is associated with well-known qualitative and quantitative changes of fibrillar and fibrous macromolecules of the vascular matrisome. However, the mechanical properties of the thin-walled microvasculature are essentially defined by components of the subendothelial matrix. Cellular communication network (CCN) 1 and 2 proteins (aka Cyr61 and CTGF, respectively) of the CCN protein family localize in and act on the pericellular matrix of microvessels and constitute primary candidate markers and regulators of microvascular compliance. CCN1 and CCN2 bind various integrin and non-integrin receptors and initiate signaling pathways that regulate connective tissue remodeling and response to injury, the associated mechanoresponse of vascular cells, and the subsequent inflammatory response. The CCN1 and CCN2 genes are themselves responsive to mechanical stimuli in vascular cells, wherein mechanotransduction signaling converges into the common Rho GTPase pathway, which promotes actomyosin-based contractility and cellular stiffening. However, CCN1 and CCN2 each exhibit unique functional attributes in these processes. A better understanding of their synergistic or antagonistic effects on the maintenance (or loss) of microvascular compliance in physiological and pathological situations will assist more broadly based studies of their functional properties and translational value.

CCN1 induces adipogenic differentiation of fibro/adipogenic progenitors in a chronic kidney disease model.

Previous studies have shown that sarcopenic obesity is highly prevalent in patients with chronic kidney disease (CKD). Here, the association between CKD and sarcopenic obesity were investigated. The 5/6 nephrectomy was performed to establish CKD in mice. Fluorescence-activated cell sorting (FACS), quantitative real-time PCR, ELISA kits assay, immunohistochemistry, and cell proliferation assay were carried out to investigate the condition of muscle loss and fatty infiltration were in CKD mice and the origin of adipocytes. Muscle atrophy occurred and adipogenic gene expression, Perilipin and FABP4 were markedly increased in the hind limb muscle of CKD mice. Results indicated that fibro/adipogenic progenitors (FAPs) are the precursor of adipocytes in the muscle of CKD mice. Meanwhile, the content of extracellular matrix protein CCN1 was notably increased in serum of CKD patients with sarcopenic obesity which was also found in muscle and serum of CKD mice. CCN1 induced the differentiation of FAPs into adipocytes. These results suggest that CKD mice are susceptible to sarcopenic obesity. CCN1 may be a novel activator of the differentiation of FAPs in CKD muscle.

Association of Cyr61-cysteine-rich protein 61 and short-term mortality in patients with acute heart failure and coronary heart disease.

Aim: The protein CCN1/CYR61 exerts critical functions in myocardial ischemic injury. We sought to investigate the prognostic value of CCN1 in patients with acute heart failure (AHF) and coronary heart disease (CAD). Methodology: We prospectively enrolled 113 patients with AHF and CAD. Patients were followed for all-cause mortality during a 30-day follow-up. Logistic models were used to estimate the association of CCN1 concentrations with 30-day mortality. Results: In multivariate logistic regression model, CCN1 was a significant predictor of 30-day mortality independent of current markers. Enhanced Feedback for Effective Cardiac Treatment risk score was recommended as one of the selected multivariable risk scores to predict outcome in AHF. CCN1 improved risk stratification for all-cause mortality when added to the Enhanced Feedback for Effective Cardiac Treatment risk scores at 30 days. Conclusion: We found CCN1 is independently associated with 30-day mortality in patients with AHF and CAD.

Prognostic Significance of Serum Cysteine-Rich Protein 61 in Patients with Acute Heart Failure.

BACKGROUND/AIMS: Cyr61-cysteine-rich protein 61 (CCN1/CYR61) is a multifunctional matricellular protein involved in the regulation of fibrogenesis. Animal experiments have demonstrated that CCN1 can inhibit cardiac fibrosis in cardiac hypertrophy. However, no study has been conducted to assess the relation between serum CCN1 and prognosis of acute heart failure (AHF). METHODS: We measured the serum CCN1 levels of 183 patients with AHF, and the patients were followed up for 6 months. The associations between CCN1 levels and some clinical covariates, especially left ventricular ejection fraction (LVEF), estimated glomerular filtration rate (eGFR), atrial fibrillation and age, were estimated. The AHF patients were followed up for 6 months. The endpoint was all-cause mortality. Kaplan-Meier curve analysis and multivariable Cox proportional hazards analysis were employed to evaluate the prognostic ability of CCN1. We used calibration, discrimination and reclassification to assess the mortality risk prediction of adding CCN1. RESULTS: Serum CCN1 concentrations in AHF patients were significantly increased compared with those in individuals without AHF (237 pg/ml vs. 124.8 pg/ml, p< 0.001). CCN1 level was associated with the level of NT-proBNP (r=0.349, p< 0.001) and was not affected by LVEF, eGFR, age or atrial fibrillation in AHF patients. Importantly, Kaplan-Meier curve analysis illustrated that the AHF patients with serum CCN1 level > 260 pg/ ml had a lower survival rate (p< 0.001). Multivariate Cox hazard analysis suggests that CCN1 functions as an independent predictor of mortality for AHF patients (LgCCN1, hazard ratio 5.825, 95% confidence interval: 1.828-18.566, p=0.003). In addition, the inclusion of CCN1 in the model with NT-proBNP significantly improved the C-statistic for predicting death (0.758, p< 0.001). The integrated discrimination index was 0.019 (p< 0.001), and the net reclassification index increased significantly after addition of CCN1 (23.9%, p=0.0179). CONCLUSIONS: CCN1 is strongly predictive of 6-month mortality in patients with AHF, suggesting serum CCN1 as a promising candidate prognostic biomarker for AHF patients.

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium.

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates ß-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.

CCN2 induces cellular senescence in fibroblasts.

The expression of Ccn2 (CTGF) has been linked to fibrosis in many tissues and pathologies, although its activities in fibroblastic cells and precise mechanism of action in fibrogenesis are still controversial. Here, we showed that CCN2 can induce cellular senescence in fibroblasts both in vitro and in vivo, whereupon senescent cells express an anti-fibrotic "senescence-associated secretory phenotype" (SASP) that includes upregulation of matrix metalloproteinases and downregulation of collagen. Mechanistically, CCN2 induces fibroblast senescence through integrin α6ß1-mediated accumulation of reactive oxygen species, leading to activation of p53 and induction of p16INK4a. In cutaneous wound healing, Ccn2 expression is highly elevated only during the initial inflammatory phase and quickly declines thereafter to a low level during the proliferation and maturation phases of healing when myofibroblasts play a major role. Consistent with this expression kinetics, knockdown of Ccn2 has little effect on the rate of wound closure, formation of senescent cells, or collagen content of the wounds. However, application of purified CCN2 protein on cutaneous wounds leads to induction of senescent cells, expression of SASP, and reduction of collagen content. These results show that CCN2 can induce cellular senescence in fibroblasts and is capable of exerting an anti-fibrotic effect in a context-dependent manner.

CCN1/CYR61 overexpression in hepatic stellate cells induces ER stress-related apoptosis.

CCN1/CYR61 is a matricellular protein of the CCN family, comprising six secreted proteins specifically associated with the extracellular matrix (ECM). CCN1 acts as an enhancer of the cutaneous wound healing process by preventing hypertrophic scar formation through induction of myofibroblast senescence. In liver fibrosis, the senescent cells are primarily derived from activated hepatic stellate cells (HSC) that initially proliferate in response to liver damage and are the major source of ECM. We investigate here the possible use of CCN1 as a senescence inducer to attenuate liver fibrogenesis by means of adenoviral gene transfer in primary HSC, myofibroblasts (MFB) and immortalized HSC lines (i.e. LX-2, CFSC-2G). Infection with Ad5-CMV-CCN1 induced large amounts of CCN1 protein in all these cells, resulting in an overload of the endoplasmic reticulum (ER) and in a compensatory unfolded protein response (UPR). The UPR resulted in upregulation of ER chaperones including BIP/Grp78, Grp94 and led to an activation of IRE1α as evidenced by spliced XBP1 mRNA with IRE1α-induced JNK phosphorylation. The UPR arm PERK and eIF2a was phosphorylated, combined with significant CHOP upregulation. Ad5-CMV-CCN1 induced HSC apoptosis that was evident by proteolytic cleavage of caspase-12, caspase-9 and the executor caspase-3 and positive TUNEL stain. Remarkably, Ad5-CMV-CCN1 effectively reduced collagen type I mRNA expression and protein. We conclude that the matricellular protein CCN1 gene transfer induces HSC apoptosis through ER stress and UPR.

Regulation of CCN1 (Cyr61) in a porcine model of intestinal ischemia/reperfusion.

Intestinal ischemia is a serious condition that may lead to both local and systemic inflammatory responses. Restoration of blood supply (reperfusion) to ischemic tissues often increases the extent of the tissue injury. Cysteine-rich angiogenic inducer 61 (Cyr61)/CCN1 is an extracellular matrix-associated signaling protein that has diverse functions. CCN1 is highly expressed at sites of inflammation and wound repair, and may modify cell responses. This study aimed to investigate regulation and cellular distribution of CCN1 in intestinal ischemia/reperfusion (I/R) injury in pigs. After intestinal I/R, increased expression of CCN1 was detected by quantitative RT-PCR, Western blot analysis and immunohistochemistry compared with non-ischemic intestine. Immunoflorescence staining revealed that CCN1 was mainly up-regulated in intestinal mucosa after intestinal I/R. Microvillus epithelial cells and vascular endothelial cells were strongly positive for CCN1 in intestinal I/R, while natural killer cells and/or subsets of neutrophils were only modestly positive for CCN1. Furthermore, blood samples taken from the portal and caval veins during ischemia and after reperfusion showed no change of the CCN1 levels, indicating that CCN1 was locally regulated. In conclusion, these observations show, for the first time, that the CCN1 molecule is up-regulated in response to intestinal I/R in a local manner.