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BackgroundPeripheral arterial disease (PAD) is the 3rdleading type of atherosclerotic disease (ASD) morbidity. Arterial stiffness is intimately connected to the onset and progression of peripheral artery disease (PAD). The role of arterial stiffening on flow-mediated atherosclerotic plaque formation is not well understood. The objective of this study is to discover endothelial cell (EC) pathways under PAD conditions and test the modifiability of these pathways on ASD.MethodsPAD conditions in mice were conferred by partial carotid ligation to induce disturbed Flow (D-flow) in pre-stiffened Fibulin-5 knockout (KO) mice that lack normal elastin function. EC pathways, including Connective tissue growth factor (CTGF/CCN) were quantified by gene analysis and histology. Atherogenic mice had PCSK9 infection + high fat diet. CTGF was inhibited in an EC-specific knockout (ECKOCTGF) and with a CTGF antibody (FG-3149). Human vascular tissue was used to validate PAD biomechanics and CTGF upregulation.ResultsBiomechanical testing demonstrated that d-flow KO arteries mimic biomechanics of PAD arteries. RNA microarray, qPCR, and immunohistochemistry identified EC plasticity in these arteries compared to WT and KO under stable flow. Under atherogenic conditions, KO arteries demonstrated vulnerable plaques not seen in WT animals. CTGF expression was increased by d-flow, in KO arteries, in aged (18 months) WT arteries, and vascular tissue under d-flow. CTGF inhibition by ECKOCTGFfavorably improved plaque characteristics in male but not female animals. FG-3149 treatment of ECWTCTGFmale animals delivered similar benefits to plaque characteristics and arterial compliance.ConclusionECs in PAD arteries exist under a complex hemodynamic environment that integrates stiffness and d-flow into an atherogenic and inflammatory environment. Stiffness + d-flow stimulates precocious onset of EC plasticity and a vulnerable plaque phenotype. CTGF is a matricellular protein that can tune fibro-inflammatory pathways. CTGF is a prominent mediator of D-flow-mediated arterial remodeling and focal atherosclerotic plaque remodeling. Inhibition of CTGF improves plaque phenotype and arterial compliance. CTGF-associated pathways hold promise as therapeutic targets for PAD patients.

The cardiac extracellular matrix plays essential roles in homeostasis and injury responses. Although the role of fibrillar collagens have been thoroughly documented, the functions of non-fibrillar collagen members remain underexplored. These include a distinct group of non-fibrillar collagens, termed, fibril-associated collagens with interrupted triple helices (FACITs). Recent reports of collagen type XIX (encoded by Col19a1) expression in adult heart and evidence of its enhanced expression in cardiac ischemia suggest important functions for this FACIT in cardiac ECM structure and function. Here, we examined the cellular source of collagen XIX in the adult murine heart and evaluated its involvement in ECM structure and ventricular function. Immunodetection of collagen XIX in fractionated cardiovascular cell lineages revealed fibroblasts and smooth muscle cells as the primary sources of collagen XIX in the heart. Based on echocardiographic and histologic analyses, Col19a1 null (Col19a1 N/N) mice exhibited reduced systolic function, thinning of left ventricular walls, and increased cardiomyocyte cross-sectional areas-without gross changes in myocardial collagen content or basement membrane morphology. Col19a1 N/N cardiac fibroblasts had augmented expression of several enzymes involved in the synthesis and stability of fibrillar collagens, including PLOD1 and LOX. Furthermore, second harmonic generation-imaged ECM derived from Col19a1 N/N cardiac fibroblasts, and transmission electron micrographs of decellularized hearts from Col19a1 N/N null animals, showed marked reductions in fibrillar collagen structural organization. Col19a1 N/N mice also displayed enhanced phosphorylation of focal adhesion kinase (FAK), signifying de-repression of the FAK pathway-a critical mediator of cardiomyocyte hypertrophy. Collectively, we show that collagen XIX, which had a heretofore unknown role in the mammalian heart, participates in the regulation of cardiac structure and function-potentially through modulation of ECM fibrillar collagen structural organization. Further, these data suggest that this FACIT may modify ECM superstructure via acting at the level of the fibroblast to regulate their expression of collagen synthetic and stabilization enzymes.

Background/Aims Scleroderma (systemic sclerosis, SSc) is an autoimmune inflammatory-fibrosis syndrome that damages the skin and internal organs and is considered a prototypic complex fibrotic disease. In SSc, persistently activated myofibroblasts are maintained by an excessive, autocrine mechanotranductive/pro-adhesive signaling loop. Drugs targeting this pathway are therefore of likely therapeutic benefit in SSc. The mechanosensitive transcriptional co-activator, yes activated protein-1 (YAP1), is activated in SSc fibroblasts. The terpenoid plant-derivative celastrol has recently been identified as a YAP inhibitor; however, if celastrol can alleviate SSc fibrosis, and its underlying mechanism of action, is as yet unclear. Methods We cultured age-, sex-, and site-matched human dermal fibroblasts sampled from healthy individuals and patients with early onset ( Results In dermal fibroblasts, celastrol impaired the ability of TGFβ1 to induce an SSc-like pattern of gene expression, including the induction of cellular communication network factor 2 (CCN2), collagen I and TGFβ1 protein (N = 6, all p Conclusion Our data are consistent with the hypothesis that compounds, such as celastrol, that antagonize the YAP pathway warrant further consideration as potential treatments for SSc skin fibrosis. Disclosure A. Leask: Grants/research support; CIHR, NSERC, Arthritis Society of Canada. R.J. Stratton: None. S. Xu: None. P. Chitturi: None.

Background: Abnormal DNA methylation is thought to contribute to the onset and progression of systemic sclerosis. Currently, the most comprehensive assay for profiling DNA methylation is whole-genome bisulfite sequencing (WGBS), but its precision depends on read depth and it may be subject to sequencing errors. SOMNiBUS, a method for regional analysis, attempts to overcome some of these limitations. Using SOMNiBUS, we re-analyzed WGBS data previously analyzed using bumphunter, an approach that initially fits single CpG associations, to contrast DNA methylation estimates by both methods. Methods: Purified CD4+ T lymphocytes of 9 SSc and 4 control females were sequenced using WGBS. We separated the resulting sequencing data into regions with dense CpG data, and differentially methylated regions (DMRs) were inferred with the SOMNiBUS region-level test, adjusted for age. Pathway enrichment analysis was performed with Ingenuity Pathway Analysis (IPA). We compared the results obtained by SOMNiBUS and bumphunter. Results: Of 8268 CpG regions of >60 CpGs eligible for analysis with SOMNiBUS, we identified 131 DMRs and 125 differentially methylated genes (DMGs; p-values less than Bonferroni-corrected threshold of 6.05-06 controlling family-wise error rate at 0.05; 1.6% of the regions). In comparison, bumphunter identified 821,929 CpG regions, 599 DMRs (of which none had >60 CpGs) and 340 DMGs (q-value of 0.05; 0.04% of all regions).The top ranked gene identified by SOMNiBUS was FLT4, a lymphangiogenic orchestrator, and the top ranked gene on chromosome X was CHST7, known to catalyze the sulfation of glycosaminoglycans in the extracellular matrix. The top networks identified by IPA included connective tissue disorders. Conclusions: SOMNiBUSis a complementary method of analyzing WGBS data that enhances biological insights into SSc and provides novel avenues of investigation into its pathogenesis.

Approximately 45% of the deaths in the developed world result from conditions with a fibrotic component. Although no specific, focused anti-fibrotic therapies have been approved for clinical use, a long-standing concept is that targeting CCN proteins may be useful to treat fibrosis. Herein, we summarize current data supporting the concept that targeting CCN2 may be a viable anti-fibrotic approach to treat scleroderma. Testing this hypothesis has been made possible by using a mouse model of inflammation-driven skin and lung fibrosis.

Fibrosis is perpetuated by an autocrine, pro-adhesive signaling loop maintained by the synthetic and contractile abilities of myofibroblasts and the stiff, highly-crosslinked extracellular matrix. Transcriptional complexes that are exquisitely responsive to mechanotransduction include the co-activator YAP1, which regulates the expression of members of the CCN family of matricellular proteins such as CCN2 and CCN1. Although selective YAP1 inhibitors exist, the effect of these inhibitors on profibrotic gene expression in fibroblasts is largely unknown, and is the subject of our current study. Herein, we use genome-wide expression profiling, real-time polymerase chain reaction and Western blot analyses, cell migration and collagen gel contraction assays to assess the ability of a selective YAP inhibitor verteporfin (VP) to block fibrogenic activities in dermal fibroblasts from healthy individual human controls and those from isolated from fibrotic lesions of patients with diffuse cutaneous systemic sclerosis (dcSSc). In control fibroblasts, VP selectively reduced expression of fibrogenic genes and also blocked the ability of TGFbeta to induce actin stress fibers in dermal fibroblasts. VP also reduced the persistent profibrotic phenotype of dermal fibroblasts cultured from fibrotic lesions of patients with dcSSc. Our results are consistent with the notion that, in the future, YAP1 inhibitors may represent a novel, valuable method of treating fibrosis as seen in dcSSc. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12079-020-00596-x.

Background Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys. Methods We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP). Results V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells. Conclusions Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.

Although cancer cells are located within a microenvironment consisting of immune cells, endothelial cells, fibroblasts and extracellular matrix (ECM), the role of the cancer-associated fibroblasts (CAFs) in driving tumorigenesis is relatively underinvestigated. Recent data suggest that a stiff ECM, generated by CAFs, and associated integrin-dependent signaling underlies the development of drug resistance to BRAF inhibitors in melanoma. Drugs targeting the matricellular protein CCN2 (centralized communication network 2, formerly termed connective tissue growth factor), are in clinical development for cancers; for example, FG-3019, an antibody targeting CCN2 has recently entered Phase III trials for pancreatic cancer. Recent data show that fibroblast-specific production of CCN2, which signals through integrins and whose overexpression in human melanomas is independent of BRAF mutational status, is essential for neovascularization, including vasculogenic mimicry, in melanoma. In clinical melanoma samples, a FAP/ITGA11/COL1A1/CCN2-expressing CAF population negatively correlates with disease-free survival. These data emphasize the essential role for a CCN2-expressing subset of CAFs in cancer progression and suggest that targeting the CAFs in the tumor microenvironment, for example by blocking the action of CCN2, may be useful in combination therapies to treat cancers.

Tumor stroma resembles a fibrotic microenvironment, being characterized by the presence of myofibroblast-like cancer-associated fibroblasts (CAFs). In wild-type mice injected with melanoma cells, we show that the stem cell transcription factor Sox2 is expressed by tumor cells and induced in CAFs derived from synthetic fibroblasts. These fibroblasts were labeled postnatally with green fluorescent protein using mice expressing a tamoxifen-dependent Cre recombinase under the control of a fibroblast-specific promoter/enhancer. Conversely, fibroblast activation was impaired in mice with a fibroblast-specific deletion of cellular communication network 2 (Ccn2), associated with reduced expression of α-smooth muscle actin and Sox2. Multipotent Sox2-expressing skin-derived precursor (SKP) spheroids were cultured from murine back skin. Using lineage tracing and flow cytometry, approximately 40% of SKPs were found to be derived from type I collagen-lineage cells and acquired multipotency in culture. Inhibition of mechanotransduction pathways prevented myofibroblast differentiation of SKPs and expression of Ccn2. In SKPs deleted for Ccn2, differentiation into a myofibroblast, but not an adipocyte or neuronal phenotype, was also impaired. In human melanoma, CCN2 expression was associated with a profibrotic integrin alpha (ITGA) 11–expressing subset of CAFs that negatively associated with survival. These results suggest that synthetic dermal fibroblasts are plastic, and that CCN2 is required for the differentiation of dermal progenitor cells into a myofibroblast/CAF phenotype and is, therefore, a therapeutic target in melanoma.

Introduction: CCN2, formerly known as CTGF, is a pro-fibrotic protein that is implicated in several diabetes-related complications. The role of CCN2 in mediating NASH fibrosis in diabetes has not been fully elucidated. We hypothesize that targeting liver CCN2 can prevent NASH fibrosis in diabetes. Aim: To examine whether hepatocyte specific homozygous gene deletion of CCN2 leads to less severe liver fibrosis compared with control mice exposed to high fat feeding and diabetes. Methods: Male control mice (B6.Cg-Tg(AlbCre)21Mgn/J (AlbCre) and homozygous CCN2fl/fl (CCN2fl/fl)), and hepatocyte specific CCN2 knockout mice (CCN2flxB6.Cg-Tg(AlbCre)21Mgn/J (CCN2KO)) were fed either standard chow or high fat diet (HFD; 45% kCal fat). HFD mice were rendered diabetic (HFD+DM) after 15 wks with low dose streptozotocin (2-3 x 0.65 mg/kg ip) then maintained for a further 10 wks. Liver fibrosis was assessed histologically with Picro Sirius Red (PSR) staining. Gene expression of fibrosis markers were measured by RT-qPCR. Biochemical assays were undertaken. Results: There was less fibrosis by PSR staining in the HFD+DM CCN2KO mice at the central vein (24% reduction, 2.20±0.84 controls vs. 1.66±0.82 CCN2-KO, p=0.03, one tailed Mann-Whitney test) and portal tract (18% reduction, 2.51±0.85 controls vs. 2.06±0.84 CCN2KO, p=0.04) compared with HFD+DM control mice. In HFD+DM CCN2KO mice, there were significant reductions in gene expression of collagen-I (44%, p=0.04), -III (44%, p=0.04) and -IVα1 (40%, p=0.01) but not in collagen-VI or fibronectin. Compared with chow fed mice, HFD+DM mice had higher body weight, BGLs, insulin levels, fat mass and AST and ALT activity but there was no difference in metabolic outcomes between control strains and CCN2KO mice. Conclusion: Our study has shown partial prevention of NASH fibrosis in diabetes in a novel model with hepatocyte specific CCN2 knockout mice. CCN2 is increasingly a potential target for limiting progression of NASH fibrosis in diabetes. Disclosure S. N. Parry: None. X. Wang: None. D. Min: None. J. S. Lourdesamy: None. P. F. Williams: None. A. Leask: Stock/Shareholder; Self; FibroGen. S. M. Twigg: Advisory Panel; Self; Abbott Diabetes, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi-Aventis, Consultant; Self; Aktivo Labs, Sanofi-Aventis, Research Support; Self; Abbott Diabetes, Speaker’s Bureau; Self; Abbott Diabetes, AstraZeneca.

The effector cells ultimately responsible for fibrosis are myofibroblasts. In the fibrotic autoimmune connective tissue disease scleroderma, myofibroblasts are autonomously activated, and retain their phenotype upon culturing. Since the 1990s, researchers have exploited this fact to use scleroderma fibroblasts as a model system to uncover the fundamental mechanisms underlying myofibroblast persistence in fibrotic conditions. These studies have suggested that an autocrine transforming growth factor (TGF)beta signaling loop is insufficient to explain the persistent myofibroblast phenotype but instead support the hypothesis that fibrotic myofibroblasts possess an intrinsically activated pro-adhesive signaling pathway, and that this contributes to the perpetuation of pathological fibrosis. This review focuses on these observations.

The CCN family of matricellular proteins are recognized bona fide targets for therapeutically targeting so-called chronic inflammatory diseases, including fibrosis and cancers. The majority of the work supporting this contention has been derived from examining CCN2, formerly, and unhelpfully, termed "connective tissue growth factor." Both CCN2, and its related protein, CCN1, formerly termed "cysteine-rich protein 61", are positively regulated by not only TGFbeta, but also by the hippo/YAP/TAZ mechanotransduction pathway that appears to drive these pathologies. Indeed, increasing evidence indicates that CCN1 also contributes to these fibrosis and cancers and, consequently, targeting both CCN2 and CCN1 simultaneously could be of therapeutic value. This commentary focuses on a recent, exciting paper (Ju et al., 2020, Scientific Reports, 10, 3201) suggesting that CCN1 is a target for non-alcoholic steatohepatitis (NASH).

The tumour microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts (CAFs) constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumourigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumour growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple negative breast cancers and that it directly induces CAF phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g. CXCL1), cytokines (e.g. IL-6) and growth factors (e.g. PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME.

CCN2 is a matricellular protein involved in several critical biological processes. In particular, CCN2 is involved in cartilage development and in osteoarthritis. CCN2 null mice exhibit a range of skeletal dysmorphisms, highlighting its importance in regulating matrix formation during development, however its role in adult cartilage remains unclear. The aim of this study was to determine the role of CCN2 in postnatal chondrocytes in models of post-traumatic osteoarthritis (PTOA). CCN2 deletion was induced in articular chondrocytes of male transgenic mice at 8 weeks of age. PTOA was induced in knees either surgically or non-invasively by repetitive mechanical loading at 10 weeks of age. Knee joints were harvested, scanned with micro-CT, and processed for histology. Sections were stained with toluidine blue and scored using the OARSI grading system. In the non-invasive model cartilage lesions were present in the lateral femur but no significant differences were observed between wildtype (WT) and CCN2 knockout (KO) mice 6 weeks post-loading. In the surgical model, severe cartilage degeneration was observed in the medial compartments but no significant differences were observed between WT and CCN2 KO mice at 2, 4, and 8 weeks post-surgery. We conclude that CCN2 deletion in chondrocytes did not modify the development of PTOA in mice, suggesting that chondrocyte expression of CCN2 in adults is not a critical player in protecting cartilage from the degeneration associated with PTOA.Summary StatementPost-natal deletion of CCN2 in chondrocytes does not affect the development of post-traumatic osteoarthritis in mice.

COVID-19 is a respiratory disease. A recent report in Lancet examined, retrospectively, 137 patients with COVD-19. Patients that died had elevated IL-6 levels and acute respiratory distress syndrome. These data have obvious implications for how to control mortality in COVID-19.

Cellular communication network (CCN) proteins are matricellular proteins that coordinate signaling among extracellular matrix, secreted proteins, and cell surface receptors. Their specific in vivo function is context-dependent, but they play profound roles in pathological conditions, such as fibrosis and cancers. Anti-CCN therapies are in clinical consideration. Only recently, however, has the function of these complex molecules begun to emerge. This review summarizes and interprets our current knowledge regarding these fascinating molecules and provides experimental evidence for their utility as therapeutic targets.

Mesenchymal progenitor cells play a key role in fibrogenesis. An exciting paper was recently published showed that blister fluid from the skin patients with the autoimmune connective tissue disease scleroderma (systemic sclerosis, SSc) preferentially activated mesenchymal progenitor cells (Taki et al. in Arthritis Rheumatol 72(8):1361–1374, 2020). These data provide new and invaluable insights into the complex interactions in the connective tissue microenvironment that ultimately result in persistent, pathological fibrosis.

Lymphangiogenesis is correlated with the degree of renal interstitial fibrosis. Pro-fibrotic transforming growth factor beta induces VEGF-C production, the main driver of lymphangiogenesis. Connective tissue growth factor (CTGF) is an important determinant of fibrotic tissue remodeling, but its possible involvement in lymphangiogenesis has not been explored. We found prominent lymphangiogenesis during tubulointerstitial fibrosis to be associated with increased expression of CTGF and VEGF-C in human obstructed nephropathy as well as in diabetic kidney disease. Using CTGF knockout mice, we investigated the involvement of CTGF in development of fibrosis and associated lymphangiogenesis in obstructive nephropathy. The increase of lymphatic vessels and VEGF-C in obstructed kidneys was significantly reduced in CTGF knockout compared to wild-type mice. Also in mouse kidneys subjected to ischemia-reperfusion injury, CTGF knockdown was associated with reduced lymphangiogenesis. In vitro, CTGF induced VEGF-C production in HK-2 cells, while CTGF siRNA suppressed transforming growth factor beta 1-induced VEGF-C upregulation. Furthermore, surface plasmon resonance analysis showed that CTGF and VEGF-C directly interact. Interestingly, VEGF-C-induced capillary-like tube formation by human lymphatic endothelial cells was suppressed by full-length CTGF but not by naturally occurring proteolytic CTGF fragments. Thus, CTGF is significantly involved in fibrosis-associated renal lymphangiogenesis through regulation of, and direct interaction with, VEGF-C

One of the first targets proposed as an anti-fibrotic therapy was CCN2. Proof of its involvement in fibrosis was initially difficult, due to the lack of appropriate reagents and general understanding of the molecular mechanisms responsible for persistent fibrosis. As these issues have been progressively resolved over the last twenty-five years, it has become clear that CCN2 is a bone fide target for anti-fibrotic intervention. An anti-CCN2 antibody (FG-3019) is in Phase III clinical trials for idiopathic pulmonary fibrosis and pancreatic cancer, and in Phase II for Duschenne’s muscular dystrophy. An exciting paper recently published by Mary Barbe and the Popoff group has shown that FG-3019 reduces established muscle fibrosis (Barbe et al., FASEB J 34:6554–6569, 2020). Intriguingly, FG-3019 blocked the decreased expression of the anti-fibrotic protein CCN3, caused by the injury model. These important data support the notion that targeting CCN2 in the fibrotic microenvironment may reverse established fibrosis, making it the first agent currently in development to do so.

Pirfenidone and nintedanib have been approved for idiopathic pulmonary fibrosis (IPF) due to their ability to statistically slow, over a year, the rate of decline in lung forced vital capacity (FVC), neither drug has been reported to have o positive effects on high-resolution computed tomography (HRCT) of the chest, symptoms, or quality of life. Moreover, pirfenidone and nintedanib have substantial gastrointestinal tolerability issues. Overall, these data highly suggest that novel therapeutic approached are needed. CCN2 has been shown to be a mediator of fibrosis in many preclinical models. Anti-CCN2 strategies are in clinical development for IPF, A recent study by Richeldi and colleagues described the recent Phase II clinical trial for FG-3019 in IPF, and the results were highly encouraging. This commentary contextualizes and summarizes these exciting findings.

© 2019, Mary Ann Liebert, Inc., publishers 2019. Impaired skin wounds represent a significant and growing complication associated with type II diabetes and effective clinical strategies to reproducibly induce wound resolution do not exist. As a class of secreted nonstructural matrix proteins that modulate many cellular aspects of skin healing, matricellular proteins could represent a novel mechanism to promote skin healing. Based on their role in the regulation of the proliferative phase of healing, we hypothesized that local delivery of periostin and CCN2 could suppress inflammation and progress wounds into the proliferative and remodeling phases of repair. Using electrospinning, scaffolds composed of collagen type I and periostin or CCN2 were fabricated. Delivery of periostin/collagen and CCN2/collagen electrospun scaffolds increased excisional wound closure rates in a murine db/db diabetic model compared with collagen alone or untreated wounds. Reduced neutrophil infiltration was evident in the presence of periostin/collagen and CCN2/collagen scaffolds, concomitant with an increase in mesenchymal cell infiltration versus empty and collagen scaffolds alone at day 7. Microarray analysis demonstrated scaffold-dependent upregulation of gene clusters associated with wound contraction, cell differentiation, and suppression of PPARγsignaling at day 7; however, no changes in mRNA levels of extracellular matrix genes were observed compared with controls. At day 11, significantly increased vascularization of the wound bed was evident. Local delivery of periostin- and CCN2-based biomaterials may represent a novel strategy to induce the proliferative phase of repair and correction of the healing process in impaired skin wounds. Nonhealing skin wounds remain a significant burden on health care systems, with diabetic patients 20 times as likely to undergo a lower extremity amputation due to impaired healing. Novel treatments that suppress the proinflammatory signature and induce the proliferative and remodeling phases are needed clinically. We demonstrate that the addition of periostin and CCN2 in a scaffold form increases closure rates of full-thickness skin wounds in diabetic mice, concomitant with enhanced angiogenesis. Our results demonstrate the efficacy of periostin- and CCN2-containing biomaterials to stimulate wound closure, which could represent a novel method for the treatment of diabetic skin wounds.

The microenvironment contributes to the excessive connective tissue deposition that characterizes fibrosis. Members of the CCN family of matricellular proteins are secreted by fibroblasts into the fibrotic microenvironment; however, the role of endogenous CCN1 in skin fibrosis is unknown. Mice harboring a fibroblast-specific deletion for CCN1 were used to assess if CCN1 contributes to dermal homeostasis, wound healing, and skin fibrosis. Mice with a fibroblast-specific CCN1 deletion showed progressive skin thinning and reduced accumulation of type I collagen; however, the overall mechanical property of skin (Young's modulus) was not significantly reduced. Real time-polymerase chain reaction analysis revealed that CCN1-deficient skin displayed reduced expression of mRNAs encoding enzymes that promote collagen stability (including prolyl-4-hydroxylase and PLOD2), although expression of COL1A1 mRNA was unaltered. CCN1-deficent skin showed reduced hydroxyproline levels. Electron microscopy revealed that collagen fibers were disorganized in CCN1-deficient skin. CCN1-deficient mice were resistant to bleomycin-induced skin fibrosis, as visualized by reduced collagen accumulation and skin thickness suggesting that deposition/accumulation of collagen is impaired in the absence of CCN1. Conversely, CCN1-deficient mice showed unaltered wound closure kinetics, suggesting de novo collagen production in response to injury did not require CCN1. In response to either wounding or bleomycin, induction of α-smooth muscle actin-positive myofibroblasts was unaffected by loss of CCN1. CCN1 protein was overexpressed by dermal fibroblasts isolated from lesional (i.e., fibrotic) areas of patients with early onset diffuse scleroderma. Thus, CCN1 expression by fibroblasts, being essential for skin fibrosis, is a viable anti-fibrotic target., Highlights • The role of endogenous CCN1 in skin biology is largely unknown • Fibroblast-specific deletion CCN1 causes thinner skin and misaligned collagen • CCN1-deficient mice were resistant to bleomycin-induced skin fibrosis • Wound healing closure kinetics was unaffected by loss of CCN1 • CCN1 may be as a target for anti-fibrotic therapy

mTOR, the target of rapamycin, has been promoted as a potential target for cancers, transplantations and even lung fibrosis. However, paradoxically, targeting mTOR has been reported to result in profibrotic side effects. Some recent publications highlight why this might be.

Tumor stroma resembles a fibrotic microenvironment, being characterized by the presence of myofibroblast-like cancer-associated fibroblasts (CAFs). In wild-type mice injected with melanoma cells, we show that the stem cell transcription factor Sox2 is expressed by tumor cells and induced in CAFs derived from synthetic fibroblasts. These fibroblasts were labeled postnatally with green fluorescent protein using mice expressing a tamoxifen-dependent Cre recombinase under the control of a fibroblast-specific promoter/enhancer. Conversely, fibroblast activation was impaired in mice with a fibroblast-specific deletion of cellular communication network 2 (Ccn2), associated with reduced expression of α-smooth muscle actin and Sox2. Multipotent Sox2-expressing skin-derived precursor (SKP) spheroids were cultured from murine back skin. Using lineage tracing and flow cytometry, approximately 40% of SKPs were found to be derived from type I collagen-lineage cells and acquired multipotency in culture. Inhibition of mechanotransduction pathways prevented myofibroblast differentiation of SKPs and expression of Ccn2. In SKPs deleted for Ccn2, differentiation into a myofibroblast, but not an adipocyte or neuronal phenotype, was also impaired. In human melanoma, CCN2 expression was associated with a profibrotic integrin alpha (ITGA) 11-expressing subset of CAFs that negatively associated with survival. These results suggest that synthetic dermal fibroblasts are plastic, and that CCN2 is required for the differentiation of dermal progenitor cells into a myofibroblast/CAF phenotype and is, therefore, a therapeutic target in melanoma.

The tumour microenvironment (TME), consisting of stromal cell types including fibroblasts, endothelial, immune and mesenchymal stromal cells (MSCs), is an important mediator of breast cancer (BCa) progression. BCa cells regulate the composition of the TME by secreting a myriad of factors; however, mechanisms governing the dynamic reciprocity between BCa and TME components are largely unknown. Cancer-associated fibroblasts (CAFs) are a major stromal component in many BCa and have been shown to be activated by BCa cells. MSCs recruited to the TME are an important source of CAFs in many cancers; therefore, characterizing the factors involved in MSC recruitment could provide additional avenues for therapeutic intervention by disrupting the TME in BCa. NODAL, an embryonic morphogen belonging to the TGF-beta superfamily, is clinically associated with BCa progression, and has been shown to sustain BCa stem cells and to promote tumour growth, metastasis, and vascularization. Herein, we report that NODAL expression positively correlates with the presence of CAFs in the stroma of triple-negative human BCa tissues, as determined by NODAL and a-smooth muscle Actin (SMA) staining. Further, our in vitro studies show that NODAL directly induces primary fibroblast activation and chemotaxis. Proteomic analysis of the conditioned media of NODAL-overexpressing MDA-MB-231 and SUM149 BCa cells by mass spectrometry reveal that NODAL reprograms BCa secretomes by simultaneously altering levels of chemokines (e.g. CXCL1), growth factors (e.g. PDGFRA) and cytokines (e.g. IL-6), and that the NODAL-regulated secretome, but not NODAL directly, impacts the ability of BCa cells to affect MSC chemotaxis. We provide evidence that IL-6 promotes MSC chemotaxis, and further discuss the context-dependent effects of NODAL in BCa cell lines. Collectively, our results suggest that factors present in NODAL-regulated secretomes may induce MSC recruitment to the breast TME, where they may contribute to CAF population, therefore demonstrating a hitherto unappreciated role of NODAL as a dynamic regulator of breast TME components. Citation Format: Dylan Dieters-Castator, Paola M. Dantonio, Matt Piaseczny, Guihua Zhang, Jiahui Liu, Miljan Kuljanin, Stephen Sherman, Michael Jewer, Katherine Quesnel, Eun-Young Kang, Martin Köbel, Gabrielle M. Siegers, Andrew Leask, David Hess, Gilles Lajoie, Lynne-Marie Postovit. Embryonic protein NODAL as a potential modulator of the tumour microenvironment: Breast cancer secretome reprogramming and fibroblast activation [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO028.