10th international workshop on the CCN family of genes, Niagara Falls, Canada, October 21–24, 2019

Annick Perbal

International CCN Society

manager@jccs-sprnature.com

PROGRAM

October 21, 2019

12:00 Registration begins

15:00–15:15

Andrew Leask

Introduction to the Workshop

ICCNS - SPRINGER AWARD CONFERENCE

15:15–15:30

Bernard Perbal

Award presentation

15:30–16:30

ICCNS - SPRINGER AWARDEE

Cynthia Kenyon

Can we stop aging?

Cynthia Kenyon, Ph.D. Vice President, Aging Research

Cynthia Kenyon is one of the world’s foremost authorities on the molecular biology and genetics of aging and life extension. She is Calico’s vice president of aging research.

Session I

Exosomes in cancer

17:00–17:30

Peter Quesenberry

Extracellular vesicles: biology and therapeutic implications

17:30–18:00

Janusz Rak

Genetic and epigenetic driver events as regulators of extracellular vesicle-mediated communication in cancer

18:00–18:30

Lynne-Marie Postovit

Epigenetics of stem cell plasticity in breast cancer

18:30–19:00

Herman Yeger

Anti-inflammatory dietary phytochemicals alter the phenotype of neuroblastoma exosomes: therapeutic implications

Introductory Cocktail/Canapes

October 22, 2019

Breakfast

Session II

Therapeutic considerations

9:00–9:30

Kunimasa Ohta

Involvement of Tsukushi in human neuronal disease

9:30–10:00

Stephen Twigg

Circulating CCNs as potential biomarkers in diabetes-related complications

10:00–10:30

Mary Barbe

Blocking CTGF/CCN2 reduces established tissue fibrosis and sensorimotor declines in a rat model of repetitive overuse injury

10:30–11:00

Coffee Break

11:00–11:30

Bruce Riser

Harnessing the power of CCN signaling for novel therapeutic and diagnostic applications

11:30–12:00

Alex Piedl

Therapeutic peptides based on CCN3 treat systemic sclerosis in a mouse model of the disease

12:00–14:30

Lunch

Session III

Cancer

14:30–15:00

Taihao Quan

CCN1-induced age-related alterations of the dermal microenvironment promote skin cancer development

15:00–15:30

Celina Kleer

CCN6 functions to regulate mitochondrial metabolism in breast cancer

15:30–16:00

Sushanta Banerjee

CCN5 pegylation: a novel approach for breast cancer therapy

16:00–16:30

Kathryn E. Meier

Role of CCN1 in growth factor responses in breast and prostate cancer cells

Ivan Topisirovic (no abstract provided)

McGill University · Department of Oncology

16:30–17:00

Coffee Break

Session IV

New looks into old problems

17:00–17:30

Bernard Perbal

Lost in JCCS

A roadmap to CCN UFOs

17:30–18:00

Havard Attramadal

CCN proteins are preproproteins that require proteolytic activation

18:00–18:30

David Brigstock (No abstract provided)

Nationwide Children’s Hospital and The Ohio State University Medical Center

Free evening

October 23, 2019

Breakfast

Session V

Stem cell activity

9:00–9:30

Nira de la Vega

Investigating the role of CCN3 during central nervous system myelination and remyelination

9:30–10:00

Sandra Romero Marquez

CTGF/CCN2 is required for recovery of hematopoiesis after stress-induction in vivo

10:00–10:30

Richard Stratton

Scleroderma disease microenvironment growth factors and metabolites induce MSCs to HER2 CCN2 expressing myofibroblast-like cells

10:30–11:00

Coffee Break

11:00–11:30

Andrew Leask

Insights into fibroblast plasticity: CCN2 is required for activation of cancer-associated fibroblasts in a murine model of melanoma

11:30–12:00

Liya Pi

CCN2 regulation by Hepatocyte Nuclear Factor 4α during liver regeneration

Zhiyong Lin (No abstract provided)

Emory University School of Medicine

12:00–14:30

Lunch

Session VI

Mechanotransduction

14:30–15:00

Katia Scotlandi

ROCK2 deprivation inhibits metastatization of osteosarcoma cells through YAP1 activity: preclinical therapeutic value of verteporin

15:00–15:30

Sunil Parapuram

Trabecular meshwork fibrosis and PI3kinase/PTEN/CCN2 pathway as drug targets

15:30–16:00

Brahim Chaqour

Role of the CCN2-YAP angiomodulatory pathway in blood vessel development and integrity

16:00–16:30

Coffee Break

Session VII

Musculoskeletal system and inflammation

16:30–17:00

Masaharu Takigawa

Role of CCN2 produced by osteocytes in bone remodeling

17:00–17:30

Satoshi Kubota

Long noncoding RNAS that regulate CCN2

17:30–18:00

Lester Lau

The role of CCN1 in inflammation and wound healing

18:00–18:30

Federica Accornero

Genetic analysis of CCN2 in skeletal muscle stress responses

Dinner

October 24, 2019

Breakfast and Departure

ABSTRACTS

Cynthia Kenyon- 7th ICCNS-SPRINGER Awardee

Calico California

Lifespan and aging were once thought to be immutable: we just wear out like old cars. This paradigm has been overturned completely by molecular genetic experiments initiated in the roundworm C. elegans and now extended to many species. Changing specific genes or cells can extend healthy lifespan dramatically while postponing age-related disease. In this lecture, the history of these discoveries and the possibilities they create for the future was discussed. Promising new research frontiers, such as the immortality of the germ lineage, were described as well.

Session I

EXOSOMES IN CANCER

Extracellular vesicles: biology and therapeutic implications

Peter Quesenberry

Brown Alpert Medical School, and Rhode Island Hospital. Providence, Rhode Island 02903

Extracellular vesicles (ECVs) are membrane enclosed biologic entities reflecting their cells of origin and variously containing protein, RNA, DNA and Lipids. Classically they have been divided into exosomes and microvesicles; the former 50–100 nm in diameter and the latter 100–1000 nm. However, because of significant overlap and heterogeneity it has been felt best to characterize them as ECVs and then describe the nature and conditions of the vesicle originating cell. They were initially thought of as cell junk but then investigated as possible vaccines especially in the cancer realm. Other work has focused on them as potential biomarkers but most recent emphasis has been on their capacity to either injure or heal normal tissue or reverse different disease states. A good example of their complex pathophysiology is represented by the actions of vesicles in experimental rodent models of pulmonary hypertension (PH). Monocrotaline injections or treatment with Sugen/Hypoxia induced PH in C57BL male mice and ECVs from lungs of these mice would induce PH in normal mice. If the mice with PH were injected with ECVs from marrow derived mesenchymal stem cells (MSC) then PH resolved. The ECV induced PH by generating “toxic” progenitors from the murine marrow which traveled to the lungs and induced vascular remodeling and PH. This opened several potential treatments for PH; administration of MSC ECV or inhibition of toxic progenitor production by the marrow cells. This latter could be obtained by administration of a Runx 1 inhibitor which blocked the endothelial to hematopoietic transition, the source of the toxic progenitors, or by administration of 100 cGy whole body irradiation (WBI) which eliminated the progenitors. Other work has demonstrated that ECVs could influence cardiac, hepatic, neural, dermatologic, bone disorders either in a positive or negative manner. Studies have shown that MSC EVs could reverse, at least in part, radiation damage to the hematopoietic and gastrointestinal systems.

Other work has focused on the use of ECVs as biomarkers. Studies of salivary vesicles from chronic concussion patients or individuals seen in an emergency room with head trauma have demonstrated biomarkers for traumatic brain injury.

Other features of ECV biology is that injury to a tissue seems to target the ECVs to that tissue, and that the evolution of ECVs show circadian rhythms.

Currently, much attention is directed to clinical trials. Major needs here are appropriate approaches to scale up production of the vesicles and to improve isolation techniques.

Genetic and epigenetic driver events as regulators of extracellular vesicle-mediated communication in cancer

Janusz Rak

Centre for Translational Biology, Faculty of Medicine, McGill University

Extracellular vesicles (EVs) constitute a unique form of intercellular communication. EVs are membranous cellular fragments released from all cell types in a manner regulated by increasingly well-defined biogenetic pathways. These pathways are implicated in creating subcellular EV generating sites (endosome, plasma membrane), their physical diversity, molecular composition (lipids, proteins, RNA and DNA) and biological activity. EVs impact cells through contact or transfer of molecular cargo. In cancer, EVs influence several key processes such as angiogenesis, myeloid and immune cell recruitment, blood clotting, metastasis and several others. Oncogenic and epigenetic driver events uniquely influence all aspect of the EV-mediated cell-cell communication in cancer (biogenesis, heterogeneity, release, molecular content, uptake by target cells). Oncogenic proteins and nucleic acids traffic between cells via EV mediated mechanisms leading to intercellular exchange of transforming signals. Circulating EVs and their educated blood cells (platelets, leukocytes) carry oncogenic molecules and have emerged as a novel liquid biopsy platform in cancer, while EV-targeting strategies and EV-inspired drug delivery nanotechnologies are being developed to treat cancer.

epigenetics of stem cell plasticity in breast cancer

Lynne-Marie Postovit

Department of Oncology, Cancer Research Institute of Northern Alberta, University of Alberta, Edmonton, Alberta, Canada T6G 2E1

Metastatic, therapy resistant breast cancers manifest a program of cellular plasticity, such that they adapt to changing microenvironments by harnessing epigenetic mechanisms. I will discuss recent work illuminating how subpopulations of breast cancer cells adapt to stresses such as hypoxia by evoking a plasticity program concomitant with the emergence of stem cell properties. This program entails broad alterations in chromatin structure and histone modifications as well as the selective mRNA translation of transcripts, such as NODAL, SNAIL and NANOG, which drive cellular reprogramming toward a more therapy resistant and metastatic stem cell-like fate. These studies provide insight into the mechanisms governing adaptive plasticity and reveal a hitherto unappreciated co-ordination between epigenetic regulation and mRNA translation.

anti-inflammatory dietary phytochemicals alter the phenotype of neuroblastoma exosomes: therapeutic implications

Herman Yeger, Reza Bayat Mokhtari, Mithuanan Ravindran, Gina Jiang, Saba Manzoor

Developmental & Stem Cell Biology, SickKids, Toronto, Ontario, Canada

Over 50% of neuroblastoma (NB) patients present with metastases at first diagnosis with the bone marrow and surrounding bone microenvironment a major site for metastasis correlating with advanced malignancy. Our earliest studies on anti-tumor phytochemicals explored flavonoids. More recently we developed a novel therapeutic approach for targeting a variety of different cancers including NB. The regimen is based on simultaneous inhibition of NB survival mechanisms with interference in pH homeostasis.

We combined a carbonic anhydrase inhibitor, acetazolamide, and the isothiocyanate,

sulforaphane (SFN), a natural HDAC inhibitor, or a semi-synthetic HDAC inhibitor, MS-275. We showed that SFN and MS-275 alone and potentiation by AZ, as in SFN + AZ and AZ + MS-275 combinations, significantly inhibited NB growth and survival in vitro and in a xenograft model. Evidence was obtained indicating targeting of the tumor initiating stem cell fraction. An exciting observation has emerged that tumors can precondition metastatic sites by disseminating microvesicular packets of proteins, miRNAs, etc., in what are called exosomes. Inflammation is now considered a critical contributor to malignant progression. Thus the anti-inflammatory activity of dietary chemopreventives, like SFN, along with its anti-tumor targeting of tumor survival mechanisms has possible implications. To isolate NB exosomes, culture supernatants from monolayer grown NB cell lines SY5Y, SKNBe2 and NUB-7 containing exosomes were differentially centrifuged to remove cells and debris, 0.22um filtered, and then purified by immunomagnetic adsorption using a primary antibody followed by a secondary antibody conjugated to magnetic beads. Primary antibodies to NB specific surface proteins GD2, NCAM and CA9 antibodies were used. CD63 served to identify plasma membranes and Bodipy TR Ceramide to label the lipid membrane component of trapped exosomes. In dose response assay using AlamarBlue all three tested anti-inflammatory agents, SFN, oleanolic acid (OA) and curcumin (Cur), showed growth inhibition of NB cells.

Treatment of NB cell lines with SFN, OA and Cur, revealed that exosomes showed a.

significant reduction in surface GD2 (disialoganglioside) after OA and SFN treatment. As membrane GD2 is the membrane target for immunotherapy of advanced NB the.

observations suggest that dietary anti-inflammatory agents may act, not only as antitumor agents, but might cooperate with immunotherapy targeting malignant progression. Since SFN is a cancer chemopreventive agent, and a chemoprotectant for normal tissues, dietary regimens offering therapeutic levels of anti-inflammatory agents may prove most valuable in eliminating and/or arresting cancer progression at earlier stages. Repurposing drugs like acetazolamide may potentiate the efficacy of dietary phytochemicals.

Session II

THERAPEUTIC CONSIDERATIONS

Involvement of Tsukushi in human neuronal disease

Kunimasa Ohta, Shah Adil Ishtiyaq Ahmad, Mohammad Badrul Anam and Naofumi Ito

Department of Developmental Neurobiology, Kumamoto University, Kumamoto 860-8556, Japan

We have been studying the molecular function of Tsukushi (TSK), which is a soluble molecule belonging to the Small Leucine-Rich Proteoglycan family (Ahmad et al., 2018). TSK is expressed in pericytes on the blood vessels and ependymal cells in the subventricular zone (SVZ) of the lateral ventricle (LV) of the mouse brain. We analyzed brain morphology and expression of marker genes in TSK knockout mice (TSK^−/−) brain. TSK^−/− mice developed hydrocephalus after birth and this phenotype became even more severe at later stages. We found aberrant cell proliferation and cell death at SVZ in TSK^−/−. Both overexpression of TSK protein using transgenic mice in TSK^−/− background and direct injection of TSK protein into the LV of TSK^−/− brain rescued the LV expansion in TSK^−/−. We performed sequencing of peripheral blood DNA from 13 hydrocephalus patients with unknown reason and found 3 heterozygous nucleotides changes within TSK coding region, which introduce missense mutations that are predicted to cause amino acid changes in TSK protein sequence. Our results suggest that TSK is involved in the pathogenesis of hydrocephalus in human patients. We would like to discuss the developmental mechanism of hydrocephalus in the absence of TSK, therapeutic potential of TSK to rescue hydrocephalus and determine the percentage of patients carrying mutated TSK gene to establish TSK as a marker for hydrocephalus diagnosis.

circulating ccns as potential biomarkers in diabetes-related complications

Sarah Parry^1,2, Xiaoyu Wang¹, Kyle WW Lloyd¹, Mark D. Gorrell³, Kathryn H. Williams^1,4, and Stephen M. Twigg^1,2

Endocrinology Dept, Central Clinical School, Faculty of Medicine and Health, University of Sydney¹; Dept of Endocrinology, Royal Prince Alfred Hospital, Sydney² Centenary Institute and Faculty of Medicine and Health, University of Sydney³, and Dept of Endocrinology, Nepean Hospital, Sydney⁴, AUSTRALIA

The metabolic disorder of type 2 diabetes commonly occurs with multiple complications, including non-alcoholic fatty liver disease (NAFLD) and its variant NASH, cardiovascular and chronic kidney disease (CKD), and distal sensorimotor polyneuropathy. Robust serological biomarkers are required to predict the development and progression of these complications. This study aimed to determine if serum levels of CCN1, CCN2 and CCN3, which are implicated in modulating inflammatory and fibrotic diseases, are potential pathophysiological biomarkers of diabetes complications. Method: Serum levels of CCN1 and CCN3 (R&D systems), and CCN2 (Cloud-Clone Corp), plus NT-proBNP, pro-fibrotic FAP and DPP4, were measured in human blood samples in n = 96 patients with type 2 diabetes, each with systematically documented complications. Serum levels of murine CCN1 (MyBioSource) were also subsequently measured in samples from n = 25 C57BL/6 male mice, subjected to high fat feeding conditions, with or without type 2 diabetes induction.

Results: In human samples, there was a significant negative correlation (Spearman) of CCN1 with waist circumference (r = −0.28, p = 0.007) and waist hip ratio (r = −0.27, p = 0.008), and a positive correlation of CCN1 with HDL-C (r = 0.32, p = 0.001). CCN1 and CCN2 each positively correlated with BNP levels (r = 0.25, p = 0.02, and r = 0.32, p = 0.002, respectively). Significantly higher CCN2 and CCN3 levels was seen in patients with lower eGFR, (each r = −0.28, p = 0.010), but not with albuminuria. CCN1 correlated with FAP (r = 0.28, p = 0.007) and DPP4 (r = 0.36, p < 0.001) measures, and CCN3 correlated with both FAP (r = 0.291, p = 0.004) and the fibrotic liver disease marker blood AST/ALT ratio (r = 0.27, p = 0.008). Furthermore, we observed a significantly lower blood murine CCN1 in type 2 diabetes-induced mice fed a high fat diet (p < 0.05).

Conclusions: These data indicate that CCN1 may be a serological biomarker of reduced metabolic syndrome presence in type 2 diabetes. Confirming work of others, increased CCN2 may reflect renal and myocardial disease in type 2 diabetes. CCN1 and CCN3 may also each have value as biomarkers for NASH. Subsequent, larger and prospective studies will be required to determine the clinical validity of these novel circulating CCN1 and CCN3 findings.

blocking ctgf/ccn2 reduces established tissue fibrosis and sensorimotor declines in a rat model of repetitive overuse injury

Barbe MF, Amin M, Michele Y Harris, Lucas J. Hobson, Geneva E. Cruz, Amanda R White, Brendan A. Hilliard, Popoff SN

Department of Anatomy and Cell Biology, Lewis Katz School of Medicine, Temple University, Philadelphia, PA

Tissue fibrosis is a pathological hallmark of overuse injuries and is considered to play key roles in the associated declines in motor and somatosensory (e.g., pain and discomfort) function. Despite this knowledge, possible treatments aimed at improving symptoms by reducing fibrosis have been understudied. Here, we tested whether inhibition of CTGF/CCN2 using this antibody reduces established muscle and nerve fibrosis and sensorimotor declines in a clinically relevant rodent model of upper-limb overuse injury. Thirty-one young adult, female, Sprague-Dawley rats performed a high repetition high force (HRHF) reaching and lever-pulling task for 18 weeks (49% of maximum pulling force, 4 reaches/min, for 2 h/day, in 30 min intervals with 1.5 h rest breaks between, 3 days/wk). Rats were then either: i) euthanised (HRHF-untreated, n = 10), ii) rested and treated twice a week for 6 weeks with an anti-CCN2 monoclonal antibody (FG-3019, FibroGen, Inc.; 40 mg/kg body wt, HRHF-Rest/anti-CCN2, n = 6), or iii) rested and treated twice a week for 6 weeks with a human IgG (FibroGen,; 50 microl/injection, i.p.; HRHF-Rest/IgG, n = 5). A separate age-matched “uninjured” (i.e., did not perform the training/task) control group (n = 10) was included for comparisons. HRHF-untreated and HRHF-Rest/IgG rats showed significantly reduced forearm grip strength, compared to Control rats Collagen type 1 and type 3, CTGF/CCN2 and TGFbeta1 were higher in the flexor digitorum muscles of HRHF-untreated and HRHF-Rest/IgG rats, compared to the age-matched control rats, as were serum PINP (pro-collagen I intact N-terminal protein) levels. These muscle fibrogenic responses were reduced in HRHF-Rest/anti-CCN2 rats. Functionally, rats treated with anti-CCN2 also showed a full restoration of HRHF-induced grip strength declines. Neuropathology was also evident in HRHF-untreated and HRHF-Rest/IgG rats’ median nerves, in the form of increased noxious cold aversion (a sign of neuropathic pain), decreased sensory conduction velocity, increased extraneural collagen deposition and increased degraded myelin basic protein immunoexpression, compared to control rats (p < 0.05 each). HRHF-Rest/anti-CCN2 rats showed a full restoration of median nerve conduction velocity and similar thermal sensitivity (aversion) as that in control rats. Sensitivity to heat was similar across groups, as were muscle MMP2, MMP9, and CCN3 levels. We found that systemic injections of a monoclonal antibody directed against CCN2, a drug known as FG-3019 or pamrevlumab, effectively reduced several tissue and serum fibrogenic protein levels, collagen deposition, restored grip strength and nerve conduction velocity declines, and ameliorated myelin degradation and somatosensory changes (i.e., noxious cold thermal sensitivity). Together, our working hypothesis is that fibrotic processes underpin the observed sensorimotor declines by compressing, and thereby impeding the excursion of tissues during movement, and that inhibiting fibrotic processes can reverse these effects.”

Acknowledgements This research was supported by NIH-NIAMS to MFB. Anti-CCN2 (FG-3019) drug was a gift from FibroGen, Inc.

harnessing the power of ccn signaling for novel therapeutic and diagnostic applications

Bruce L. Riser BLR Bio LLC, Kenosha, WI and North Chicago IL, USA, Rosalind Franklin University of Medicine and Science, Center for Cancer Cell Biology, Immunology and Infectious Disease, North Chicago, Illinois, USA

Nearly half of the deaths in the developed world are associated with fibrosis, which can affect any organ; and the fibrotic stroma in solid tumors is likely key to cancer progression and metastases. In spite of this knowledge, there is no FDA approved drug specifically designed to totally arrest or reverse fibrotic disease, or to treat cancer by attacking the stroma specifically. Over the last two decades, the matricellular signaling protein CCN2, as an established pro-fibrotic factor, has emerged as a highly promising target for the treatment of fibrotic diseases and cancer and has been our focus. In 2009 we showed that another CCN family member, CCN3, is downregulated by TGF-beta and acts as a natural antagonist of CCN2 and fibrosis development using a kidney disease cell model (Riser, et al., Amer. J. Path., 2009), work also supported by others. More recently we published a proof-of-principle study in a “best” rodent model of human type 2 diabetic nephropathy and obesity, where treatment with rhCCN3 both halted and reversed key hallmarks of established renal fibrosis and disease progression (Riser, et al., Amer. J. Path, 2014). To minimize the potential off-target effects of a multifunctional protein therapeutic, we identified key regions of the CCN3 protein responsible for the specific anti-fibrotic and anti-cancer activity, then developed stable therapeutic small peptide drugs. Using animal models of multiple fibrotic diseases including diabetic kidney disease, non-alcoholic fatty liver disease, scleroderma, and solid tumor cancers, we have shown the potential of our lead peptides to threat and even reverse fibrosis, in some cases regaining organ function. We have also shown the safety of these drugs in animals. BLR Bio has also capitalized on the criticality of these molecules in the initiation and development of disease and has developed a novel diagnostic assay focused on accomplishing: 1) earlier diagnosis, 2) more accurate staging of disease, and 3) early determination of the response to therapy. We think such a diagnostic will also have high value in clinical trials for new drugs in chronic kidney disease, scleroderma and liver fibrosis, and possibly in solid tumor cancers. Current studies with our collaborators are focused on not only proof-of-efficacy, but also the mechanisms of action, and the similarities between different fibrotic diseases and cancers. Our next goal is the demonstration of safety and efficacy in patient clinical trials.

therapeutic peptides based on ccn3 treat systemic sclerosis in a mouse model of the disease

Alex Peidl¹, Bruce Riser^2,3, and Andrew Leask¹

¹Department of Physiology and Pharmacology University of Western Ontario London, ON, Canada

²Department of Physiology Rosalind Franklin University of Medicine and Science. Chicago, IL, USA ³BLR Bio LLC Kenosha, WI, USA

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs, often resulting in severe disability and high mortality. Additionally, the etiology of the disease is largely unknown, hence, no effective disease therapy has been developed. In the connective tissue of fibrotic lesions, fibroblasts differentiate into myofibroblasts, which are characterized by excessive collagen secretion and remodelling, abnormally active adhesive signalling, and overexpression of highly contractile α-smooth muscle actin (αSMA). Substantial evidence suggests the CCN family of matricellular proteins are important contributors to the activation and sustained activity of myofibroblasts in human fibrotic disease. CCN2 is upregulated during wound healing and is characteristically overexpressed in fibrotic disease, as illustrated by the presence of CCN2 in biological fluids of SSc patients. CCN2 is a key selective downstream mediator of the fibrotic response in many organs including the skin, lung, kidney and liver. Furthermore, CCN2 expression by fibroblasts is required in mouse models of SSc skin fibrosis. CCN3 has been shown to be reciprocally regulated to CCN2 in certain cell types including mesangial cells and dermal fibroblasts, suggesting that CCN3 may have effects opposing CCN2. Our corporate partner, BLR Bio, has shown that intraperitoneal injections of recombinant human CCN3 blocks fibrosis in a mouse model of diabetic nephropathy. These discoveries led BLR Bio to develop a proprietary peptide (designated as BLR-200) based on CCN3 and its interaction with other CCN proteins, which might be useful in treating SSc fibrosis. Therefore, the goal of this study was to determine if this peptide is useful in treating mouse models of SSc fibrosis. Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin once daily for 28 days. Alongside bleomycin injections, mice were either treated with a scrambled vehicle control or BLR-200 peptide. At the end of the experiment, mice were investigated for changes in dermal thickness, collagen content, and expression of fibrotic markers such as αSMA, CCN2, and integrin alpha 11 (ITGA11). Results indicate that BLR-200 treatment was able to prevent increases in dermal thickness and changes in collagen content induced by bleomycin treatment. Furthermore, treatment with BLR-200 was able to reduce the number of αSMA-positive cells, as well as prevent induction of CCN2 and ITGA11. These data indicate that peptides based on CCN3 and its interaction with pro-fibrotic CCNs may represent a novel therapeutic approach for treatment of SSc fibrosis.

Session III

CANCER

ccn1-induced age-related alterations of the dermal microenvironment promote skin cancer development

Taihao Quan, Yaping Xiang, Yingchun Liu, Zhaoping Qin, Andrzej A Dlugosz, John J Voorhees, Gary J Fisher

Department of Dermatology, University of Michigan, Ann Arbor, MI USA

Keratinocyte skin cancer, the most common type of carcinoma in the United States, is increasingly common in older individuals. Although accumulation of mutations caused by chronic exposure to solar ultraviolet (UV) radiation is a significant contributor to skin cancer, it does not fully explain the high prevalence of skin cancer in the elderly. One emerging explanation for this enigma is that initiation and/or progression of epithelial skin cancer requires both UV irradiation-induced mutations and a pro-tumor dermal microenvironment.

Human skin is composed of dense, collagen-rich extracellular matrix (ECM) that is essential for skin structure and function. The ECM is synthesized and remodeled by dermal fibroblasts. During aging, fibroblasts produce less collagen and other ECM proteins, more enzymes that degrade the ECM (matrix metalloproteinases, MMPs), and more pro-inflammatory mediators. These alterations of collagen-rich ECM impair the structural integrity and function of the skin.

We have previously reported that many age-related, deleterious alterations of the dermal ECM are driven by the actions of the matricellular protein CCN1. Elevated expression of CCN1 by dermal fibroblasts reduces production and increases degradation of the ECM, thereby promoting age-related skin thinning and fragility. Elevated CCN1 also increases expression of pro-inflammatory cytokines, which contributes to the generation of an inflammatory dermal microenvironment (inflammaging).

We have generated a transgenic mouse model in which CCN1 is expressed in fibroblasts, the source of elevated CCN1 in human skin, under the control of a fibroblast-specific promoter Col1a2 promoter and enhancer. Col1a2-CCN1 transgenic mice exhibit significantly accelerated appearance of changes seen in aged human skin, characterized by thin and fragmented dermal ECM. Mechanistically, the aged dermal phenotype reflects reduced collagen production, due to impaired TGF-β signaling, and increased collagen fragmentation, due to elevated expression of multiple MMPs. Importantly, CCN1 transgenic mice or CCN1-expressing dermal fibroblasts display a high propensity for skin tumor formation in three skin tumor models; 1) two-stage chemical carcinogenesis, 2) Transgenic HRas-driven epithelial skin tumorigenesis, and 3) xenograft tumor. Our Col1a2-CCN1 mouse model demonstrates that alterations of the dermal microenvironment, which occur in aged human skin, promote tumorigenesis in the overlying epidermis. We propose that age-related alterations in the dermal ECM, leading to a pro-tumorigenic microenvironment, partially explain why skin cancer is so common in the elderly.

ccn6 functions to regulate mitochondrial metabolism in breast cancer

Mai Tran, Shoshana Leflein, Boris Burman, Maria E. Gonzalez, Celina G. Kleer

Department of Pathology and Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109

Metaplastic breast carcinoma (mBrCA) is an aggressive subtype of triple negative breast carcinoma with frequent distant metastasis. Our lab has shown that CCN6 is a secreted protein that regulates breast epithelial cell phenotype. Mammary epithelial-specific CCN6 knockout (KO) mice (MMTV-cre;ccn6^fl/fl) develop mammary tumors recapitulating human MBC, suggesting the critical role of CCN6 in suppressing the formation of mBrCA. We set out to test the hypothesis mammary glands from MMTV-cre;ccn6^fl/fl mice may exhibit gene expression changes before tumor develops that may promote neoplastic transformation. Gene expression profiles of normal mammary tissues from MMTV-cre;ccn6^fl/fl with wild type virgin mice at 8 weeks of age showed deregulated expression of mitochondrial metabolism genes. We validated the expression of the top deregulated genes in MMTV-cre;ccn6^fl/fl mammary epithelium compared to controls by quantitative RT-PCR and Western blot. Mitochondria stress tests (Seahorse) studies showed that CCN6 downregulation in human mammary epithelial cells led to a significant reduction of maximal respiratory capacity measured as oxygen consumption rate compared to controls. The opposite effect was observed upon CCN6 overexpression in MDA-MB-231 breast cancer cells. Using immunofluorescence and cell fractionation experiments, we found that CCN6 localizes to the mitochondria of breast cancer cells, in addition to being secreted in the extracellular medium. Treatment of CCN6 KO mice with recombinant CCN6 protein was sufficient to rescue the expression of important mitochondrial function regulators. Our data highlight a novel function of CCN6 in mitochondrial activity, which is being currently investigated in the lab. Ongoing experiments are designed to examine the consequences of the mitochondrial regulatory effect of CCN6 in mBrCA development and progression.

role of ccn1 in growth factor responses in breast and prostate cancer cells

Pravita Balijepalli, Mary P. Nivison, and Kathryn E. Meier

Department of Pharmaceutical Sciences College of Pharmacy and Pharmaceutical Sciences Washington State University Spokane, WA, USA

Multiple studies have established that the matricellular protein CCN1/Cyr61 is rapidly induced at the transcriptional level when certain cancer cells are incubated with growth factors. In addition, it is also clear that CCN1 itself, which is secreted, can act in an autocrine manner to promote cancer cell proliferation and migration. However, it has not yet been established to what extent CCN1 induction is required for the actions of growth factors. To this end, our group is examining the role of CCN1 in growth factor-mediated signal transduction in breast and prostate cancer cells. We have previously shown that CCN1 protein expression is induced by both lysophosphatidic acid (LPA) and epidermal growth factor (EGF) in human prostate and breast cancer cell lines. We have also demonstrated that activation of free fatty acid receptors (FFARs) by omega-3 fatty acids and synthetic FFAR agonists can suppress responses to LPA and EGF in the same cell lines. Recent data confirm that LPA and EGF induce CCN1 in MDA-MB-231 breast cancer cells, and show that this response can be detected using immunolocalization of CCN1 by confocal microscopy. Co-incubation of cells with FFAR agonists inhibits EGF-induced CCN1 induction in these cells. These studies are being extended to additional breast cancer cell lines. The overall role of CCN1 in growth factor-mediated proliferation remains to be elucidated; the establishment of multiple cellular models is anticipated to facilitate this. Mechanistic investigation. (Supported by the ASPET David Lehr Award and by the WSU College of Pharmacy and Pharmaceutical Sciences).

Session IV

NEW LOOKS INTO OLD PROBLEMS:

LOST IN JCCS: A ROADMAP TO CCN UFOs

Bernard Perbal

International CCN Society, Paris France

Each module of the CCN proteins is derived from exon shuffling that distributed four module-encoding sequences in a set of proteins that evolved separately, as shown by the fact that they are not playing redundant functions and are not expressed at the same time in the same tissues.

Yet, the modules contained in each of the proteins, have maintained the capacity to interact with some common ligands and receptors. Furthermore, other regulatory proteins functionally unrelated to the CCN family of proteins, do contain the same type of module, albeit showing significant physical differences.

Two modules in the CCN proteins contain motifs that have been involved in dimerization (cystine knot within the C-terminal module), and mutimerization (VWC motif in the second module). It is therefore possible that the modules present in CCN proteins not only allow the CCN protein to dimerize, as experimentally shown, but also to engage into heterotypic dimers and multimers with other regulatory proteins (Perbal 2013).

As previously discussed the combination of modules from CCN proteins with functionally similar modules in other proteins might influence, or modulate the activities of the complex. Furthermore, the CCN5 protein that is lacking the CT module, present in the other CCN proteins, might play the role of a dominant inhibitor, by combining with other CCN proteins or other CCN-like-containing regulators.

Altogether, the temporal regulation of CCN protein expression added to the potential to interact with others in a homotypic or heterotypic fashion, constitutes a very flexible set of combinatorial events that likely dictate the robust functionality of these protein complexes.

From the point of view of quantum mechanics, a group of Qubits that can exist simultaneously in multiple states provides way more processing power when they are connected than the same number of binary bits. Similarly, the hierarchy of complexity levels supplied by the CCN protein layers would provide significantly more regulatory biological power than the same number of individual activities.The complexity of CCN biological properties needs to be tackled from this kind of new angle.

Although it may seem a wish made to Santa Clause, I strongly believe in the strength of the combinatorial events within this kind of integrated system.

One can predict that global approaches that will take into account the six CCN proteins as a whole integrated system, should prove much more fruitful than the present way of considering isolated CCN proteins on their own.

Perbal B (2013) CCN proteins: A centralized communication network. J Cell Commun Signal 7(3):169–177.

ccn proteins are preproproteins that require proteolytic activation

Ole J. Kaasbøll, Vivi T. Monsen, Sima Zolfaghari, Else Marie V. Hagelin, and Håvard Attramadal

Institute for Surgical Research, Oslo University Hospital and University of Oslo, NO-0424 OSLO, Norway

CCN proteins are defined as matricellular proteins conceptually interacting with a wide array of proteins in the extracellular matrix as well as acting as independent signaling molecules. However, the biologically active signaling entities of CCN proteins have been elusive. We recently reported that CCN2 is synthesized and secreted as a preproprotein that is auto-inhibited by its two N-terminal domains and requires proteolytic processing in order to become biologically active. Furthermore, we demonstrated that that the proteolytic fragment comprising domains III (thrombospondin type 1 repeat homology domain) and IV (cystine knot homology domain) appears to convey all biologically relevant activities of CCN2. The C-terminal fragment of CCN2 (domains III and IV) also formed homo-dimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. Here we provide evidence that the carboxyl terminal fragments of CCN1 (domains III and IV), CCN3 (domains III and IV), and CCN5 (domain III) are also biologically active signaling entities. CCN5 (domain III) was shown to inhibit AKT (Ser 473) phosphorylation and inhibit mammosphere formation of MCF-7 mammary cancer cells, i.e. the converse actions of CCN1, CCN2 and CCN3. Thus, CCN5 (domain III) is an antagonist of 4-domain CCN proteins. Identification of the biologically active entities of CCN proteins may facilitate research on the functions of these proteins in physiologic as well as in pathophysiologic processes. We also demonstrate use of fusion protein technology that may simplify production and purification of biologically active CCN proteins as well as facilitate physiologic studies in vivo.

Session V

STEM CELL ACTIVITY

investigating the role of ccn3 during central nervous system myelination and remyelination

Nira de la Vega Gallardo¹, Rosana Penalva¹, Marie Dittmer¹, Michelle Naughton¹, John Falconer¹, Jill Moffat¹, Zhiyong Lin², Bernard Perbal³, Rebecca J. Ingram¹, Emma Evergren¹, Denise C. Fitzgerald¹

¹ The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Northern Ireland, UK

² Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA; Harrington Heart and Vascular Institute, University Hospitals Cleveland Medical Centre, Cleveland, Ohio, USA

³ Université Côte d’Azur, CNRS, GREDEG, Nice, France; International CCN Society, Paris, France

Multiple sclerosis is a demyelinating disease of the central nervous system (CNS) characterised by immune-mediated destruction of myelin-producing oligodendrocytes. While there is a plethora of disease-modifying therapies to reduce relapses, there are currently no available treatments to boost oligodendrocyte and myelin regeneration. Regulatory T cells secrete CCN3, a matricellular protein that promotes oligodendrocyte progenitor cell (OPC) differentiation and myelination in murine brain slice cultures. These findings identified CCN3 as a candidate of interest in CNS myelination and therefore we sought to investigate the expression and role of CCN3 during myelin development and regeneration. Oligodendrocytes were quantified in the corpus callosum in the brain, and the ventral white matter in the spinal cord of wild type (wt) and ccn3-deficient (ko) mice. We found no difference in the number of oligodendrocytes between groups, suggesting that ccn3 is not necessary for normal oligodendrocyte development. To investigate the role of ccn3 in opc differentiation during remyelination, wt and ccn3 ko mice were injected with lysolecithin in the ventral white matter of the spinal cord, creating a focal demyelinated lesion. There was no difference in the number of proliferating opcs at 5 days post lesion (dpl), or differentiated oligodendrocytes at 14 dpl in wt and KO groups. In a model of cuprizone-induced demyelination, there was no significant difference in the number of oligodendroglia or differentiated oligodendrocytes in the corpus callosum during remyelination in WT and CCN3 KO mice. Overall, these results suggest that CCN3 is not necessary for efficient OPC proliferation or differentiation in the CNS. However, interestingly CCN3 was transiently upregulated in the brain and spinal cord during demyelination and remyelination respectively. Furthermore, around 40% of CCN3-expressing cells in the remyelinating spinal cord were oligodendroglia. Ongoing work is examining whether CCN3 is required for efficient myelin wrapping in the spinal cord, as well as identifying the other cellular sources of CCN3 during CNS demyelination and remyelination.

ctgf/ccn2 is required for recovery of hematopoiesis after stress-induction in vivo

Sandra Romero Marquez¹, Franziska Hettler¹, Anna Hasenkopf¹, Theresa Sippenauer¹, Christina Schreck¹, Renate Hausinger¹, Andrew Leask², Roel Goldschmeding³, Rouzanna Istvanffy¹, and Robert A.J. Oostendorp¹

¹Medical Clinic and Policlinic for Internal medicine III, Klinikum Rechts der Isar der Technischen Universität München, Germany

²Departments of Physiology and Pharmacology and Dentistry, University of Western Ontario, London, ON, Canada

³Department of Pathology, University Medical Center Utrecht, the Netherlands

In the bone marrow (BM) reside hematopoietic stem cells (HSCs) which are on top of the hierarchy of the hematopoietic system. Proper signaling is essential in maintaining HSCs during hematopoietic stress as such caused by infection, blood loss or therapeutic irradiation. HSC maintenance, self-renewal and differentiation are controlled by interactions with the BM microenvironment, also termed the niche. In studies aimed at dissecting the mechanisms of cross-talk between niche cells and HSC we found Connective Tissue Growth Factor (CTGF) to be required for the quality and self-renewal of HSCs in vitro.

Here, we investigated the in vivo function of CTGF during adult hematopoiesis. As Ctgf knockout mice are embryonically lethal we studied a tamoxifen inducible Ctgf-knockout mouse model based on Rosa-CreERT2;Ctgf-lox mice.

In accordance with the role of CTGF as an early response gene to stress we found decreased myeloid progenitors (MPs) as well as Gr1+ granulocytes in CtgfD/D mice compared to control mice five and 14 days after sublethal irradiation (4.5 Gy). More importantly, the HSCs present in the mice recovered from irradiation, showed strongly reduced ability to engraft lethally irradiated recipients. These results indicate that CTGF is required for recovery of full HSC activity after (irradiation-induced) stress in vivo.

To determine whether this loss of HSC activity was due to environmental CTGF rather than Ctgf expressed by HSCs and their progeny, we transplanted WT HSCs into a CtgfD/D microenvironment. These experiments showed that CTGF from the niche is necessary for normal HSC regeneration in vivo. As in the HSCs recovered from irradiated CtgfD/D mice, we found that in CtgfD/D recipients, decreased engraftment was accompanied by decreased generation of activated HSCs, as well as decreased numbers of myeloid cells, particularly Gr1+ granulocytes.

Molecular mechanisms involved in loss of HSC activity and granulocytic exhaustion, like increased G0/G1 state of cell cycle in HSCs from CtgfD/D mice with increased acetylated P53, increased P27KIP1 and decreased β-CATENIN levels strongly suggest aberrant proliferation of HSCs after sublethal irradiation in CtgfD/D.

Our results demonstrate that CTGF is required for regeneration of active HSCs after induction of severe stress. Furthermore, our findings suggest, that extrinsic CTGF is essential for granulopoiesis and is contingent on β-CATENIN and P27KIP1-dependent cell cycle control. Our results help to unravel mechanisms of physiological tissue regeneration and show that CTGF and its signaling pathway are possible targets with the aim to improve regeneration.

insights into fibroblast plasticity: ccn2 is required for activation of cancer-associated fibroblasts in a murine model of melanoma

Matthew Tsang¹, Katherine Quesnel², Krista Vincent^3,4, James Hutchenreuther¹, Lynne-Marie Postovit³and Andrew Leask^1,2

¹Departments of Physiology and Pharmacology and ²Dentistry, Dental Sciences Building, University of Western Ontario, London, ON, Canada N6A 5C1³Department of Oncology, University of Alberta, Edmonton, AB, Canada⁴ Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada

Tumor stroma resembles a fibrotic microenvironment in that it is characterized by the presence of myofibroblast-like cancer-associated fibroblasts (CAFs) that express alpha-smooth muscle actin (α-SMA) and CCN2. In wild type mice injected with melanoma, we show that the stem cell transcription factor Sox2 is not only expressed by tumor cells but is also induced in tumor stroma CAFs derived from synthetic fibroblasts, which were labeled postnatally with green fluorescent protein using mice expressing a tamoxifen-dependent Cre recombinase expressed under the control of a fibroblast-specific Col1a2-derived promoter/enhancer. Conversely, fibroblast activation is impaired in mice with a fibroblast-specific deletion of CCN2, associated with a marked reduction in expression of α-SMA and Sox2. As an in vitro model, we cultured multipotent Sox2-expressing skin-derived precursor (SKP) spheroids from murine back skin. Using lineage tracing and flow cytometry, we found that ~40% of SKPs are derived from a Col1a2 origin and acquire 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 ITGA11-expressing subset of cancer-associated fibroblasts that negatively associated with survival. Collectively, these results suggest that synthetic dermal fibroblasts possess a previously unheralded in vivo and in vitro plasticity, 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.

connective tissue growth factor (ctgf/ccn2) regulation by hepatocyte nuclear factor 4α during liver regeneration

Junmei Zhou^1,2,&, Xiaowei Sun^1,3,&, Lu Yang⁴, Liqun Wang⁵, Jinhui Wang⁴, Qi Cao⁶, Lizi Wu⁷, Andrew Bryant⁵, Chen Ling^1,8#, Liya Pi^1,#

¹Department of Pediatrics, ⁵Department of Medicine, ⁷Department of Microbiology& Molecular Genetics, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.²Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, University of South China, Hengyang 421001, China. ³Institute of Pathology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. ⁴Integrative Genomics Core, Beckman Research Institute of the City of Hope, Duarte, CA, USA. ⁶Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA. ⁸State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China

The liver is a critical metabolic and digestive organ. It exposes to exogenous and endogenous toxins daily, such as alcohol, viruses and drugs. Thus, an extraordinary ability to regenerate has been evolved. However, abnormal liver repair characterized myofibroblast activation and excessive collagen deposition often occurs as a common and final pathway in chronic liver disease. Understanding molecular mechanism governing the balance between regeneration and fibrosis is pre-requisite for many therapeutic interventions that optimize regenerative outcome and avoid scar formation after liver injury. It has been known that liver regeneration requires dynamic expression of hepatocyte nuclear factor (hnf)4α, yes-associated protein (yap), and transforming growth factor (tgf)-β. Failure to regenerate causes loss of hnf4α + hepatocytes and overproduces fibrotic signals including yap, tgf-β, and their target gene connective tissue growth factor (ctgf/ccn2) in the cyr61/ctgf/nov protein family. Here, we investigated hnf4α regulation of ctgf/ccn2 during liver regeneration. Gene expression was examined following three types of liver injuries, including partial hepatectomy as well as carbon tetrachloride intoxication with or without ethanol pre-exposure. Liver regeneration was compared in animals that contained sirna-mediated hnf4α knockdown and cre-mediated ctgf/ccn2 conditional deletion. We observed that ctgf/ccn2 induction was associated with hnf4α decline, nuclear yap accumulation, and tgf-β upregulation during early stage of liver regeneration. The ctgf/ccn2 promoter contained hnf4α binding sequences that overlapped with cis-regulatory elements for yap and tgf-β. Ctgf/ccn2 loss attenuated inflammation, hepatocyte proliferation and collagen synthesis, whereas hnf4α knockdown enhanced ctgf/ccn2 induction and liver fibrogenesis. These findings demonstrated critical roles of ctgf/ccn2 in liver regeneration, and highlighted the fine-tuned regulation of ctgf/ccn2 by hnf4α antagonism of yap and tgf-β activities to balance regenerative and fibrotic signals after liver injury. The knowledge we obtained would provide deep insights into molecular mechanism of liver regeneration and may help therapeutic intervention against fibrotic reactions during chronic liver disease.

ccn4 levels are induced by cytokines in rotator cuff disease tenofibroblasts

David O’Gorman

Lawson Research Institute, University of Western Ontario, London, ON, Canada

Rotator cuff disease (RCD) is a common and debilitating disorder characterized by the spontaneous tearing of rotator cuff tendons. The pathophysiology of this condition remains poorly understood and definitive identifiers of tenofibroblasts derived from diseased tendons have remained elusive. Mechanosensitive tenofibroblast progenitors have been previously reported to facilitate tendon repair. While tendons under homeostasis are relatively homogenous at the cellular level, torn tendons contain widely variable levels of inflammatory infiltrate, making the cellular sources of gene expression changes in these tissues difficult to discern. We have set out to develop physiologically relevant, reproducible 3D in vitro models of RCD to study its pathogenesis. In this study, we derived and compared low passage primary human tenofibroblasts derived from the torn supraspinatus tendons of patients with RCD, intact subscapularis tendons in either age-matched (ARC) or young and healthy (NRC) controls, cultured on type-1 collagen substrates and treated with pro- and/or anti-inflammatory cytokines. Analyses were performed under either routine laboratory (21%) or physiological (2%) oxygen levels to assess any confounding effects of oxygen on tenofibroblast metabolic activity and gene expression. The expression levels of a subset of tissue repair-associated genes were assessed before and after exposure to pro- and anti-inflammatory cytokine mixtures, separately and in combination. Tenofibroblast cultures in 21% oxygen consistently exhibit decreased metabolic activity and increased gene expression levels relative to cultures under 2% oxygen. ARC, NRC and RCD tenofibroblasts expressed the progenitor cell biomarkers NANOG and POU5F1 (OCT4) under either oxygen condition. While no overt differences in gene expression were observed between unstimulated tenofibroblasts derived from healthy or diseased tendons under either oxygen level, sequential pro- and anti-inflammatory cytokine treatments significantly increased the expression levels of WISP1, encoding CCN4, in RCD cells relative to ARC and/or NRC cells. Parallel increases in ACTA2 expression, encoding the myofibroblast-associated protein α-smooth muscle actin and decrease in SCX expression, encoding the tenocyte-associated basic helix-loop-helix transcription factor Scleraxis, were also evident after cytokine challenges. We interpret our findings to indicate that challenging RCD tenofibroblasts with pro- and/or anti-inflammatory cytokines can reveal changes in gene and protein expression in RCD tenofibroblasts that are not evident under unstimulated conditions. A detailed understanding of the roles of CCN4 in tendon homeostasis and repair may shed new light on the pathophysiology of RCD.

Session VI

MECHANOTRANSDUCTION

role of the ccn2-yap angiomodulatory pathway in blood vessel development and integrity

Brahim Chaqour

Department of Cell Biology and Ophthalmology, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA

The formation of a functional vascular network during development involves coordinated signaling between vascular cells and the extracellular matrix (ECM), a network of proteins and glycans providing not only anchorage points to the cells but also chemical and mechanical cues for directional migration, morphogenesis, and stability. Dysregulation of these signals plays an important role in the pathogenesis of vascular diseases associated with diabetes, inflammation and ischemia. The cellular communication network factor 2 (aka connective tissue growth factor, CTGF) is a secreted ECM protein essential for skeletal development and perinatal viability. Despite its prominent expression in blood vessel cells, the mechanisms whereby CCN2/CTGF regulates vessel formation and regeneration are incompletely understood. Herein, we used mouse genetics to study the regulation and function of CCN2/CTGF in the postnatal retinal vascular development mouse model. Using CCN2/CTGF-GFP BAC transgenic mouse, as a proxy marker for endogenous CCN2/CTGF, we found a heterogeneous spatiotemporal expression pattern of CCN2/CTGF in developing vessels. CCN2/CTGF is predominantly produced by endothelial and pericytes of sprouting and fully formed blood vessels. CCN2/CTGF expression is also dynamic within subsets of retinal glial and microglial cells as well as differentiating retinal ganglion neurons. Global and endothelial-specific deletion of CCN2/CTGF using a Cre-lox system markedly reduced vessel area, microvessel density, and vascular branching. While arteriovenous differentiation and vascular expansion to the retinal edge is not affected, the vascular network becomes partially shaped into rudimentary arterioles, venules, and capillaries. Importantly, systemic injection of fluorescently-labeled albumin to CCN2/CTGF mutant mice leads to diffuse and patchy hyperfluorescence in the extravascular space indicating breach of the blood retinal barrier. Genome-wide analysis of the retina transcriptome of wild-type and CCN2/CTGF mutant mice identified numerous CCN2/CTGF target genes that include cytoskeletal, extracellular matrix, integrin and transcription regulators including the yes-associated protein YAP, a transcriptional coactivator that regulates cell growth and organ size. YAP deficiency in mice altered vascular growth and barrier function mimicking a CCN2/CTGF-like loss of function phenotype. Adeno-associated virus-mediated repression of YAP, at least in part, rescued the vascular phenotype associated with CCCN2/CTGF deficiency. Thus, the CCN2/CTGF and YAP “regulomes” promotes normal expansion, cell-cell interactions and mechanical stability of developing blood vessels.

trabecular meshwork fibrosis and pi3-kinase/pten/ccn2 pathway as drug targets

Geetha Subramanian, Venkat Arutla, Sebastian Aniol, Sandra Gijidharan, Sunil Parapuram, and Andrew Leask

Departments of Dentistry, Pathology and Laboratory Medicine. University of Western Ontario, London, ON Canada

Increased intraocular pressure (IOP) triggers optic nerve degeneration and loss of vision in high-tension primary open-angle glaucoma (POAG) patients. The increase in IOP is mainly due to increased resistance to the aqueous humor outflow through the trabecular meshwork (TM) tissue caused by fibrosis [excess deposition of extracellular matrix (ECM)] in the TM tissue. An increase in the levels of active transforming growth factor-β (TGF-β) in the aqueous humor of high-tension POAG patients is implicated in the fibrosis of the TM. We have previously found that conditional deletion of phosphatase and tensin homolog (PTEN) gene in fibroblasts of mice caused fibrosis of the skin (Parapuram et al., 2011) and that the fibrosis induced by the deletion of the Pten gene is mediated by CCN2 (Liu et al., 2013; Parapuram et al., 2015). Based on these studies, we recently demonstrated that the TGF-β induced fibrosis in TM cells by phosphorylating PTEN, thereby rendering PTEN inactive. We also found that the TGF-β-induced fibrosis and inhibition of PTEN activity can be prevented by inhibiting PI3-kinase signaling (Tellios et al., 2017). Given the importance of PI3-kinase in cellular biology, pan-inhibition of PI3-kinase could have toxic side effects. Thus, in this study we have identified the specific PI3-kinase p110 catalytic unit isoform(s) that regulate PTEN activity as it could lead to a more efficient and less toxic treatment to attenuate fibrosis of the TM tissue.

Human TM cells in low serum medium were pre-treated with a p110 isoform (α, β, γ, δ) specific inhibitor for 30 min before treatment with active TGF-β2. Protein was extracted at 24 h and the effect of inhibition of a specific p110 isoform on the expression and phosphorylation of PTEN and the expression of fibrotic marker collagen I was determined by Western blot. We have found that AS605240, a p110γ specific inhibitor, prevented TGF-β-induced collagen expression and PTEN phosphorylation in TM cells at 24 h. TGX-221, a p110β inhibitor also had a similar effect. However, inhibition of p110α with A66 or p110δ with CAL101 did not prevent the TGF-β-induced increase in collagen expression or PTEN phosphorylation.

Only PI3-kinase isoforms p110β and p110γ significantly affected PTEN activity and collagen expression in TM cells. Inhibition of these PI3-kinase isoforms could attenuate fibrosis in the TM of high-tension POAG patients and reduce IOP. Further studies are required to elucidate the effect of these isoforms on downstream targets of PTEN such as CCN2.

Session VII

MUSCULOSKELETAL SYSTEM AND INFLAMMATION

role of ccn2 produced by osteocytes in bone remodeling

Takashi Nishida,^1,2 Satoshi Kubota,^{1, 2} Hideki Yokoi,³ Masashi Mukoyama,⁴ and Masaharu Takigawa²

¹Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; ²Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan; ³Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; ⁴Department of Nephrology, Kumamoto University Graduate School of Medical Science, Kumamoto, Japan

Bone is composed of three types of cells (bone-forming osteoblasts, bone-resorbing osteoclasts and osteocytes) and bone matrix. Among the cells, osteocytes are the most abundant and embedded in bone matrix and are believed to control bone remodeling. We previously reported that CCN2 promoted both osteoblast differentiation and osteoclastogenesis, but it was unknown how CCN2 plays in osteocytes in bone. In this study, we evaluated the possible involvement of osteocyte-derived CCN2 on bone remodeling. To mimic the bone matrix environment, osteocytic MLO-Y4 cells had been embedded in collagen-gel with recombinant CCN2 (rCCN2), and mouse macrophage-like RAW264.7 cells were inoculated on the gel and treated with receptor activator of NF-κB ligand (RANKL). As a result, NFATc1 and cathepsin K (CTSK) productions were increased in the combination of RAW264.7 and MLO-Y4 cells treated with rCCN2, compared with the combination without rCCN2. Next, we isolated an osteocyte-enriched population of cells and osteoclast progenitor cells from wild type and tamoxifen-inducible Ccn2-deficient (KO) mice and performed similar analysis. NFATc1 and CTSK productions were decreased in the KO osteocyte-enriched population at 6 months after the tamoxifen injection, regardless of the origin of the osteoclast progenitor cells. CTSK production was rather increased in KO osteocytes at 1 year after the tamoxifen injection. Finally, osteoblastic MC3T3-E1 on collagen-gel containing MLO-Y4 cells together with rCCN2 revealed the up-regulation of osteoblastic marker genes. These findings indicate that CCN2 supplied by osteocytes regulates both osteoclastogenesis and osteoblast differentiation, suggesting that CCN2 plays essential roles in bone remodeling.

long noncoding rnas that regulate ccn2

Satoshi Kubota¹, Takanori Ishikawa¹, Tomomi Mizukawa¹, Sei Kondo¹, Abdellatif El-Seoudi¹, Takashi Nishida¹, Takako Hattori¹, Kazumi Kawata¹, Takayuki Furumatsu², Takeshi Takarada³, Mitsuaki Ono⁴, Masaharu Takigawa⁵

¹Department of Biochemistry and Molecular Dentistry, ²Orthopaedic Surgery, ³Regenerative Science, ⁴Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, ⁵Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan

Long noncoding RNAs (lncRNAs) are defined as RNAs with more than 200 nucleotides, which do not possess significant protein information. More than 30,000 active lncRNA genes are present in a human genome, and at least 20,000 lncRNAs are estimated to play significant biological roles in human cells. Indeed, the number of lncRNA genes is more than that of protein-encoding genes, suggesting their critical roles in human biology and evolution. Therefore, we suspected the contribution of lncRNAs to human skeletal development as well.

Urothelial cancer associated 1 (UCA1) is an lncRNA transcribed from one of the human endogenous retroviral proviruses. As represented by its name, this lncRNA was found overexpressed in human malignancies including bladder and breast cancers and thus has been investigated mainly in the context of cancer biology. However, we uncovered its physiological role in skeletal development. In fact, UCA1 is induced at an early stage of the differentiation of human bone marrow stem cells towards chondrocytes. Furthermore, gene silencing of UCA1 in human chondrocytic cells resulted in the repression of chondrocytic phenotype, decreasing CCN2 production. A previous study suggests that UCA1 may act as a competing endogenous RNA (ceRNA) against miR-18a. Since we found that miR-18a repressed CCN2 in human chondrocytic cells, the observed CCN2 repression by UCA1 gene silencing may be ascribed, at least in part, to the increase in active miR-18a molecules in the absence of this RNA sponge. However, concomitant induction of SOX9, a master transcription factor of chondrogenesis, indicates another molecular function of UCA1 inducing CCN2 indirectly. It should be also noted that CCN2 is regulated by at least 7 miRNAs through its 3′-untranslated region (UTR), which may be sponged by a variety of other ceRNAs. Therefore, the fate of CCN2 mRNA is supposed to be controlled by a complex regulatory network of short and long noncoding RNAs.

Apart from UCA1, we discovered an antisense lncRNA against CCN2. Interestingly, this lncRNA covers almost the whole 3’-UTR of CCN2. Functional characterization of this lncRNA is also underway.

the role of ccn1 in inflammation and wound healing.

Lester Lau

University of Illinois, Chicago, IL

The matricellular protein CCN1 plays diverse roles during wound healing, which occurs through three phases: inflammation, proliferation, and maturation. We have shown that CCN1 stimulates phagocytic removal of apoptotic cells by macrophages. In the context of wound healing, this activity promotes the clearance of neutrophils, thus allowing wound healing to progress through the inflammatory phase onto the proliferation phase. CCN1 also promotes matrix remodeling and dampens fibrosis during the maturation phase by inducing senescence in myofibroblasts. In recent studies, we have found that CCN1 promotes the clearance of bacterial infections, which poses a threat to the organism during wounding. Moreover, CCN1 activates a genetic program controlling inflammatory responses. These findings reveal new roles for CCN1 in combating bacterial infections during wound healing and tissue repair.

genetic analysis of ccn2 in skeletal muscle stress responses

Federica Accornero

Departments of Physiology and Cell Biology, Ohio State University, Columbus, OH - USA

The matricellular secreted protein CCN2 is upregulated in response to muscle injury, where it has been suggested to function as a fibrotic effector. Here we adopted transgenic mice with myofiber- and fibroblast- specific deletion of Ccn2 to dissect the contribution of these major muscle cell types for Ccn2-dependent muscle remodeling post injuries. We also studied the effect of genetic overexpression of Ccn2 in skeletal muscle. Remarkably, we found that Ccn2 plays critical roles in determining how muscle hypertrophies when overloaded. We also found that fibroblast-derived Ccn2 regulates myofiber regeneration and growth. Overall, our data suggest pivotal roles for Ccn2 in skeletal muscle that could be independent from its previously recognized role as pro-fibrotic molecule.