Biochemistry and Molecular Biology MicroRNA Signature in Wound Healing Following Excimer Laser Ablation: Role of miR-133b on TGFb1, CTGF, SMA, and COL1A1 Expression Levels in Rabbit Corneal Fibroblasts

PURPOSE. The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes. METHODS. Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3 0 untranslated region (UTR) of transforming growth factor b1 (TGFb1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative realtime PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration. RESULTS. At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at À14.33. Pre-miR-133b targeted the 3 0 UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor b1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFb1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF. CONCLUSIONS. Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation. Keywords: CTGF, microRNA, corneal wound healing, gene expression A fter corneal trauma, stromal wound healing is the result of a complex cascade of multiple factors including growth factors, cytokines, chemokines, proteases, and, most recently discovered, microRNAs (miRNAs). Directly after epithelial damage, the process of healing is initiated by multiple cytokines and growth factors released by the epithelial cells, keratocytes/ corneal fibroblast, and/or the lacrimal gland

Abstract 2388: Interaction between connective tissue growth factor and a disintegrin and metalloproteinase with thrombospondin type I repeats 7 during cancer development

Abstract Introduction: Connective tissue growth factor (CTGF) is a secreted pro-angiogenic protein within the CCN (Cyr61/CTGF/Nov) family and modulates multiple angiogenic pathways through its broad binding ability to cysteine knot motifs presents in key angiogenic factors including vascular endothelial growth factor (VEGF)-A. Overexpression of CTGF has been found in many cancers such as Lewis lung carcinoma (LLC), cholangiocarcinoma (CC), and hepatocellular carcinoma (HCC). In an effort to understand the molecular action of CTGF, we identified a disintegrin and metalloproteinase with thrombospondin type I repeat 7 (ADAMTS7) as a CTGF binding protein in yeast two-hybrid analysis. This enzyme belongs to the ADAMTS family capable of cleaving extracellular matrix (ECM) components and extracellular regulatory molecules. Here, we show that ADAMTS7 binds to and degrades CTGF during cancer development. Methods: Adamts7 knockout mice were used to investigate the importance of ADAMTS7 in CTGF interaction and cleavage during LLC development. Adamts7+/+ and Adamts7−/− mice were subcutaneously implanted with LLC cells. Tumor growth was quantitatively measured within two weeks after implantation. Tumor angiogenesis was assessed utilizing a microvascular density assay. In addition, Adamts7 expression was tracked via X-gal staining of LLC tumors grown in Adamts7−/− mice that contained a LacZ trap-in cassette in the targeted Adamts7 allele. VEGF-A, CTGF, and ADAMTS7 were determined at both the mRNA and protein levels. Results: Adamts7 induction as indicated by the β-galactosidase activity in X-gal staining was found in intra-tumor stromal cells of LLC tumors grown in Adamts7−/− mice. The association of CTGF and ADAMTS7 proteins in protein lysates of LLC tumors grown in Adamts7+/+ mice was confirmed in immunoprecipitation assays. Higher levels of VEGF-A, CTGF and ADAMTS7 mRNAs were found in LLC tumors from Adamts7−/− animals as compared to Adamts7+/+ controls. Slower rate of CTGF turnover, faster growth of LLC tumors, and higher microvascular density were also found in Adamts7−/− animals than controls. Conclusion: These observations confirmed the binding and processing of CTGF by ADAMTS7 during LLC growth. The host-derived ADAMTS7 appears to regulate CTGF turnover and provides a protective effect towards aberrant LLC tumorigenesis and angiogenesis. Citation Format: Liya Pi, Bryon E. Petersen. Interaction between connective tissue growth factor and a disintegrin and metalloproteinase with thrombospondin type I repeats 7 during cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2388. doi:10.1158/1538-7445.AM2015-2388</jats:p

The SLIT/ROBO Pathway in Liver Fibrosis and Cancer

Liver fibrosis is a common outcome of most chronic liver insults/injuries that can develop into an irreversible process of cirrhosis and, eventually, liver cancer. In recent years, there has been significant progress in basic and clinical research on liver cancer, leading to the identification of various signaling pathways involved in tumorigenesis and disease progression. Slit glycoprotein (SLIT)1, SLIT2, and SLIT3 are secreted members of a protein family that accelerate positional interactions between cells and their environment during development. These proteins signal through Roundabout receptor (ROBO) receptors (ROBO1, ROBO2, ROBO3, and ROBO4) to achieve their cellular effects. The SLIT and ROBO signaling pathway acts as a neural targeting factor regulating axon guidance, neuronal migration, and axonal remnants in the nervous system. Recent findings suggest that various tumor cells differ in SLIT/ROBO signaling levels and show varying degrees of expression patterns during tumor angiogenesis, cell invasion, metastasis, and infiltration. Emerging roles of the SLIT and ROBO axon-guidance molecules have been discovered in liver fibrosis and cancer development. Herein, we examined the expression patterns of SLIT and ROBO proteins in normal adult livers and two types of liver cancers: hepatocellular carcinoma and cholangiocarcinoma. This review also summarizes the potential therapeutics of this pathway for anti-fibrosis and anti-cancer drug development

Spatiotemporal Evolution and Influencing Factors of Electricity Consumption in the Yangtze River Delta Region

Electricity consumption accounts for a considerable part of the final energy consumption, and it is important for economic development and human life. This study explores the spatiotemporal evolution characteristics and influencing factors of electricity consumption in the Yangtze River Delta region in China from 2006 to 2019, using the gravity model and Logarithmic Mean Divisia Index method, respectively. The results show that: (1) The centers of gravity for the total final, industrial and residential electricity consumptions have a trend of migration towards the west. (2) The distance of migration of the center of gravity for residential electricity consumption is the highest, and the trend of migration of the center of gravity for industrial and total final electricity consumptions are synchronous. (3) Economic development is the main reason for the growth in regional electricity consumption, and the decrease in the investment electricity consumption intensity inhibits the growth of electricity consumption. This study provides references to restrain the excessive increase in electricity consumption and improve the layout of power facilities at the regional level

Human Parathyroid Hormone (1–34) accelerates skin wound healing through inducing cell migration via up-regulating the expression of Rac1

Abstract Delayed wound healing is a public issue that imposes a significant burden on both society and the patients themselves. To date, although numerous methods have been developed to accelerate the speed of wound closure, the therapeutic effects are partially limited due to the complex procedures, high costs, potential side effects, and ethical concerns. While some studies have reported that the in-vivo application of Human Parathyroid Hormone (1–34) (hPTH(1–34)) promotes the wound-healing process, the definitive role and underlying mechanisms through which it regulates the behavior of fibroblasts and keratinocytes remains unclear. Herein, hPTH(1–34)’s role in cell migration is evaluated with a series of in-vitro and in-vivo studies, whereby hPTH(1–34)’s underlying mechanism in activating the two types of cells was detected. The in-vitro study revealed that hPTH(1–34) enhanced the migration of both fibroblasts and HaCaT cells. Ras-associated C3 botulinum toxin subunit 1 (Rac1), a classical member of the Rho family, was upregulated in hPTH(1–34)-treated fibroblasts and HaCaT cells. Further study by silencing the expression of Rac1 with siRNA reversed the hPTH(1–34)-enhanced cell migration, thus confirming that Rac1 was involved in hPTH(1–34)-induced cell behavior. In-vivo study on rat wound models confirmed the effects of hPTH(1–34) on fibroblasts and keratinocytes, with increased collagen deposition, fibroblasts accumulation, and Rac1 expression in the hPTH(1–34)-treated wounds. In summary, the present study demonstrated that hPTH(1–34) accelerated wound healing through enhancing the migration of cells through the up-regulation of Rac1 expression

Spatiotemporal Evolution and Influencing Factors of Electricity Consumption in the Yangtze River Delta Region

Electricity consumption accounts for a considerable part of the final energy consumption, and it is important for economic development and human life. This study explores the spatiotemporal evolution characteristics and influencing factors of electricity consumption in the Yangtze River Delta region in China from 2006 to 2019, using the gravity model and Logarithmic Mean Divisia Index method, respectively. The results show that: (1) The centers of gravity for the total final, industrial and residential electricity consumptions have a trend of migration towards the west. (2) The distance of migration of the center of gravity for residential electricity consumption is the highest, and the trend of migration of the center of gravity for industrial and total final electricity consumptions are synchronous. (3) Economic development is the main reason for the growth in regional electricity consumption, and the decrease in the investment electricity consumption intensity inhibits the growth of electricity consumption. This study provides references to restrain the excessive increase in electricity consumption and improve the layout of power facilities at the regional level.</jats:p

Abstract 5107: The involvement of connective tissue growth factor in nonalcoholic steatohepatitis and liver cancer development under diabetic condition

Abstract Introduction: Obesity and type 2 diabetes are risk factors for liver cancer due to a transition from steatosis to nonalcoholic steatohepatitis (NASH), resulting in liver fibrosis. If left untreated, liver fibrosis can progress into cirrhosis leading to hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF) is a pro-fibrotic matricellular protein that is highly expressed during hepatocarcinogenesis. This study aims to investigate the involvement of CTGF in nonalcoholic steatohepatitis (NASH) and liver cancer development during diabetic conditions induced by streptozotocin (STZ) treatment and the feeding of a high fat diet (HFD) in mice. Methods: Transgenic male mice expressing green fluorescent protein (GFP) under the control of Ctgf promoter (Ctgfp-GFP), liver specific CTGF knockouts (CtgfΔhep/Δhep), and control mice that contained two alleles of floxed Ctgf (Ctgffloxed/floxed) were given STZ (200 ng/pup) at postnatal day 2 (P2) through subcutaneous injection and were fed HFD at postnatal week four to induce NASH and HCC. The treated animals were harvested at postnatal week 5, 12, and 20 for early steatosis, fibrosis and liver cancer development respectively. CTGF expression was analyzed by immunofluorescent staining, Western analysis, and qRT-PCR. In addition, the mouse liver cancer RT2 profiler PCR array was screened to identify cancer-related genes that were differentially expressed during NASH and HCC development comparing CtgfΔhep/Δhep and Ctgffloxed/floxed mice. Results: All three types of animals developed NASH and HCC in response to STZ and HFD feeding. Liver pathologies ranging from steatosis, NASH, liver fibrosis, and nodule formation, to intratumoral angiogenesis were observed as discrete molecular and histological stages in the STZ/HFD induced NASH-HCC model. Immunofluorescent analysis of Ctgfp-GFP reporter mice showed induction of the Ctgf gene in vascular endothelial cells, biliary epithelial cells, hepatocytes, and inflammatory cells in STZ/HFD livers. We utilized the mouse liver cancer RT2 profiler PCR array and compared the expression of 90 liver cancer related genes between CtgfΔhep/Δhep and Ctgff/f tumors that developed after 12-week HFD feeding. 12 upregulated genes and 10 downregulated genes were identified in Ctgfk/k mice compared to Ctgff/f animals. Conclusion: We successfully established a NASH-HCC model combining STZ and HFD treatment. CTGF expression was found in multiple cell types including endothelial cells, cholangiocytes, inflammatory cells, and hepatocytes. Liver specific deletion of CTGF was associated with differential expression of groups of genes involved in cell proliferation and apoptosis. The functional relationship between these genes and CTGF in liver cancer development in the setting of metabolic syndrome needs to be characterized in future studies. Citation Format: Liya Pi, Marda Jorgensen, Seh-Hoon Oh, Altin Gjymishka, Bryon E. Petersen. The involvement of connective tissue growth factor in nonalcoholic steatohepatitis and liver cancer development under diabetic condition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5107.</jats:p

Members of the Cyr61/CTGF/NOV Protein Family: Emerging Players in Hepatic Progenitor Cell Activation and Intrahepatic Cholangiocarcinoma

Hepatic stem/progenitor cells (HPC) reside quiescently in normal biliary trees and are activated in the form of ductular reactions during severe liver damage when the replicative ability of hepatocytes is inhibited. HPC niches are full of profibrotic stimuli favoring scarring and hepatocarcinogenesis. The Cyr61/CTGF/NOV (CCN) protein family consists of six members, CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3, which function as extracellular signaling modulators to mediate cell-matrix interaction during angiogenesis, wound healing, fibrosis, and tumorigenesis. This study investigated expression patterns of CCN proteins in HPC and cholangiocarcinoma (CCA). Mouse HPC were induced by the biliary toxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Differential expression patterns of CCN proteins were found in HPC from DDC damaged mice and in human CCA tumors. In addition, we utilized reporter mice that carried Ccn2/Ctgf promoter driven GFP and detected strong Ccn2/Ctgf expression in epithelial cell adhesion molecule (EpCAM)+ HPC under normal conditions and in DDC-induced liver damage. Abundant CCN2/CTGF protein was also found in cytokeratin 19 (CK19)+ human HPC that were surrounded by α-smooth muscle actin (α-SMA)+ myofibroblast cells in intrahepatic CCA tumors. These results suggest that CCN proteins, particularly CCN2/CTGF, function in HPC activation and CCA development

Abstract 3474: Connective tissue growth factor promotes tumor angiogenesis by modulating multiple angiogenic factors via binding to cystine knot motifs

Abstract Introduction Connective tissue growth factor (CTGF), a member of the CCN (Cyr61/CTGF/Nov) protein family, may function as both a positive and negative regulator of angiogenesis. However, the mechanism(s) underlying the regulatory behavior of CTGF is poorly understood. Here, we have undertaken a multipronged approach to provide insight into how CTGF enhances tumor angiogenesis. Methods Lewis lung carcinomas (LLC) cells were subcutaneously injected in syngeneic transgenic mice expressing green fluorescent protein (GFP) under the control of CTGF promoter (CTGFp-GFP). Recombinant lentivirus was generated to overexpress FLAG tagged CTGF while recombinant adenovirus expressing CTGF shRNA was used to knock down CTGF expression in LLCs. The levels of CTGF mRNA were examined by RT-PCR analysis. Tumor sizes were measured within two weeks and intratumoral microvessel density was quantified by immunochemical detection of Meca-32+ blood vessels. In addition, CTGF binding proteins were identified using the yeast two-hybrid approach. Immunoprecipitation assay was performed to verify these interactions. In vitro angiogenesis activity was indicated by tubule length formation by human umbilical vein endothelial cells (HUVECs). The migratory activity was measured in Boyden chamber assay. Activation of downstream signal effectors including cell division cycle 42 GTP binding protein (Cdc42) and extracellular signal-regulated kinase (ERK)1/2 was detected in Western blotting analysis. Results CTGF was expressed in Meca-32+ endothelial cells of intratumoral vessels growing in CTGFp-GFP transgenic mice. CTGF expression was also highly induced in hypoxic cultured LLC cells. Ectopic expression of CTGF promoted LLC tumor growth (n=3) and the microvessel density. Knocking down CTGF expression in LLC tumors (n=3) inhibited the growth of LLC tumors and decreased the microvessel density compared to those with scramble shRNA. Several proangiogenic factors were identified and shown to bind CTGF via cystine knot motifs. The factors include vascular endothelial growth factor (VEGF)-A, von Willebrand factor (vWF), platelet derived growth factor (PDGF)-B and Slit3. The combination of CTGF and Slit3 together resulted in an increased tubule length and an enhanced activation of Cdc42. Elevated cell migration and enhanced activation of ERK1/2 were observed when NIH3T3 fibroblasts were cultured with both CTGF and PDGF-B proteins. Although CTGF has been shown to bind to VEGF-A and inhibits VEGF-A mediated angiogenesis, our findings demonstrate that CTGF functions in concert with angiogenic factors Slit3 and PDGF-B to promote angiogenesis. Conclusion This study shows a critical role for CTGF in LLC tumor angiogenesis. CTGF binding to cystine knot motifs of key angiogenic factors represent a novel mechanism to regulate angiogenesis through PDGF-B and Slit3 signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3474. doi:10.1158/1538-7445.AM2011-3474</jats:p