Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo.

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

A short peptide encoded by long non-coding RNA small nucleolar RNA host gene 6 promotes cell migration and epithelial-mesenchymal transition by activating transforming growth factor-beta/SMAD signaling pathway in human endometrial cells.

BACKGROUND: Endometrial dysfunction is closely correlated with the development of multiple severe gynecological disorders including intrauterine adhesion. Accumulating evidence supports that some long non-coding RNAs (lncRNAs) have peptide-coding potential. In this text, the peptide-coding ability of lncRNA SNHG6 was examined. Also, the effects of an SNHG6-encoded peptide on the viability and migration of human endometrial stromal cells (hESCs) and human endometrial epithelial cells (hEECs) and related molecular mechanisms were explored. METHODS: The peptide-encoding potential of SNHG6 was predicted by FuncPEP and getorf databases and validated by western blot assay. Cell viability was tested by cell counting kit-8 assay. Cell migratory ability was examined by wound healing and transwell migration assays. Protein levels of genes were measured by western blot assay. RESULTS: Prediction analysis suggested that SNHG6 had the potential peptide-coding ability and multiple open-reading frames (ORFs). Western blot validated that SNHG6 ORF#1 and ORF#2 could translate into short peptides. SNHG6 ORF#2 overexpression facilitated cell migration and epithelial-mesenchymal transition (EMT) in hESCs and hEECs, while these effects were abrogated by transforming growth factor-beta (TGF-ß)/SMAD signaling inhibitor GW788388. Moreover, GW788388 inhibited the increase of p-SMAD2 and p-SMAD3 levels induced by SNHG6 ORF#2 in hESCs. SNHG6 ORF#2-encoded peptide did not influence endometrial stromal and epithelial cell viability. CONCLUSIONS: LncRNA SNHG6 ORF#1 and ORF#2 could translate into small peptides and SNHG6 ORF#2 overexpression promoted cell migration and EMT by activating the TGF-ß/SMAD pathway in hESCs and hEECs, suggesting the potential roles of SNHG6-encoded peptides in the development of endometrial stromal and epithelial cells and related gynecological diseases.

Integration of transcriptomics and metabolomics reveals pathways involved in MDSC supernatant attenuation of TGF-ß1-induced myofibroblastic differentiation of mesenchymal stem cells.

Overexposure to transforming growth factor b1 (TGF-ß1) induces myofibroblastic differentiation of mesenchymal stem cells (MSCs), which could be attenuated by myeloid-derived suppressor cell (MDSC) supernatant. However, the promyofibroblastic effects of TGF-ß1 and the antimyofibroblastic effects of MDSC supernatant in MSCs have not been fully elucidated. To further clarify the latent mechanism and identify underlying therapeutic targets, we used an integrative strategy combining transcriptomics and metabolomics. Bone marrow MSCs were collected 24 h following TGF-ß1 and MDSC supernatant treatment for RNA sequencing and untargeted metabolomic analysis. The integrated data were then analyzed to identify significant gene-metabolite correlations. Differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) were assessed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for exploring the mechanisms of myofibroblastic differentiation of MSCs. The integration of transcriptomic and metabolomic data highlighted significantly coordinated changes in glycolysis/gluconeogenesis and purine metabolism following TGF-ß1 and MDSC supernatant treatment. By combining transcriptomic and metabolomic analyses, this study showed that glycolysis/gluconeogenesis and purine metabolism were essential for the myofibroblastic differentiation of MSCs and may serve as promising targets for mechanistic research and clinical practice in the treatment of fibrosis by MDSC supernatant.

TRPV2 Promotes Cell Migration and Invasion in Gastric Cancer via the Transforming Growth Factor-ß Signaling Pathway.

BACKGROUND: Transient receptor potential vanilloid 2 (TRPV2) is a highly Ca2+-permeable ion channel that is involved in a number of cellular processes. It is expressed in various human cancers; however, the role of TRPV2 in gastric cancer (GC) remains poorly understood. METHODS: TRPV2 gene expression was knocked down in GC cell lines by small-interfering RNA (siRNA), and the biological roles of TRPV2 in the proliferation, migration, and invasion of GC cells were then investigated. The gene expression profile of GC was elucidated using a microarray analysis. TRPV2 expression in tumor tissue sections was analyzed by immunohistochemistry. RESULTS: The migration and invasion abilities of GC cells were inhibited by the knockdown of TRPV2. Moreover, the microarray assay revealed that TRPV2 was associated with the transforming growth factor (TGF)-ß signaling pathway. Immunohistochemical staining showed that the strong expression of TRPV2 correlated with lymphatic invasion, venous invasion, pathological T (pT), pathological N (pN), and a poor prognosis in GC patients. CONCLUSIONS: TRPV2 appeared to promote tumor migration and invasion via the TGF-ß signaling pathway, and the strong expression of TRPV2 was associated with a worse prognosis in GC patients.

Circ-POLR3A accelerates TGF-ß2-induced promotion in cell viability, migration, and invasion of lens epithelial cells via miR-31/TXNIP signaling cascade.

Posterior capsular opacification (PCO) is the major complication after cataract surgery and can result in secondary vision loss. Circular RNAs (circRNAs) are reported to play critical regulatory roles in multiple cell biological processes. The most common working mechanism of circRNAs is by acting as microRNA sponges. Here, we analyzed the role and mechanism of circRNA RNA polymerase III subunit A (POLR3A) in PCO. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell motility was assessed by transwell and wound healing assays. Dual-luciferase reporter and RNA-pull-down assays were performed to verify the interaction between microRNA-31 (miR-31) and circ-POLR3A or thioredoxin interacting protein (TXNIP). PCO cell model was established by treating SRA01/04 cells with transforming growth factor-ß2 (TGF-ß2). We found that TGF-ß2 enhanced SRA01/04 cell viability, migration, and invasion abilities. Circ-POLR3A expression was upregulated in PCO tissues and TGF-ß2-induced SRA01/04 cells. TGF-ß2 promoted the viability and motility of SRA01/04 cells largely by upregulating circ-POLR3A. Circ-POLR3A negatively regulated the miR-31 level by directly interacting with it. Circ-POLR3A absence-induced influences in TGF-ß2-induced SRA01/04 cells were partly reversed by silencing miR-31. miR-31 is directly bound to the 3'-untranslated region of TXNIP. TXNIP overexpression largely attenuated miR-31 overexpression-mediated effects in TGF-ß2-induced SRA01/04 cells. Circ-POLR3A could elevate the protein expression of TXNIP by sponging miR-31. Exosomes were involved in mediating the delivery of circ-POLR3A in SRA01/04 cells. In conclusion, circ-POLR3A contributed to TGF-ß2-induced promotion of cell viability, migration, and invasion of SRA01/04 cells by targeting miR-31/TXNIP axis.

Hsa-microRNA-27b-3p inhibits hepatocellular carcinoma progression by inactivating transforming growth factor-activated kinase-binding protein 3/nuclear factor kappa B signalling.

BACKGROUND: MicroRNAs (miRNAs) play crucial roles in the development of hepatocellular carcinoma (HCC). Hsa-microRNA-27b-3p (hsa-miR-27b) is involved in the formation and progression of various cancers, but its role and clinical value in HCC remain unclear. METHODS: The expression of hsa-miR-27b in HCC was examined by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) assays of clinical samples. Cell Counting Kit-8 assays (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, Transwell assays, filamentous actin (F-actin) staining and western blot analyses were used to determine the effects of hsa-miR-27b on HCC cells in vitro. Subcutaneous xenograft and lung metastatic animal experiments were conducted to verify the role of hsa-miR-27b in HCC in vivo. In silico prediction, qRT-PCR, western blot, anti-Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) and dual luciferase reporter assays were applied to identify the target genes of hsa-miR-27b. To detect the impacts of hsa-miR-27b on nuclear factor kappa B (NF-кB) signalling cascades mediated by transforming growth factor-activated kinase-binding protein 3 (TAB3), we performed qRT-PCR, western blot assays, immunofluorescence staining, immunohistochemistry (IHC) and dual-luciferase reporter assays. Recombinant oncolytic adenovirus (OncoAd) overexpressing hsa-miR-27b was constructed to detect their therapeutic value in HCC. RESULTS: The expression of hsa-miR-27b was lower in HCC than in adjacent non-tumourous tissues (ANTs), and the reduced expression of hsa-miR-27b was associated with worse outcomes in patients with HCC. Hsa-miR-27b significantly inhibited the proliferation, migration, invasion, subcutaneous tumour growth and lung metastasis of HCC cells. The suppression of hsa-miR-27b promoted the nuclear translocation of NF-κB by upregulating TAB3 expression. TAB3 was highly expressed in HCC compared with ANTs and was negatively correlated with the expression of hsa-miR-27b. The impaired cell proliferation, migration and invasion by hsa-miR-27b overexpression were recovered by ectopic expression of TAB3. Recombinant OncoAd with overexpression of hsa-miR-27b induced anti-tumour activity compared with that induced by negative control (NC) OncoAd in vivo and in vitro. CONCLUSIONS: By targeting TAB3, hsa-miR-27b acted as a tumour suppressor by inactivating the NF-кB pathway in HCC in vitro and in vivo, indicating its therapeutic value against HCC.

Salmonella pathogenicity island 1 knockdown confers protection against myocardial fibrosis and inflammation in uremic cardiomyopathy via down-regulation of S100 Calcium Binding Protein A8/A9 transcription.

BACKGROUND/AIM: Uremic cardiomyopathy (UCM) is a characteristic cardiac pathology that is commonly found in patients with chronic kidney disease. This study dissected the mechanism of SPI1 in myocardial fibrosis and inflammation induced by UCM through S100A8/A9. METHODS: An UCM rat model was established, followed by qRT-PCR and western blot analyses of SPI1 and S100A8/A9 expression in myocardial tissues. After alterations of SPI1 and S100A8/A9 expression in UCM rats, the blood specimens were harvested from the cardiac apex of rats. The levels of creatine phosphokinase-MB (CK-MB), blood creatinine, blood urea nitrogen (BUN), and inflammatory cytokines (interleukin [IL]-6, IL-1ß, and tumor necrosis factor-α [TNF-α]) were examined in the collected blood. Collagen fibrosis was assessed by Masson staining. The expression of fibrosis markers [transforming growth factor (TGF)-ß1, α-smooth muscle actin (SMA), Collagen 4a1, and Fibronectin], IL-6, IL-1ß, and TNF-α was measured in myocardial tissues. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were conducted to test the binding relationship between SPI1 and S100A8/A9. RESULTS: S100A8/A9 and SPI1 were highly expressed in the myocardial tissues of UCM rats. Mechanistically, SPI1 bound to the promoter of S100A8/A9 to facilitate S100A8/A9 transcription. S100A8/A9 or SPI1 knockdown reduced myocardial fibrosis and inflammation and the levels of CK-MB, blood creatinine, and BUN, as well as the expression of TGF-ß1, α-SMA, Collagen 4a1, Fibronectin, IL-6, TNF-α, and IL-1ß in UCM rats. CONCLUSION: SPI1 knockdown diminished S100A8/A9 transcription, thus suppressing myocardial fibrosis and inflammation caused by UCM.

The TGF-ß Receptor Gene Saxophone Influences Larval-Pupal-Adult Development in Tribolium castaneum.

The transforming growth factor-ß (TGF-ß) superfamily encodes a large group of proteins, including TGF-ß isoforms, bone morphogenetic proteins and activins that act through conserved cell-surface receptors and signaling co-receptors. TGF-ß signaling in insects controls physiological events, including growth, development, diapause, caste determination and metamorphosis. In this study, we used the red flour beetle, Tribolium castaneum, as a model species to investigate the role of the type I TGF-ß receptor, saxophone (Sax), in mediating development. Developmental and tissue-specific expression profiles indicated Sax is constitutively expressed during development with lower expression in 19- and 20-day (6th instar) larvae. RNAi knockdown of Sax in 19-day larvae prolonged developmental duration from larvae to pupae and significantly decreased pupation and adult eclosion in a dose-dependent manner. At 50 ng dsSax/larva, Sax knockdown led to an 84.4% pupation rate and 46.3% adult emergence rate. At 100 ng and 200 ng dsSax/larva, pupation was down to 75.6% and 50%, respectively, with 0% adult emergence following treatments with both doses. These phenotypes were similar to those following knockdowns of 20-hydroxyecdysone (20E) receptor genes, ecdysone receptor (EcR) or ultraspiracle protein (USP). Expression of 20E biosynthesis genes disembodied and spookier, 20E receptor genes EcR and USP, and 20E downstream genes BrC and E75, were suppressed after the Sax knockdown. Topical application of 20E on larvae treated with dsSax partially rescued the dsSax-driven defects. We can infer that the TGF-ß receptor gene Sax influences larval-pupal-adult development via 20E signaling in T. castaneum.

Insights into the evolution of metazoan regenerative mechanisms: roles of TGF superfamily members in tissue regeneration of the marine sponge Chondrosia reniformis.

Tissue repair is an adaptive and widespread metazoan response. It is characterised by different cellular mechanisms and complex signalling networks that involve numerous growth factors and cytokines. In higher animals, transforming growth factor-ß (TGF-ß) signalling plays a fundamental role in wound healing. In order to evaluate the involvement of TGF superfamily members in lower invertebrate tissue regeneration, sequences for putative TGF ligands and receptors were isolated from the transcriptome of the marine sponge Chondrosia reniformis We identified seven transcripts that coded for TGF superfamily ligands and three for TGF superfamily receptors. Phylogenetically, C. reniformis TGF ligands were not grouped into any TGF superfamily clades and thus presumably evolved independently, whereas the TGF receptors clustered in the Type I receptor group. We performed gene expression profiling of these transcripts in sponge regenerating tissue explants. Data showed that three ligands (TGF1, TGF3 and TGF6) were mainly expressed during early regeneration and seemed to be involved in stem cell maintenance, whereas two others (TGF4 and TGF5) were strongly upregulated during late regeneration and thus were considered pro-differentiating factors. The presence of a strong TGF inhibitor, SB431542, blocked the restoration of the exopinacoderm layer in the sponge explants, confirming the functional involvement of the TGF pathway in tissue regeneration in these early evolved animals.

Gastric Acid and Pepsin Work Together in Simulated Gastric Acid Inhalation Leading to Pulmonary Fibrosis in Rats.

BACKGROUND The clinical association between gastroesophageal reflux disease (GERD) and idiopathic pulmonary fibrosis (IPF) has been known for many years, but it is still unclear. The present study investigated the association between experimentally simulated aspiration and pulmonary fibrosis. MATERIAL AND METHODS A total of 120 male Sprague-Dawley rats were randomly divided into a negative control group, a bleomycin group, and 3 simulated aspiration groups. The bleomycin group was administered a one-time intratracheal injection of bleomycin, whereas the 3 simulated aspiration groups were treated either with an intratracheal instillation of gastric fluid combined with pepsin, with pepsin alone, or with hydrochloric acid, all twice a week, and the negative control group was administered normal saline twice a week. Lung tissues were collected to evaluate pathological changes and the mRNA expression levels of connective tissue growth factor (CTGF), type I collagen, and transforming growth factor. RESULTS The results demonstrated that the degree of fibrosis in the early stage was low in each of the 3 simulated aspiration groups, but gradually increased over time. The expression levels of the downstream factor of fibrosis, CTGF, and type I collagen also reflected this trend. CONCLUSIONS The study demonstrates that aspiration of gastric contents can cause pulmonary fibrosis, and mixed aspiration of pepsin and gastric fluid can accelerate this process. This study provides strong evidence in support of a potential association between human GERD and IPF.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-ß3 alone showed promising result but the previously tested association of BMP-2 and TGF-ß1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-ß1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

Role of miR-24, Furin, and Transforming Growth Factor-ß1 Signal Pathway in Fibrosis After Cardiac Infarction.

BACKGROUND Cardiac fibrosis after primary infarction is a type of pathological phenomena as shown by increased collagen in myocardial cells. Transforming growth factor (TGF)-ß1 is a critical factor participating in myocardial fibrosis. A previous study has shown the inhibitory role on TGF-ß1 by microRNA-24 (miR-24) via targeting Furin. This study thus investigated the role of miR-24 and Furin/TGF-ß1 in rat myocardial fibrosis. MATERIAL AND METHODS A total of 40 adult SD rats (both males and females) were prepared for myocardial infarction model by ligating the descending branch of left coronary artery after anesthesia. HE staining was performed to observe myocardial fibrosis after 1, 2, and 4 weeks. Tissue RNA was extracted to detect mRNA levels of Furin, TGF-ß1, and miR-24 by real-time PCR. Western blotting was used to quantify protein expression of Furin and TGF-ß1 in myocardial tissues. RESULTS Increased connective tissues were observed in myocardial tissues at 4 weeks after infarction by HE staining, which also revealed widening of the intra-myocardial cleft, along with more inflammatory cells and fibroblast hypertrophy. miR-24 expression was significantly depressed at 2 and 4 weeks after cardiac infarction (p<0.05). mRNA levels of Furin and TGF-ß1 were elevated after infarction (p<0.05). With prolonged time periods of myocardial infarction, protein levels of Furin and TGF-ß1 were further increased. The level of miR-24 was positively correlated with left ventricular end-diastolic diameter, left ventricular systolic diameter, and left ventricular ejection fraction. However, the level of Furin or TGF-ß1 was negatively correlated with the above parameters. CONCLUSIONS This study demonstrated the important role of abnormal expression of miR-24 in myocardial fibrosis after infarction, and may provide drug targets for treating myocardial fibrosis.

The immunotranscriptome of the Caribbean reef-building coral Pseudodiploria strigosa.

The viability of coral reefs worldwide has been seriously compromised in the last few decades due in part to the emergence of coral diseases of infectious nature. Despite important efforts to understand the etiology and the contribution of environmental factors associated to coral diseases, the mechanisms of immune response in corals are just beginning to be studied systematically. In this study, we analyzed the set of conserved immune response genes of the Caribbean reef-building coral Pseudodiploria strigosa by Illumina-based transcriptome sequencing and annotation of healthy colonies challenged with whole live Gram-positive and Gram-negative bacteria. Searching the annotated transcriptome with immune-related terms yielded a total of 2782 transcripts predicted to encode conserved immune-related proteins that were classified into three modules: (a) the immune recognition module, containing a wide diversity of putative pattern recognition receptors including leucine-rich repeat-containing proteins, immunoglobulin superfamily receptors, representatives of various lectin families, and scavenger receptors; (b) the intracellular signaling module, containing components from the Toll-like receptor, transforming growth factor, MAPK, and apoptosis signaling pathways; and (3) the effector module, including the C3 and factor B complement components, a variety of proteases and protease inhibitors, and the melanization-inducing phenoloxidase. P. strigosa displays a highly variable and diverse immune recognition repertoire that has likely contributed to its resilience to coral diseases.

Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-ß1, TGF-ß2, and TGF-ß3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-ß family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell interactions, and the results suggest that stromal cells and epithelial cells may act in concert in the cornea.

[Association study between candidate genes on transforming growth factor-ß signaling pathway and the risk of non-syndromic cleft lip with or without cleft palate in Chinese populations].

OBJECTIVE: To explore the association between 10 candidate genes on transforming growth factor-ß (TGFB) signaling pathway and non-syndromic cleft lip with or without cleft palate (NSCL/P) among Chinese populations, and to study the gene-environment interaction. METHODS: A total of 806 Chinese Han NSCL/P trios were ascertained from an international consortium, which conducted a genome-wide association study using a case-parent trio design to investigate the genes affecting risk to NSCL/P. The transmission disequilibrium test (TDT) was used to test for effects of 343 single nucleotide polymorphisms (SNPs) in 10 genes on TGFB signaling pathway including DCN, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, BAMBI, SMAD2, SMAD3 and SMAD4. The conditional regression models were used to test for gene-environment interaction. RESULTS: For TDT, although 19 SNPs showed nominal significant association with NSCL/P, no significant evidence of association was seen for all SNPs in 806 NSCL/P trios after Bonferroni correction. The interactions between genes and maternal smoking, environmental tobacco smoke, alcohol consumption and multi-vitamin supplementation during pregnancy did not attain statistical significance after correction for multiple comparisons. CONCLUSION: No evidence for SNP effect of genes on TGFB signaling pathway and significant gene-environment interaction was seen in our data.

Activation of FGF2-FGFR signaling in the castrated mouse prostate stimulates the proliferation of basal epithelial cells.

The prostate gland is unique in that it undergoes rapid regression following castration but regenerates completely once androgens are replaced. Residual ductal components play an important role in the regeneration of a fully functional prostate. In this study, to examine how androgen status affects prostate structure and components, we conducted histopathological studies of the involuted and regenerated mouse dorsolateral prostate (DLP). In the castrated mouse DLP, the number of luminal epithelial cells decreased in a time-dependent manner. On Day 14 postandrogen replacement, the number of luminal epithelial cells was completely restored to the baseline level. In contrast, the number of basal epithelial cells gradually increased in the castrated mouse prostate. The Ki67-labeling index of prostate basal epithelial cells was significantly increased after castration. The number of basal epithelial cells decreased to baseline after androgen replacement. After castration, mRNA expression levels of specific growth factors, such as Fgf2, Fgf7, Hgf, Tgfa, and Tgfb, were relatively abundant in whole mouse DLPs. In organ culture experiments, basal epithelial proliferation was recapitulated in the absence of dihydrotestosterone (DHT). The proliferation of basal epithelial cells in the absence of DHT was suppressed by treatment with an FGF receptor inhibitor (PD173074). Moreover, FGF2 treatment directly stimulated the proliferation of basal epithelial cells. Taken together, these data indicated that the FGF2-FGF receptor signal cascade in the prostate gland may be one of the pathways stimulating the proliferation of basal epithelial cells in the absence of androgens.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral.

OBJECTIVES: The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS: Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS: EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION: The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE: The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Association Between Genetic Diagnosis and Clinical Outcomes in Patients With Heritable Thoracic Aortic Disease.

Background Differences in the clinical course of heritable thoracic aortic disease based on the disease-causing gene have not been fully evaluated. To clarify the clinical relevance of causative genes in heritable thoracic aortic disease, we assessed the clinical course of patients categorized based on genetic diagnosis. Methods and Results We investigated cardiovascular events and mortality in 518 genetically diagnosed patients in 4 groups: Group 1, FBN1 (n=344); Group 2, TGFBR1, TGFBR2, SMAD3, or TGFB2 (n=74); Group 3, COL3A1 (n=60); and Group 4, ACTA2 or MYH11 (n=40). The median age at the first cardiovascular event ranged from 30.0 to 35.5 years (P=0.36). Patients with gene variants related to transforming growth factor-ß signaling had a significantly higher rate of subsequent events than those with FBN1 variants (adjusted hazard ratio, 2.33 [95% CI, 1.60-3.38]; P<0.001). Regarding the incidence of aortic dissection, there were no significant differences among the 4 groups in male patients (36.3%, 34.3%, 21.4%, and 54.2%, respectively; P=0.06). Female patients with COL3A1 variants had a significantly lower incidence than female patients in the other 3 groups (34.2%, 59.0%, 3.1%, and 43.8%, respectively; P<0.001). Conclusions Gene variants related to transforming growth factor-ß signaling are associated with a higher incidence of subsequent cardiovascular events than FBN1 variants. COL3A1 variants might be related to a lower incidence of aortic dissection than other gene variants in women only. Identifying the genetic background of patients with heritable thoracic aortic disease is important for determining appropriate treatment.

Single-Cell Analysis Reveals Transcriptomic Features of Drug-Tolerant Persisters and Stromal Adaptation in a Patient-Derived EGFR-Mutated Lung Adenocarcinoma Xenograft Model.

INTRODUCTION: Targeted therapies require life-long treatment, as drug discontinuation invariably leads to tumor recurrence. Recurrence is mainly driven by minor subpopulations of drug-tolerant persister (DTP) cells that survive the cytotoxic drug effect. In lung cancer, DTP studies have mainly been conducted with cell line models. METHODS: We conducted an in vivo DTP study using a lung adenocarcinoma patient-derived xenograft tumor driven by an EGFR mutation. Daily treatment of tumor-bearing mice for 5 to 6 weeks with the EGFR inhibitor erlotinib markedly shrunk tumors and generated DTPs, which were analyzed by whole exome, bulk population transcriptome, and single-cell RNA sequencing. RESULTS: The DTP tumors maintained the genomic clonal architecture of untreated baseline (BL) tumors but had reduced proliferation. Single-cell RNA sequencing identified a rare (approximately 4%) subpopulation of BL cells (DTP-like) with transcriptomic similarity to DTP cells and intermediate activity of pathways that are up-regulated in DTPs. Furthermore, the predominant transforming growth factor-ß activated cancer-associated fibroblast (CAF) population in BL tumors was replaced by a CAF population enriched for IL6 production. In vitro experiments indicate that these populations interconvert depending on the levels of transforming growth factor-ß versus NF-κB signaling, which is modulated by tyrosine kinase inhibitor presence. The DTPs had signs of increased NF-κB and STAT3 signaling, which may promote their survival. CONCLUSIONS: The DTPs may arise from a specific preexisting subpopulation of cancer cells with partial activation of specific drug resistance pathways. Tyrosine kinase inhibitor treatment induces DTPs revealing greater activation of these pathways while converting the major preexisting CAF population into a new state that may further promote DTP survival.

Immune characterization of mesenchymal stem cells in human umbilical cord Wharton's jelly and derived cartilage cells.

Mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hWJMSCs) became prospective seed cell candidate for tissue engineering and cell-based therapy because of its variety source, easy procurement, robust proliferation, and high purity compared with bone marrow- and adipose-derived MSCs. Such neonatal stem cells can be isolated from a variety of extraembryonic tissues and appear to be more primitive and have greater multi-potentiality than their adult counterparts. In this study, we investigated the immune characters of hWJMSCs and its derived cartilage cells (hWJMSC-Cs) by detecting the expression of major histocompatibility complex I/I(MHC-I/II), costimulatory molecules (CD40, CD80 and CD86) and immune inhibitors including human leukocyte antigen G (HLA-G), indoleamine-2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2). We found that hWJMSCs did not express MHC-II and costimulatory molecules, but moderately expressed MHC-I, and positively expressed immune inhibitors as HLA-G, IDO, PGE2, demonstrating their very low immunogenicity and potential to induce immune tolerance microenvironment in hosts. The results of chondrogenic differentiated hWJMSCs(hWJMSC-Cs) are similar to those of undifferentiated cells, except for the slightly elevated MHC-II and costimulators expression. Additionally, we detected cytokine profile of hWJMSCs through cytokine antibody array and verified by western blot the positive expression of immune suppression-related molecules, HGF, VEGF, TGF, and IL-10. Furthermore, to investigate the in vivo immune response of the cells, hWJMSCs-scaffold constructs were implanted into rabbits and rats, and the result showed that hWJMSCs did not elicit immune rejection in the animals. Their intermediate state between adult and embryonic stem cells makes them an ideal candidate for reprogramming to the pluripotent status. Additional studies are necessary to clarify the potential of hWJMSCs to be used in cartilage and other tissue regeneration and cell-based therapies.