Decorin Gene Expression Research Articles

Effect of aerobic and resistance training on secreted protein acidic rich in cysteine, decorin, and myonectin muscle genes expression in rats fed with a high-fat diet

Background and aims: Fatty diet can affect the expression of myokines. Thus, the aim of this study was to evaluate the effect of aerobic and resistance training secreted protein acidic rich in cysteine (SPARC), decorin (DCN), and myonectin muscle gene expression in rats fed with a high-fat diet. Methods: In this experimental study, 32 rats were randomly divided into healthy control, obese control, obese+aerobic exercise, and obese+resistance exercise groups. The training program was implemented for 4 weeks at aerobic moderate intensity (50% VO2max in the first week and 65% VO2max in the last week). For resistance training, the animals were also trained to climb the ladder (height 110 cm, slope 80%, and the distance between the bars of the ladder 2 cm), which is based on the determination of one-repetition maximum (1RM). Obese rats received a dose of 0.5 mL/100 g of body weight orally by gavage for 4 weeks and 5 days a week. Results: There was no significant difference between the groups in terms of DCN expressions (P=0.702). However, the expression of SPARC (P=0.039) and myonectin in the obese control group was significantly increased compared to the healthy control (P=0.038). The expression of SPARC and myonectin in the aerobic training group (P=0.038, P=0.042) and the resistance training group (P=0.048, P=0.049) was significantly decreased in comparison to obesity control. Resistance training caused a greater decrease in myonectin compared to aerobic training (P=0.049). Conclusion: The findings revealed that aerobic and resistance exercises are effective in reducing SPARC and myonectin expression in the skeletal muscle of rats fed with a high-fat diet.

Mild heat stimulation facilitates muscle hypertrophy in C2C12 and mouse satellite cells through myokine release to the culture medium

Mild heat stimulation is defined as a stimulation that occurs several degrees above optimal cell culture or body temperatures. Muscle hypertrophy in C2C12 cells is reportedly facilitated by 39 °C mild heat stimulation. However, the underlying molecular mechanisms by which 39 °C mild heat stimulation promotes muscle hypertrophy remains elusive. In this study, we aimed at understanding the details of these mechanisms. First, we found that 39 °C mild heat stimulation has little or no effect on Myogenesis-Related Factor (MRF) expression both in C2C12 and mouse primary satellite cells. Therefore, we hypothesized that 39 °C mild heat stimulation promotes muscle hypertrophy through myokines, essential components in myogenesis. Interestingly, we observed muscle hypertrophy in the group cultured at 37 °C in conditioned medium from 39 °C compared to the group cultured at 37 °C in conditioned medium from 37 °C in the case of both C2C12 and mouse primary satellite cells. These results suggest that 39 °C mild heat stimulation promotes muscle hypertrophy through myokines, once released in the culture medium. Finally, we identified Decorin as a hypertrophy-inducing myokine candidate. Therefore, in this study, we demonstrated that 39 °C mild heat stimulation contributes to muscle hypertrophy through enhancing Decorin gene expression in C2C12 and mouse primary satellite cells.

Extracellular Matrix Synthesis and Remodeling by Mesenchymal Stromal Cells Is Context-Sensitive.

Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-β1 (TGFβ1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGFβ1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.

Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.

Maintenance of Ligament Homeostasis of Spheroid-Colonized Embroidered and Functionalized Scaffolds after 3D Stretch.

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.

The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model.

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

Effect of Eight Weeks of Exercise with Different Intensities on the Gene Expression of Decorin and Muscular TGF-β in the Male Adult Rats

Effect of Eight Weeks of Exercise with Different Intensities on the Gene Expression of Decorin and Muscular TGF-β in the Male Adult Rats

Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry.Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Tendon and Cytokine Marker Expression by Human Bone Marrow Mesenchymal Stem Cells in a Hyaluronate/Poly-Lactic-Co-Glycolic Acid (PLGA)/Fibrin Three-Dimensional (3D) Scaffold.

We developed a (three-dimensional) 3D scaffold, we named HY-FIB, incorporating a force-transmission band of braided hyaluronate embedded in a cell localizing fibrin hydrogel and poly-lactic-co-glycolic acid (PLGA) nanocarriers as transient components for growth factor controlled delivery. The tenogenic supporting capacity of HY-FIB on human-Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) was explored under static conditions and under bioreactor-induced cyclic strain conditions. HY-FIB elasticity enabled to deliver a mean shear stress of 0.09 Pa for 4 h/day. Tendon and cytokine marker expression by hBM-MSCs were studied. Results: hBM-MSCs embedded in HY-FIB and subjected to mechanical stimulation, resulted in a typical tenogenic phenotype, as indicated by type 1 Collagen fiber immunofluorescence. RT-qPCR showed an increase of type 1 Collagen, scleraxis, and decorin gene expression (3-fold, 1600-fold, and 3-fold, respectively, at day 11) in dynamic conditions. Cells also showed pro-inflammatory (IL-6, TNF, IL-12A, IL-1β) and anti-inflammatory (IL-10, TGF-β1) cytokine gene expressions, with a significant increase of anti-inflammatory cytokines in dynamic conditions (IL-10 and TGF-β1 300-fold and 4-fold, respectively, at day 11). Mechanical signaling, conveyed by HY-FIB to hBM-MSCs, promoted tenogenic gene markers expression and a pro-repair cytokine balance. The results provide strong evidence in support of the HY-FIB system and its interaction with cells and its potential for use as a predictive in vitro model.

Abstract P3-01-06: Decorin-mediated suppression of tumorigenesis and skin invasion in inflammatory breast cancer via inhibition of the E-cadherin/EGFR axis

Abstract Background: Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of primary breast cancer. Although considered rare, IBC accounts for 10% of breast cancer-related deaths owing to its rapid proliferation and strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC remain elusive. Through transcriptome profiling we identified Decorin (DCN) as being significantly altered in metastatic IBC cell sublines. DCN is a secreted, small leucine-rich proteoglycan known to function as a tumor/metastasis suppressor by inhibiting several signaling pathways including EGFR, TGFβ, and c-MET. However, whether or how DCN regulates IBC tumorigenesis or metastasis is unknown. The aim of this study was to investigate the function and mechanism of DCN in IBC tumorigenesis and metastasis. Methods: Three IBC cell lines [ER-/HER2+ (MDA-IBC3, SUM190) and ER-/HER2- (SUM149)] were used. DCN gene expression in clinical samples was analyzed from publically available datasets and the IBC Consortium dataset. DCN was stably expressed in IBC cell lines by using lentiviral vectors. For in vivo studies, DCN-overexpressing stable cell lines were injected into cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. Reverse phase protein array analysis was used for proteomic profiling. Protein-protein interactions were analyzed by reciprocal immunoprecipitation of exogenous or endogenous proteins. Results: DCN expression was significantly lower in breast cancer samples than in normal breast (p&amp;lt;0.0001), in more aggressive breast cancer subtypes (p&amp;lt;0.0001), and in metastatic tumors relative to primary tumors (p&amp;lt;0.0001). High DCN expression correlated with improved overall survival (p&amp;lt;0.0001) and relapse-free survival (p=0.0003). In vitro, DCN overexpression in IBC cell lines inhibited colony formation (MDA-IBC3, p=0.0115; SUM149, p=0.0068), migration (SUM149, p=0.0165), invasion (SUM149, p=0.0159), and primary and secondary mammosphere formation (primary: MDA-IBC3, p=0.0209; SUM149, p=0.0232; secondary: MDA-IBC3, p=0.01; SUM149, p=0.0031). In vivo, DCN overexpression in MDA-IBC3 cells inhibited primary tumor growth (p=0.0092) and reduced skin invasion (89.9% DCN control vs 33.3% DCN-overexpressed, p=0.017, Chi-square test). Our proteomics data in DCN-overexpressing SUM149 and MDA-IBC3 cells showed downregulated EGFR and E-cadherin protein levels. Mechanistically, DCN reduced expression of E-cadherin and EGFR and reduced phosphorylation of EGFR in IBC cells. Exogenous and endogenous co-immunoprecipitation experiments showed direct physical binding between DCN and E-cadherin proteins in all IBC cell lines. Moreover, overexpression of DCN downregulated E-cadherin via protein instability rather than decreased E-cadherin mRNA expression. E-cadherin knockdown in IBC cells decreased, whereas its overexpression increased, activation of EGFR signaling without affecting DCN expression. Finally, restoring E-cadherin in DCN-overexpressing IBC cell lines rescued the inhibitory effect of DCN on EGFR signaling. Conclusions: We found that DCN inhibited tumorigenesis and skin invasion in IBC via its direct interaction with and stabilization of E-cadherin and its suppression of EGFR signaling. Our findings provide new insights and a novel mechanism for IBC pathobiology that may be therapeutically targetable. Future studies will determine the role of DCN in IBC metastasis and the detailed mechanism of DCN-mediated suppression of tumorigenesis and metastasis in IBC. Citation Format: Xiaoding Hu, Emilly Schlee Villodre, Richard Larson, Omar M. Rahal, Xiaoping Wang, Savitri Krishnamurthy, Debu Tripathy, Naoto T Ueno, Wendy A Woodward, Bisrat Godefay Debeb. Decorin-mediated suppression of tumorigenesis and skin invasion in inflammatory breast cancer via inhibition of the E-cadherin/EGFR axis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-06.

Decorin Expression Is Associated With Diffusion MR Phenotypes in Glioblastoma

Abstract INTRODUCTION Significant evidence from multiple phase II trials have suggested diffusion-weighted imaging estimates of apparent diffusion coefficient (ADC) are a predictive imaging biomarker for survival benefit for recurrent glioblastoma when treated with anti-VEGF therapies, including bevacizumab, cediranib, and cabozantinib. Despite this observation, the underlying mechanism linking anti-VEGF therapeutic efficacy with diffusion MR characteristics remains unknown. We hypothesized that a high expression of decorin, a small proteoglycan that has been associated with sequestration of pro-angiogenic signaling as well as reduction in the viscosity of the extracellular environment, may be associated with elevated ADC. METHODS A differential gene expression analysis was carried out in human glioblastoma samples in whom preoperative diffusion imaging was obtained. ADC histogram analysis was carried out to calculate preoperative ADCL values, the average ADC in the lower distribution using a double Gaussian mixed model. The Cancer Imaging Archive (TCIA) and The Cancer Genome Atlas (TCGA) databases were queried to identify diffusion imaging and levels of decorin protein expression. Patients with recurrent glioblastoma who undergo resection prospectively had targeted biopsies based on the ADC analysis collected. These samples were stained for decorin and quantified using whole-slide image analysis software. RESULTS Differential gene expression analysis between tumors associated with high and low preoperative ADCL showed that patients with high ADCL had increased decorin gene expression. Patients from the TCGA database with elevated ADCL had a significantly higher level of decorin gene expression (P = .01). These patients had a survival advantage with a log-rank analysis (P = .002). Patients with preoperative diffusion imaging had multiple targeted intraoperative biopsies stained for decorin. Patients with high ADCL had increased decorin expression on immunohistochemistry (P = .002). CONCLUSION Increased ADCL on diffusion MR imaging is associated with high decorin expression as well as increased survival in glioblastoma. Decorin may play an important role the imaging features on diffusion MR and anti-VEGF treatment efficacy. Decorin expression may serve as a future therapeutic target in patients with favorable diffusion MR characteristics.

Placental and serum levels of human Klotho in severe preeclampsia: A potential sensitive biomarker

IntroductionThe Klotho (KL) gene, initially defined as an anti-aging gene in mice, shares 86% of the amino acid sequence withthe human KL protein. The KL gene plays roles in endothelial nitric oxide production, angiogenesis, antioxidant enzyme production and protecting against endothelial dysfunction, all of which may be associated with preeclampsia (PE). Human KL is the precursor of the gene products: α-KL and β-KL. In this study, we evaluated the gene expression, serum and placental levels of human KL in women with severe PE, pregnant women with chronic hypertension and healthy pregnant controls. Also, the gene expression, serum and placental levels of human decorin (DCN) were evaluated. MethodsA total of 36 patients with severe PE, 10 with chronic hypertension, and 28 with healthy controls were enrolled. Placental and serum levels together with of KL and DCN were measured by ELISA and alsogene expression of these were evaluated. ResultsPlacental and serum KL levels were significantly higher in the PE than in the controls and in women with chronic hypertension. Serum DCN levels were significantly higher in the PE women compared to controls and pregnant women with chronic hypertension. Placental DCN was similar in PE and healthy controls. There was no significant difference in the gene expression of KL and DCN in the groups. The best cut-off level for human KL to identify the presence of PE was calculated as 12.48 pg/ml with a sensitivity of 100% and and specificity of 96%, whereas for DCN 62.33 ng/ml to assess the presence of PE with a sensitivity of 86.1% and a specificity of 88%. ConclusionHuman KL may be a valuable marker for PE, with high sensitivity and specificity. It also appears to be more sensitive and specific than human DCN.

Functional polymorphisms within the inflammatory pathway regulate expression of extracellular matrix components in a genetic risk dependent model for anterior cruciate ligament injuries

ObjectivesTo investigate the functional effect of genetic polymorphisms of the inflammatory pathway on structural extracellular matrix components (ECM) and the susceptibility to an anterior cruciate ligament (ACL) injury. DesignLaboratory study, case–control study. MethodsEight healthy participants were genotyped for interleukin (IL)1B rs16944 C>T and IL6 rs1800795 G>C and classified into genetic risk profile groups. Differences in type I collagen (COL1A1), type V collagen (COL5A1), biglycan (BGN) and decorin (DCN) gene expression were measured in fibroblasts either unstimulated or following IL-1β, IL-6 or tumor necrosis factor (TNF)-α treatment.Moreover, a genetic association study was conducted in: (i) a Swedish cohort comprised of 116 asymptomatic controls (CON) and 79 ACL ruptures and (ii) a South African cohort of 100 CONs and 98 ACLs. Participants were genotyped for COL5A1 rs12722 C>T, IL1B rs16944 C>T, IL6 rs1800795 G>C and IL6R rs2228145 G>C. ResultsIL1B high-risk fibroblasts had decreased BGN (p=0.020) and COL5A1 (p=0.012) levels after IL-1β stimulation and expressed less COL5A1 (p=0.042) following TNF-α treatment. Similarly, unstimulated IL6 high-risk fibroblasts had lower COL5A1 (p=0.012) levels than IL6 low-risk fibroblasts.In the genetic association study, the COL5A1-IL1B-IL6 T–C–G (p=0.034, Haplo-score 2.1) and the COL5A1-IL1B-IL6R T–C–A (p=0.044, Haplo-score: 2.0) combinations were associated with an increased susceptibility to ACL injury in the Swedish cohort when only male participants were evaluated. ConclusionsThis study shows that polymorphisms within genes of the inflammatory pathway modulate the expression of structural and fibril-associated ECM components in a genetic risk depended manner, contributing to an increased susceptibility to ACL injuries.

Incorporation of types I and III collagen in tunable hyaluronan hydrogels for vocal fold tissue engineering

Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21 days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. Statement of SignificanceVoice disorders affect about 1/3rd of the US population and significantly reduce quality of life. Patients with vocal fold fibrosis have few treatment options. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. Various studies have used collagen or thiolated hyaluronan (HA) with gelatin as potential tissue engineering therapies. However, there is room for improvement in providing cells with more relevant biological cues that mimic the native tissue microenvironment and sustain regeneration. The present study introduces the use of type I collagen and type III collagen along with thiolated HA as a natural biomimetic hydrogel for vocal fold tissue engineering applications.

Cell biological profiling of reprogrammed cancer stem cell-like colon cancer cells maintained in culture.

Cancer stem cells (CSCs) are specific targets for therapeutic applications, but the rarity of CSCs within tumors makes the isolation of CSCs difficult. To overcome these problems, we generated CSCs in vitro using established reprogramming techniques. We transduced four previously established reprogramming factors, Oct3/4, Sox2, Klf4, and L-myc, into the colon cancer cell lines LoVo and OUMS-23, and investigated the biological characteristics of these lines. Tra-1-60+ cells were obtained from reprogrammed induced pluripotent stem (iPS) cell-like colonies and showed CSC properties, including colony formation, maintenance of colonies by repeated passages, and feeder cell dependency, as well as increased expressions of CSC markers such as CD133 and ALDH1. The CSC-like cells showed increased chemoresistance to 5-fluorouracil and elevated tumorigenicity upon transplantation into kidneys of immune-deficient mice. These tumors shifted to a poorly differentiated stage with many atypical cells, cytoplasmic mucin, and focal papillary components, with demonstrated dedifferentiation. The principal component analysis from DNA microarrays showed that though both cell lines moved to iPS cells after reprogramming, they were not completely identical to iPS cells. Significantly elevated gene expression of Decorin and CD90 was observed in CSC-like cells. Together, these results show that reprogramming of cancer cells produced not pluripotent stem cells but CSC-like cells, and these findings will provide biological information about genuine CSCs and help establish new CSC-targeted therapies.

Effect of starvation on expression of troponin complex genes and ER stress associated genes in skeletal muscles

Little is known about the mechanism of gene expression changes in skeletal muscle during starvation. This study investigated the influence of starvation on gene expression of troponin complex (TnC, TnI and TnT), tropomyosin (Tpm), and endoplasmic reticulum (ER) stress associated proteins (BiP, calreticulin, PDI, and ATF6) in three kinds of skeletal muscles of Gryllus bimaculatus. Dorsal ventral flight muscle (DVM), dorsal longitudinal, and dorsal wing flight muscles (DWM) were identified as typical skeletal muscles by H&E staining. Obvious gene expression was detected in the DVM, but not in the DLM or DWM. In addition to gene expression in DVM, gene expression of decorin, a type of myokine, was also significantly increased by starvation. This is the first study that reports the effect of starvation on expression of troponin complex genes and ER stress associated genes in three types of skeletal muscles.

Effects of aging and resistance training in rat tendon remodeling.

In elderly persons, weak tendons contribute to functional limitations, injuries, and disability, but resistance training can attenuate this age-related decline. We evaluated the effects of resistance training on the extracellular matrix (ECM) of the calcaneal tendon (CT) in young and old rats and its effect on tendon remodeling. Wistar rats aged 3 mo (young, n = 30) and 20 mo (old, n = 30) were divided into 4 groups: young sedentary, young trained, old sedentary (OS), and old trained (OT). The training sessions were conducted over a 12-wk period. Aging in sedentary rats showed down-regulation in key genes that regulated ECM remodeling. Moreover, the OS group showed a calcification focus in the distal region of the CT, with reduced blood vessel volume density. In contrast, resistance training was effective in up-regulating connective tissue growth factor, VEGF, and decorin gene expression in old rats. Resistance training also increased proteoglycan content in young and old rats in special small leucine-rich proteoglycans and blood vessels and prevented calcification in OT rats. These findings confirm that resistance training is a potential mechanism in the prevention of aging-related loss in ECM and that it attenuates the detrimental effects of aging in tendons, such as ruptures and tendinopathies.-Marqueti, R. C., Durigan, J. L. Q., Oliveira, A. J. S., Mekaro, M. S., Guzzoni, V., Aro, A. A., Pimentel, E. R., Selistre-de-Araujo, H. S. Effects of aging and resistance training in rat tendon remodeling.

Scar formation following excisional and burn injuries in a red Duroc pig model.

Scar research is challenging because rodents do not naturally form excessive scars, and burn depth, size, and location cannot be controlled in human longitudinal studies. The female, red Duroc pig model has been shown to form robust scars with biological and anatomical similarities to human hypertrophic scars. To more closely mimic the mode of injury, recreate the complex chemical milieu of the burn wound environment and enhance scar development, an animal model of excessive burn-induced scarring was developed and compared with the more commonly used model, which involves excisional wounds created via dermatome. Standardized, full-thickness thermal wounds were created on the dorsum of female, red Duroc pigs. Wounds for the dermatome model were created using two different total dermatome settings: ∼1.5 mm and ≥ 1.9 mm. Results from analysis over 150 days showed that burn wounds healed at much slower rate and contracted more significantly than dermatome wounds of both settings. The burn scars were hairless, had mixed pigmentation, and displayed fourfold and twofold greater excess erythema values, respectively, compared with ∼1.5 mm and ≥ 1.9 mm deep dermatome injuries. Burn scars were less elastic, less pliable, and weaker than scars resulting from excisional injuries. Decorin and versican gene expression levels were elevated in the burn group at day 150 compared with both dermatome groups. In addition, transforming growth factor-beta 1 was significantly up-regulated in the burn group vs. the ∼1.5 mm deep dermatome group at all time points, and expression remained significantly elevated vs. both dermatome groups at day 150. Compared with scars from dermatome wounds, the burn scar model described here demonstrates greater similarity to human hypertrophic scar. Thus, this burn scar model may provide an improved platform for studying the pathophysiology of burn-related hypertrophic scarring, investigating current anti-scar therapies, and development of new strategies with greater clinical benefit.

A Comparison of Gene Expression of Decorin and MMP13 in Hypertrophic Scars Treated With Calcium Channel Blocker, Steroid, and Interferon: A Human-Scar-Carrying Animal Model Study

The formation of hypertrophic scaring (HSc) is an abnormal wound-healing response. In a previous study, an animal model with human scar tissue implanted into nude mice (BALB/c) has been successfully established. The effects of verapamil as well as combination therapy with verapamil and kenacort have been studied and compared. To treat persistent hypertrophic scars, local injection of drugs composed of steroids, calcium channel blockers (CCBs), and interferon might be a good method. What is the best dose of the regimen and what are the mechanisms are also a worthwhile study. Scar specimens were harvested from patients with HSc or Keloid resulting from burn injury, and then implanted to BALB/c-nu nude mice for 4 weeks. Before implantation, the specimen was either injected with or without drugs such as steroids (kenacort), CCBs (verapamil), and interferons (INFα2b), respectively. After the removal of implants, quantitative gene expressions of decorin and collagenase (MMP13) were measured using a real-time polymerase chain reaction to detect their mRNAs. Two way-ANOVA and Post Hoc were used for statistical analysis using the software SPSS 15.0. All drug-treated groups increased the expressions of decorin and MMP13 in comparison with those in noninjected group (p < .001) in a dose-dependent manner. Comparing equal amounts of individual drugs, gene expression of decorin was increased with increasing injection amount, and the best result in low amount of injection (0.02 mL of each) was shown in the group injected with INFα2b followed by kenacort and verapamil. However, the results were changed while injection amount was up to 0.04 mL and the strongest decorin gene expression was found in kenacort injection. Regarding MMP-13 expression, low-amount injection (0.02 mL) of INFα2b has strongest gene expression followed by kenacort and verapamil, but in the large-amount regimes (0.04 mL), verapamil had strongest gene expression followed by INFα2b and kenacort. This study showed that the kenacort, verapamil, and INFα2b all inhibited HSc in a dose-dependent manner through the evidence of gene expression of decorin and MMP13. In comparison with the injections between small amounts of drugs, INFα2b potentiated the strongest decorin and MMP13 expression. On the contrary, among the large-amount injection regimes, kenacrot was more effective on decorin expression as verapamil to MMP13 expression. To decrease side effects from the drugs and produce promising results for the clinical practice, it is suggested to maintain the dose of INFα2b along with an increased dose of verapamil for HSc improvement.

Decorin expression is decreased in first trimester placental tissue from pregnancies with small for gestation age infants at birth

Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. FGR pregnancies are often associated with histological evidence of placental vascular thrombosis. The proteoglycans are important components and regulators of vascular homeostasis. Previous studies from our laboratory highlighted mRNA and protein expression differences in placental proteoglycan decorin (DCN), within a clinically well-characterised cohort of third-trimester idiopathic FGR compared with gestation-matched uncomplicated control pregnancies. We also showed that decorin contributes to abnormal angiogenesis and increased thrombin generation in vitro. These observations suggest that DCN gene expression may contribute to the etiology of FGR. Small for gestational age (SGA) is frequently used as a proxy for FGR and is defined as a birth weight below the 10th percentile of a birth weight curve. We therefore made use of a unique resource of first trimester tissues obtained via chorionic villus sampling during the first trimester to investigate the temporal relationship between altered DCN expression and any subsequent development of SGA. We hypothesized that placental DCN expression is decreased early in gestation in SGA pregnancies. Surplus chorionic villus specimens from 15 women subsequently diagnosed with FGR and 50 from women with uncomplicated pregnancies were collected. DCN mRNA and DCN protein were determined using real-time PCR and immunoblotting, respectively. Both DCN mRNA and protein were significantly decreased in placentae from first-trimester SGA-pregnancies compared with controls (p < 0.05). This is the first study to report a temporal relationship between altered placental DCN expression and subsequent development of SGA.