BACKGROUND Bronchopulmonary dysplasia (BPD) is a chronic lung disease mostly affecting premature infants. Long non-coding RNA (lncRNA) X inactive specific transcript (Xist) is actively involved in pulmonary disease development. The present study explored the potential mechanism of Xist in BPD development. MATERIAL AND METHODS First, newborn BPD mouse models were successfully established. lncRNAs and genes with differential expression were identified using microarray analysis. Various injuries and radial alveolar counts of lung tissues of BPD mice were detected by hematoxylin-eosin staining. Functional assays were utilized to detect alterations of superoxide dismutase (SOD), malondialdehyde (MDA), vascular endothelial growth factor, collagen I, alpha-smooth muscle Actin, TGF-ß1, and Smad3. Then, dual-luciferase reporter gene assay and RNA pull-down assay were performed to clarify the targeting relationship between Xist and miR-101-3p and between miR-101-3p and high-mobility group protein B3 (HMGB3). RESULTS In BPD mice, radial alveolar counts value and SOD activity declined while MDA level increased. Results of microarray analysis found that Xist and HMGB3 were highly expressed in BPD mice. Next, silenced Xist alleviated lung damage in BPD mice. Xist competitively bound to miR-101-3p to activate HMGB3, and overexpressed miR-101-3p mitigated lung damage in BPD mice. Additionally, silenced Xist downregulated the TGF-ß1/Smad3 axis. CONCLUSIONS Our study demonstrated that silencing of Xist suppressed BPD development by binding to miR-101-3p and downregulating HMGB3 and the TGF-b1/Smad3 axis. Our results may provide novel insights for BPD treatment.

Recombinant human interferon beta (rhIFN-β) is a glycoprotein produced by genetically engineered cells and has anti-virus, anti-tumor and immunoregulation functions. Although studies have shown that other subtypes of IFN such as IFN-γ affects cell proliferation and differentiation to some extent, the effect of rhIFN-β on chondrogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) is less known. In this study we studied the effect of rhIFN-β on the chondrogenic differentiation of hMSCs by inducing hMSCs into cartilage pellet via adding IFN-β1a into regular TGF-β3 chondrogenic differentiation medium. We collected the induced pellets and then detected GAG content, assessed pellets size, observed agreecan using alcian blue staining, and analyzed the expression of Sox and CollangenⅡusing real-time PCR and Western blotting. Addition of 100 ng/mL IFN-β1a to regular TGF-β3 chondrogenic differentiation medium could improve the concentration of GAG, increase the size of pellets, promote the formation of aggrecan and up-regulate the expression of CollangenII and Sox9. IFN-β1a combined with TGF-β3 could promote chondrogenic differentiation of hMSCs.

Metastasis is a major risk for lung adenocarcinoma-related mortality. Accumulating evidence raises the possibility that anticancer therapies might be more sensitive by targeting premetastatic niches in addition to the cancer cells themselves. Here, we identified a subpopulation of metastatic lung adenocarcinoma, which was characterized by EMT-related markers such as E-cadherin, Twist, SMAD, and β-catenin. EMT⁺ cases exhibited poorer prognosis than EMT^- patients, reflecting the pro-metastatic features of EMT. Immunohistochemical staining decorated CD15⁺ PMN-MDSCs surrounding EMT⁺ cancer cells in lymph nodes. Metastatic tissues secreted high levels of chemokines, including CXCL1, CXCL5, and CCL2, into the circulation to recruit histidine decarboxylase (Hdc)-positive PMN-MDSCs into metastatic colonies through upregulated CXCR2. The percentage of Hdc⁺ PMN-MDSCs increased in the setting of metastasis. Hdc⁺ PMN-MDSCs obtained from EMT⁺ metastatic masses expressed a higher level of TGF-β1, rather than TGF-β2 and TGF-β3, compared to EMT^- counterparts. The depletion of Hdc⁺ PMN-MDSCs or downregulation of TGF-β1 significantly decreased EMT⁺ percentage and, thus, hampered the metastasis process in murine models. Together, our findings suggest that metastatic tumor secretes high levels of chemokines to recruit Hdc⁺ PMN-MDSCs, which, in turn, express TGF-β1 to induce cancer cells to undergo EMT at metastatic sites.

Keywords: MDSCs; TGF-β; epithelial-mesenchymal transition; lung adenocarcinoma; metastasis.

MicroRNAs (miRNAs) have been shown to be involved in the pathophysiological processes of pain. At present, the roles and mechanisms of miRNAs in neonatal repetitive pain are largely unknown. In our research, the expression of miR-140-3p was increased in premature infants who received repetitive painful stimuli since admission, and in rat pups after repetitive needlestick stimulation. As a result of behavioral testing, the inhibition of miR-140-3p significantly suppressed abnormal mechanical and thermal hyperalgesia in rats after needlestick. Furthermore, the inhibition decreased the expression of the inflammatory cytokines IL-1β, TNF-α, and IL-6, as well as glucocorticoid receptor expression in rats after needlestick. Using bioinformatic analyses, the 3'-untranslated region of TGF-β3 was predicted to be a target of miR-140-3p. Down-regulation of miR-140-3p significantly promoted the expression of TGF-β3 in vitro and in vivo. Mechanistic investigations revealed that TGF-β3 is a direct target of miR-140-3p, and is involved in the miR-140-3p-mediated effects on neonatal repetitive pain and neuroinflammation. In summary, our current research suggests that down-regulation of miR-140-3p can inhibit painful tactile stimulation of rat pups by inhibiting TGF-β3. Our results suggest that miR-140-3p may provide a new regulatory target for preventing the effects of neonatal repetitive pain.

Osteoarthritic degeneration of cartilage is a major social health problem. Tissue engineering of cartilage using combinations of scaffold and mesenchymal stem cells (MSCs) is emerging as an alternative to existing treatment options such as microfracture, mosaicplasty, allograft, autologous chondrocyte implantation, or total joint replacement. Induction of chondrogenesis in high density pellets of MSCs is generally attained by soluble exogenous TGF-β3 in culture media which requires lengthy in vitro culture period during which pellets gain mechanical robustness. On the other hand, a growth factor delivering and a mechanically-robust scaffold material that can accommodate chondroid pellets would enable rapid deployment of pellets after seeding. Delivery of the growth factor from the scaffold locally would drive the induction of chondrogenic differentiation in the post-implantation period. Therefore, we sought to develop a biomaterial formulation that will induce chondrogenesis in situ, and compared its performance to soluble delivery in vitro. In this vein, a heparin-conjugated mechanically-robust collagen fabric was developed for sustained delivery of TGF-β3. The amount of conjugated heparin was varied to enhance the amount of TGF-β3 uptake and release from the scaffold. The results showed that the scaffold delivered TGF-β3 for up to 8 days of culture which resulted in 15-fold increase in GAG production, and 6-fold increase in collagen synthesis with respect to the No TGF-β3 group. The resulting matrix was cartilage-like, in that type II collagen and aggrecan were positive in the spheroids. Enhanced chondrogenesis under in situ TGF-β3 administration resulted in a Young's modulus of ~600 kPa. In most metrics, there were no significant differences between the soluble delivery group and in situ heparin-mediated delivery group. In conclusion, heparin-conjugated collagen scaffold developed in this study guides chondrogenic differentiation of hMSCs in a mechanically competent tissue construct, which showed potential to be used for cartilage tissue regeneration.

Regeneration of large-sized acute and chronic wounds provoked by severe burns and diabetes is a major concern worldwide. The availability of immunocompatible matrix with a wide range of regenerative medical applications, more specifically, for nonhealing chronic wounds is an unmet clinical need. Extrapolating the in vitro tissue engineering knowledge for in vivo guided wound regeneration could be a meaningful approach. This study aimed to develop a completely human-derived and minimally immune-responsive scaffold comprising of acellular amniotic membrane (AM), fibrin (FIB) and hyaluronic acid (HA), termed AMFIBHA. The potential for in vivo guidance of skin regeneration was validated through in vitro dermal tissue assembly on the combination scaffold by growing human fibroblasts, differentiated from human adipose tissue-derived mesenchymal stem cells (hADMSCs). An effective method was standardized for obtaining decellularized amnion (dAM) for assuring better immuno-compatibility. The biochemical stability of dAM upon plasma sterilization (pdAM) confirms its suitability for both in vitro and in vivo tissue engineering. The problem of poor handling characteristics was solved by combining the dried dAM with fibrin derived from a clinically used fibrin sealant kit. An additional constituent HA, derived from human umbilical cord tissue, imparts the required water absorption and retention property for better cell migration and growth. Post sterilization, the combination scaffold AMFIBHA demonstrated hemo-/cytocompatibility, confirming the absence of detergent residuals. Upon long-term (20 days/40 days) culture of hADMSC-derived fibroblasts, the suppleness of generated tissue was established by demonstrating regulated deposition of collagen, elastin, and glycosaminoglycans using both qualitative and quantitative measurements. Regulated expressions of transforming growth factors-beta 1 (TGF-β1) & TGF-β3, alpha smooth muscle actin (α-SMA), fibrillin-1, collagen subtypes, and elastin suggest non-fibrotic fibroblast phenotype, which could be an effect of microenvironment endowed by the AM, FIB, and HA. In burn wound model experiments, immune response to cellular AM was prominent as compared to untreated/sham control wounds and decellularized AM-treated and AMFIBHA-treated wounds, ensuring biocompatibility. Wound regeneration with complete epithelialization, angiogenesis, development of rete pegs, and other skin appendages were clearly visualized in 28 days after treating large-sized (4 × 4 cm²), debrided, full-thickness third-degree burn wounds, indicating guided wound regeneration potential of AMFIBHA dermal substitute.

Keywords: decellularized amnion; dermal construct; fibrin; hyaluronic acid; skin tissue engineering; wound care.

C1q/TNF-related protein 9 (CTRP9) is implicated in diverse cardiovascular diseases, but its role in viral myocarditis (VMC) is not well explored. This study is aimed at investigating the role and potential mechanism of CTRP9 in VMC. Herein, we found that the peripheral blood collected from children with VMC had lower CTRP9 levels than that from children who had recovered from VMC. H9c2 cardiomyocytes treated with coxsackievirus <em>B3</em> (CV<em>B3</em>) were applied to establish a VMC model <i>in vitro</i>, and the expression of CTRP9 was significantly decreased in CV<em>B3</em>-induced H9c2 cells. The overexpression of CTRP9 attenuated CV<em>B3</em>-induced apoptosis, inflammation, and fibrosis reactions in H9c2 cells by promoting cell proliferation, reducing the cell apoptosis rate, and inhibiting inflammatory cytokine levels and fibrosis-related gene expression. Moreover, we found that thrombospondin 1 (THBS1) levels were increased in children with VMC, and CTRP9 negatively regulated THBS1 expression by interacting with THBS1. The downregulation of THBS1 inhibited CV<em>B3</em>-induced apoptosis, inflammation, and fibrosis in H9c2 cells. In addition, our mechanistic investigation indicated that the overexpression of THBS1 impaired the inhibitory effect of CTRP9 on CV<em>B3</em>-induced H9c2 cells. The results further revealed that the CV<em>B3</em>-induced NF-<i>κ</i>B and <em>TGF</em>-<i>β</i>1/Smad2/3 signaling pathways of H9c2 cells were blocked by CTRP9 yet activated by THBS1. In conclusion, CTRP9 protected H9c2 cells from CV<em>B3</em>-induced injury via the NF-<i>κ</i>B and <em>TGF</em>-<i>β</i>1/Smad2/3 signaling pathways by modulating THBS1.

Background: Early rhinovirus (RV) infection is a strong risk factor for asthma development. Airway remodeling factors play a key role in the progression of the asthmatic condition. We hypothesized that RV infection in young children elicits the secretion of growth factors implicated in airway remodeling and asthma progression. Methods: We examined the nasal airway production of remodeling factors in children ( ≤ 2 years old) hospitalized due to PCR-confirmed RV infection. Airway remodeling proteins included: MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, TIMP-1, TIMP-2, EGF, Angiopoietin-2, G-CSF, BMP-9, Endoglin, Endothelin-1, Leptin, FGF-1, Follistatin, HGF, HB-EGF, PLGF, VEGF-A, VEGF-C, VEGF-D, FGF-2, TGF-β1, TGF-β2, TGF-β3, PDGF AA, PDGF BB, SPARC, Periostin, OPN, and TGF-α. Results: A total of 43 young children comprising RV cases (n = 26) and uninfected controls (n = 17) were included. Early RV infection was linked to (1) enhanced production of several remodeling factors (e.g., HGF, TGFα), (2) lower MMP-9/TIMP-2 and MMP-2/TIMP-2 ratios, and (3) increased MMP-10/TIMP-1 ratios. We also found that relative to term infants, severely premature children had reduced MMP-9/TIMP-2 ratios at baseline. Conclusion: RV infection in young children elicits the airway secretion of growth factors implicated in angiogenesis, fibrosis, and extracellular matrix deposition. Our results highlight the potential of investigating virus-induced airway remodeling growth factors during early infancy to monitor and potentially prevent chronic progression of respiratory disorders in all ages.

Keywords: airway remodeling; growth factor; infancy; prematurity; rhinovirus (RV).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental teratogenic agent for cleft palate. But transforming growth factor β3 (TGF-β3) is an essential growth factor for palatogenesis. This study is to clarify effects of TCDD and TGF-β3 in mouse embryonic palatal mesenchymal (MEPM) cells. The result showed that with increase of TCDD (0.5 nM-10 nM), the expression of TGF-β3 increased, but after 10 nM TCDD, the expression of TGF-β3 reduced. The viabilities of MEPM cells decreased in 10 nM TCDD-treated group. But the viabilities increased in 10 ng/mL TGF-β3-treated group, or the viabilities were between that of them in combination of 10 nM TCDD and 10 ng/mL TGF-β3-treated group. This phenomenon was the same as the motilities. In addition, we found that the expression of phosphorylated Smad2/3 and Smad7 was increased by 10 nM TCDD, 10 ng/mL TGF-β3, or combination of 10 nM TCDD and 10 ng/mL TGF-β3 induced, but the expression of Smad4 was decreased. These data revealed that the TGF-β/Smad signaling pathway affected TCDD and TGF-β3 in MEPM cells.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification with decreased numbers of alveoli and increased airspace. BPD, frequently suffered by very low birth weight infants, has been closely associated with intrauterine infection. However, the underlying mechanisms of BPD remain unclear. In the present study, it was identified that administration of intra-amniotic lipopolysaccharide (LPS) to pregnant rats on embryonal day 16.5 (E16.5) induced significant alveolarization arrest similar to that of BPD in neonatal pups, and theophylline injected subcutaneously into the newborns improved the pathological changes. To further investigate the underlying mechanism of the morphogenesis amelioration of theophylline, cytokine antibody arrays were performed with the lung lysates of neonatal rats. The results indicated that LPS upregulated a series of pro-inflammatory cytokines and theophylline significantly attenuated the expression levels of pro-inflammatory cytokines tumor necrosis factor‑α, macrophage inflammatory protein (MIP)-1α and MIP-2, and markedly elevated the production of tumor growth factor (TGF)-β family members TGF-β1, TGF-β2 and TGF-β3, which are anti‑inflammatory cytokines. Accordingly, it was hypothesized that theophylline may protect against BPD and improve chorioamnionitis‑induced alveolar arrest by regulating the balance between pro‑and anti-inflammatory cytokine expression.

OBJECTIVE

Antidepressants are known to positively influence several factors in patients with depressive disorders, resulting in increased neurogenesis and subsequent relief of depressive disorders. To study the effects of venlafaxine during neural differentiation at the cellular level, we looked at its effect on protein expression and regulation mechanisms during neural differentiation.

METHODS

After exposing NCCIT cell-derived EBs to venlafaxine during differentiation (1 day and 7 days), changes in protein expression were analyzed by 2-DE and MALDI-TOF MS analysis. Gene levels of proteins regulated by venlafaxine were analyzed by real-time RT-PCR.

RESULTS

Treatment with venlafaxine decreased expression of prolyl 4-hydroxylase (P4HB), ubiquitin-conjugating enzyme E2K (HIP2) and plastin 3 (T-plastin), and up-regulated expression of growth factor beta-3 (TGF-β3), dihydropyrimidinase-like 3 (DPYSL3), and pyruvate kinase (PKM) after differentiation for 1 and 7 days. In cells exposed to venlafaxine, the mRNA expression patterns of HIP2 and PKM, which function as negative and positive regulators of differentiation and neuronal survival, respectively, were consistent with the observed changes in protein expression.

CONCLUSIONS

Our findings may contribute to improve understanding of molecular mechanism of venlafaxine.

The existence of diabetes and microbial infection in burn wounds makes the healing process more complex. Herein, we synthesize a collagen based hybrid scaffold incorporated with a silica-resorcinol composite and cross-linked with an oxidized fenugreek seed polysaccharide to stimulate scar-less healing in chronic wounds with type-I diabetes and microbial infection. The spectroscopic analyses of the hybrid scaffolds reveal the chemical and structural integrity of collagen. The hybrid scaffolds are shown to be appropriate for in vivo tissue regeneration through cytocompatibility and hemocompatibility studies. Scaffolds were applied to diabetic albino rats induced with chronically infected burn wounds with respective controls. Histological and immunohistochemical analyses of the granulation tissue collected from the hybrid scaffold treated animal groups showed improved angiogenesis, reepithelialization and TGF-β3 expression, which eventually led to scar-less wound healing. The results confirm that the prepared hybrid collagen scaffold could be used for effective scar-less wound healing in chronic burn wounds.

BACKGROUND

In peritoneal dialysis (PD), the peritoneum is exposed to intermediate Amadori adducts (AmAs) and advanced (AGE) glycated products of proteins. The aim of this study was to test the capacity of AmAs created in different PD solutions (PDSs) to elicit a fibroblast-like transdifferentiation of human peritoneal mesothelial cells (HPMCs) in culture.

METHODS

HPMCs were incubated for 12 hours with AmA obtained by human serum albumin (HSA) incubated for 6 days with commercial 3.86% glucose (Glu), 1.36% Glu and 7.5% icodextrin (Ico) PDS. Mesenchymal (vimentin), epithelial (cadherin) and myofibroblastic (Type I collagen and alpha smooth muscle cell actin [ASMA]) markers were evaluated (RT-PCR, immunostaining and Western blot), as well as TGF-b3 synthesis (ELISA and Western blot).

RESULTS

Ico-PDS was less active than 3.86% and 1.36% Glu-PDS in glycating albumin (p<0.001). AmA-HSA-Glu 3.86% and 1.36% induced a significantly higher increase in vimentin and Type I collagen mRNA expression than AmA-HSA-Ico (p<0.0001). By contrast, AmA-HSA-Glu 3.86% and 1.36% induced a reduction in cadherin mRNA expression which was significantly different from AmA- HSA-Ico (p<0.0001). RT-PCR data were confirmed by immunostaining and Western blot analysis. AmA-HSA-Glu 3.86% and 1.36% induced a significantly higher increase in ASMA mRNA expression than AmA-HSA-Ico (p<0.0001). AmA-HSA-Glu 3.86% and 1.36% stimulated ASMA and TGF-b3 synthesis which were significantly higher than AmA-HSA-Ico (p<0.001 and p<0.01, respectively).

CONCLUSIONS

Our data suggest that Glu-PDS, but not Ico-PDS, can turn on the fibroblastic-like transdifferentiation in HPMCs, and this mechanism may result in peritoneal sclerosis.

BACKGROUND

Atherosclerosis occurs in arterial curvatures and branches, where the flow is disturbed with low and oscillatory shear stress (OSS). The remodeling and alterations of extracellular matrices (ECMs) and their composition is the critical step in atherogenesis. In this study, we investigated the effects of different ECM proteins on the regulation of mechanotransduction in vascular endothelial cells (ECs) in response to OSS.

METHODS

Through the experiments ranging from in vitro cell culture studies on effects of OSS on molecular signaling to in vivo examinations on clinical specimens from patients with coronary artery disease (CAD), we elucidated the roles of integrins and different ECMs, i.e., fibronectin (FN) and laminin (LM), in transforming growth factor (TGF)-β receptor (TβR)-mediated Smad2 activation and nuclear factor-κB (NF-κB) signaling in ECs in response to OSS and hence atherogenesis.

RESULTS

OSS at 0.5±12 dynes/cm2 induces sustained increases in the association of types I and II TβRs with β1 and β3 integrins in ECs grown on FN, but it only transient increases in ECs grown on LM. OSS induces a sustained activation of Smad2 in ECs on FN, but only a transient activation of Smad2 in ECs on LM. OSS-activation of Smad2 in ECs on FN regulates downstream NF-κB signaling and pro-inflammatory gene expression through the activation of β1 integrin and its association with TβRs. In contrast, OSS induces transient activations of β1 and β3 integrins in ECs on LM, which associate with type I TβR to regulate Smad2 phosphorylation, resulting in transient induction of NF-κB and pro-inflammatory gene expression. In vivo investigations on diseased human coronary arteries from CAD patients revealed that Smad2 is highly activated in ECs of atherosclerotic lesions, which is accompanied by the concomitant increase of FN rather than LM in the EC layer and neointimal region of atherosclerotic lesions.

CONCLUSIONS

Our findings provide new insights into the mechanisms of how OSS regulates Smad2 signaling and pro-inflammatory genes through the complex signaling networks of integrins, TβRs, and ECMs, thus illustrating the molecular basis of regional pro-inflammatory activation within disturbed flow regions in the arterial tree.

Background: Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A.

Methods: MenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-β3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture.

Results: MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro.

Conclusion: These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

Keywords: Activin A; Bone marrow; Chondrogenic differentiation; Human mesenchymal stem cells; Menstrual blood; TGF-β3.

Cyclophosphamide (CP) is commonly used as an anticancer agent but has been associated with high toxicity in several organs, including the testes. In this study, we aimed to evaluate the effects of CP-induced testicular toxicity, using glial cell line-derived neurotrophic factor (GDNF), occludin and transforming growth factor beta 3 (TGF-β3) primary antibodies, and miR-34b and miR-34c expressions. Eighteen young Balb/c male mice were divided into three groups. The control group received no treatment. The mice of CP group were injected 100 mg kg^-1 day^-1 CP for 5 days, and the same amount of saline was injected in the sham group. The animals were sacrificed 24 hr after the last injection. Immunohistochemical analysis of testicular tissues showed a decrease in both spermatogenic germ cell count and also GDNF, occludin expressions, but an increase in TGF-β3 expression in the CP group compared to the others group. The expressions of miR-34b and miR-34c were examined by qPCR technique, a significant decrease was observed in tissue samples in the CP-treated group. The expression of GDNF, occludin and TGF-β3 plays an important role in testicular injury caused by CP, and the decrease in the expression of miR-34b/c in tissue samples may be an important marker for the detection of testicular damage.

Keywords: Cyclophosphamide; GDNF; TGF-β3; Testes; miR-34b/c; occludin.

Understanding how the local cellular environment influences cell metabolism, phenotype and matrix synthesis is crucial to engineering functional tissue grafts of a clinically relevant scale. The objective of this study was to investigate how the local oxygen environment within engineered cartilaginous tissues is influenced by factors such as cell source, environmental oxygen tension and the cell seeding density. Furthermore, the subsequent impact of such factors on both the cellular oxygen consumption rate and cartilage matrix synthesis were also examined. Bone marrow derived stem cells (BMSCs), infrapatellar fat pad derived stem cells (FPSCs) and chondrocytes (CCs) were seeded into agarose hydrogels and stimulated with transforming growth factor-β3 (TGF- β3). The local oxygen concentration was measured within the center of the constructs, and numerical modeling was employed to predict oxygen gradients and the average oxygen consumption rate within the engineered tissues. The cellular oxygen consumption rate of hydrogel encapsulated CCs remained relatively unchanged with time in culture. In contrast, stem cells were found to possess a relatively high initial oxygen consumption rate, but adopted a less oxidative, more chondrocyte-like oxygen consumption profile following chondrogenic differentiation, resulting in net increases in engineered tissue oxygenation. Furthermore, a greater reduction in oxygen uptake was observed when the oxygen concentration of the external cell culture environment was reduced. In general, cartilage matrix deposition was found to be maximal in regions of low oxygen, but collagen synthesis was inhibited in very low (less than 2%) oxygen regions. These findings suggest that promoting an oxygen consumption profile similar to that of chondrocytes might be considered a key determinant to the success of stem cell-based cartilage tissue engineering strategies.

Keywords: cartilage; cellular metabolism; chondrogenesis; oxygen consumption; stem cell differentiation; tissue engineering.

Objectives: We aimed to investigate the radiological, biomechanical, histopathological, histomorphometric, and immunohistochemical effects of different doses of vardenafil on fracture healing.

Materials and methods: Fifty-one rats were divided into three groups. Group V5 was given 5 mg/kg/day of vardenafil; Group V10 was given 10 mg/kg/day of vardenafil; and the control group was given the same volume of saline. Six rats from each group were sacrificed on Day 14 (early period) and the remaining rats were sacrificed on Day 42 (late period). Callus/femoral volume and bone mineral density were measured using micro-computed tomography. Five femurs from each group in the late period were examined by biomechanical tests. In addition to the histopathological and histomorphometric evaluations, immunohistochemical analyses were performed to examine the levels of inducible nitric oxide synthase (iNOS), transforming growth factor-3 (TGF-β3), and nuclear factor kappa B (NF-κB) proteins.

Results: Both doses of vardenafil increased primary bone volume and maximal bone fracture strength in late period, compared to the control group (p<0.05). Histological healing scores of vardenafil groups were significantly higher in early period (p<0.001). While cartilaginous callus/total callus ratio in early period was higher, callus diameter/femoral diameter ratio in late period was lower in vardenafil groups (p<0.01). The NF-κB immunopositivity in V10 group decreased in early period, compared to control group (p<0.001). The TGF-β3 and iNOS immunopositivity increased in both V5 and V10 groups, compared to the control group in early period, but returned to normal in late period.

Conclusion: During the first period of fracture healing process in which vasodilation is mostly required with increasing inflammation, vardenafil has ameliorating effects on the bone union and supports fracture healing.

Objective: Approximately 30% of preeclamptic pregnancies exhibit abnormal liver function tests. We assessed liver injury-associated enzyme levels and circulating transforming growth factor beta (TGF-β) levels in an arginine vasopressin (AVP)-induced pregnant Sprague-Dawley rat model.

Methods: Pregnant and non-pregnant Sprague-Dawley rats (n=24) received AVP (150 ng/hr) subcutaneously via mini-osmotic pumps for 18 days. Blood pressure was measured, urine samples were collected, and all animals were euthanized via isoflurane. Blood was collected to measure circulating levels of TGF-β1-3 isomers and liver injury enzymes in pregnant AVP (PAVP), pregnant saline (PS), non-pregnant AVP (NAVP), and non-pregnant saline (NS) rats.

Results: The PAVP group showed significantly higher systolic and diastolic blood pressure than both saline-treated groups. The weight per pup was significantly lower in the AVP-treated group than in the saline group (p<0.05). Circulating TGF-β1-3 isomer levels were significantly higher in the PAVP rats than in the NS rats. However, similar TGF-β1 and TGF-β3 levels were noted in the PS and PAVP rats, while TGF-β2 levels were significantly higher in the PAVP rats. Circulating liver-type arginase-1 and 5'-nucleotidase levels were higher in the PAVP rats than in the saline group.

Conclusion: This is the first study to demonstrate higher levels of TGF-β2, arginase, and 5'-nucleotidase activity in PAVP than in PS rats. AVP may cause vasoconstriction and increase peripheral resistance and blood pressure, thereby elevating TGF-β and inducing the preeclampsia-associated inflammatory response. Future studies should explore the mechanisms through which AVP dysregulates liver injury enzymes and TGF-β in pregnant rats.

Keywords: Arginase 1; Arginine vasopressin; Aspartate transaminase 1; Liver injury; Transforming growth factor beta.

Background: Unexplained graft fibrosis and inflammation are common after pediatric liver transplantation (LT).

Objective: We investigated the graft expression of fibrogenic genes and correlated the findings with transplant histopathology and outcome.

Methods: Liver biopsies from 29 recipients were obtained at a median of 13.1 (IQR: 5.0-18.4) years after pediatric LT. Control samples were from six liver-healthy subjects. Hepatic expression of 40 fibrosis-related genes was correlated to histological findings; normal histology, fibrosis with no inflammation, and fibrosis with inflammation. Liver function was evaluated after a subsequent follow-up of 9.0 years (IQR: 8.0-9.4).

Results: Patients with fibrosis and no inflammation had significantly increased gene expression of profibrotic TGF-β3 (1.17 vs 1.02 p=0.005), CTGF (1.64 vs 0.66 p=0.014), PDGF-α (1.79 vs 0.98 p=0.049), PDGF -β (0.99 vs 0.76 p=0.006), integrin-subunit-β1 (1.19 vs 1.02 p=0.045), α-SMA (1.12 vs 0.58 p=0.013), type I collagen (0.82 vs 0.53 p=0.005) and antifibrotic decorin (1.15 vs 0.99 p=0.045) compared to patients with normal histology. mRNA expression of VEGF A (0.84 vs 1.06 p=0.049) was lower. Only a few of the studied genes were upregulated in patients with both fibrosis and inflammation. The gene expression levels showed no association with later graft outcome.

Conclusions: Altered hepatic expression of fibrosis-related genes is associated with graft fibrosis without concurrent inflammation.

Keywords: Fibrosis; Gene expression; Inflammation; Liver transplantation; Pediatrics.

Biomimetic scaffolds that provide a tissue-specific environment to cells are particularly promising for tissue engineering and regenerative medicine applications. The goal of this study was to integrate emerging additive manufacturing and biomaterial design strategies to produce articular cartilage (AC) mimetic scaffolds that could be used as 'off-the-shelf' implants for joint regeneration. To this end alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize porous alginate-based scaffolds and to support the sustained release of transforming growth factor-β3 (TGF-β3). Covalent crosslinking dramatically improved the elasticity of the alginate/alginate sulfate scaffolds, while scaffold architecture could be tailored using a directional freezing technique. Introducing such an anisotropic architecture was found to promote mesenchymal stem cell (MSC) infiltration into the scaffold and to direct the orientation of the deposited extracellular matrix, leading to the development of cartilage tissue with a biomimetic zonal architecture. In vitro experiments also demonstrated the capacity of the sulfated scaffolds to both enhance chondrogenesis of MSCs and to control the release of TGF-β3, leading to the development of a tissue rich in sGAG and type II collagen. The scaffolds were further reinforced with a 3D printed PLCL framework, leading to composite implants that were more elastic than those reinforced with PCL, and which better mimicked the bulk mechanical properties of native cartilage tissue. The ability of this composite scaffold to support chondrogenesis was then confirmed within a dynamic culture system. Altogether, these findings demonstrate the potential of such biomimetic scaffolds as putative 'single-stage' or 'off-the-shelf' strategies for articular cartilage regeneration.

Keywords: additive manufacturing; cartilage; growth factor; off-the-shelf; scaffold.

Purpose: This study performs to evaluate the Hydrophobic and Hydrosmart 360°square-edge intraocular lens drug delivery of Aspirin using an in vitro lens capsular model.

Methods: Cell counting kit-8 assay was used to calculate 50% inhibiting concentration values in both SRA01/04 and HLE-B3 cells. Hoechst staining and transwell assay were used to detect cell proliferation and cell migration. The in vitro lens capsule model was established mainly with a special transwell-col and cell climbing sheet, in which an intraocular lens and the TGF-β2 were added. The ultraviolet spectrophotometer was used to measure the drug concentrations released in vitro. Cell-exclusion zone assay was used to detect the cell migration in the in vitro capsular model.

Results: It shows that cell morphology and distribution of SRA01/04 in the in vitro lens capsular model were closer to those in vivo. The results revealed that there could be significant inhibiting effects on cell migration of the hydrosmart intraocular lens with a sustained drug release in vitro in 7 days, while the hydrophobic intraocular lens drug delivery of Aspirin was mainly performed only from day 1 to day 3.

Conclusions: Results showed the developed hydrosmart intraocular lens could release Aspirin continuously in vitro to inhibit the cell migration of lens epithelial cells.

Keywords: Aspirin; Posterior capsule opacification; cell migration; in vitro capsular model; intraocular lens.

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has largely remained unelucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for the proper MEE removal. Ras Responsive Element Binding Protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental EMT, in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Keywords: EMT; MEE cells; Palatogenesis; Ras; Rreb1; TGF-β3.

Objective: To investigate the molecular mechanism by which pirfenidone inhibits scar formation through the TGF-β/Smad pathway.

Methods: Cultured rabbit tenon fibroblasts (RTFs) were treated with different concentrations of pirfenidone to determine its initial active concentration and optimum concentration of pirfenidone for inhibiting RTF proliferation using CCK-8 assay. In RTFs treated with pirfenidone at the initial and optimal concentrations, expressions of TGF-β3, collagen I and collagen III were examined with both immunofluorescence assay and Western blotting, and their mRNA expression levels were detected using RT-PCR.

Results: The initial and optimal concentrations of pirfenidone for inhibiting RTF proliferation were 0.1 mg/mL and 0.27 mg/mL, respectively. In RTFs treated with pirfenidone at the two concentrations for 24 h, both immunofluorescence assay and Western blotting showed significantly lowered protein expressions of TGF-β3, collagen I and collagen III as compared with those in the control group (P < 0.05). The mRNA expressions of TGF-β3, collagen I and collagen III in the RTFs were also significantly lowered after treatment with pirfenidone at the initial and optimal concentrations (P < 0.05).

Conclusions: Pirfenidone concentration-dependently inhibits the proliferation of RTFs possibly by down-regulating the expression of TGF-β3 in the TGF-β/Smad pathway.

Keywords: fibrosis; pirfenidone; rabbit tenon fibroblasts; transforming growth factor-β.