The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVβ6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVβ6 affinity, in contrast to β1 integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVβ6 on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αVβ6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVβ6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.

Background: Reendothelialisation is the natural pathway that inhibits neointimal hyperplasia and in-stent restenosis. Circulating endothelial progenitor cells (EPCs) derived from bone marrow (BM) might contribute to endothelial repair. However, the temporal and spatial distributions of reendothelialisation and neointimal hyperplasia after EPC transplantation in injured arteries are currently unclear.

Methods: A carotid balloon injury (BI) model was established in Sprague-Dawley rats, and PKH26-labelled BM-derived EPCs were transplanted after BI. The carotid arteries were harvested on the first, fourth, seventh, and 14th day post-injury and analysed via light-sheet fluorescence microscopy and pathological staining (n = 3). EPC and human umbilical vein endothelial cell culture supernatants were collected, and blood samples were collected before and after transplantation. The paracrine effects of VEGF, IGF-1, and TGF-β1 in cell culture supernatants and serum were analysed by enzyme-linked immunosorbent assay (n = 4).

Results: Transplanted EPCs labelled with PKH26 were attached to the injured luminal surface the first day after BI. In the sham operation group, the transplanted EPCs did not adhere to the luminal surface. From the fourth day after BI, the mean fluorescence intensity of PKH26 decreased significantly. However, reendothelialisation and inhibition of neointimal hyperplasia were significantly promoted by transplanted EPCs. The degree of reendothelialisation of the EPC^7d and EPC^14d groups was higher than that of the BI^7d and BI^14d groups, and the difference in neointimal hyperplasia was observed between the EPC^14d and BI^14d groups. The number of endothelial cells on the luminal surface of the EPC^14d group was higher than that of the BI^14d group. The number of infiltrated macrophages in the injured artery decreased in the EPC transplanted groups.

Conclusions: Transplanted EPCs had chemotactic enrichment and attached to the injured arterial luminal surface. Although decreasing significantly after the fourth day at the site of injury after transplantation, transplanted EPCs could still promote reendothelialisation and inhibit neointimal hyperplasia. The underlying mechanism is through paracrine cytokines and not differentiation into mature endothelial cells.

Keywords: Angioplasty; Endothelial progenitor cells; Light-sheet fluorescence microscopy; Neointimal hyperplasia; Reendothelialisation.

Importin 8, encoded by IPO8, is a ubiquitously expressed member of the importin-β protein family that translocates cargo molecules such as proteins, RNAs, and ribonucleoprotein complexes into the nucleus in a RanGTP-dependent manner. Current knowledge of the cargoes of importin 8 is limited, but TGF-β signaling components such as SMAD1-4 have been suggested to be among them. Here, we report that bi-allelic loss-of-function variants in IPO8 cause a syndromic form of thoracic aortic aneurysm (TAA) with clinical overlap with Loeys-Dietz and Shprintzen-Goldberg syndromes. Seven individuals from six unrelated families showed a consistent phenotype with early-onset TAA, motor developmental delay, connective tissue findings, and craniofacial dysmorphic features. A C57BL/6N Ipo8 knockout mouse model recapitulates TAA development from 8-12 weeks onward in both sexes but most prominently shows ascending aorta dilatation with a propensity for dissection in males. Compliance assays suggest augmented passive stiffness of the ascending aorta in male Ipo8^-/- mice throughout life. Immunohistological investigation of mutant aortic walls reveals elastic fiber disorganization and fragmentation along with a signature of increased TGF-β signaling, as evidenced by nuclear pSmad2 accumulation. RT-qPCR assays of the aortic wall in male Ipo8^-/- mice demonstrate decreased Smad6/7 and increased Mmp2 and Ccn2 (Ctgf) expression, reinforcing a role for dysregulation of the TGF-β signaling pathway in TAA development. Because importin 8 is the most downstream TGF-β-related effector implicated in TAA pathogenesis so far, it offers opportunities for future mechanistic studies and represents a candidate drug target for TAA.

Keywords: Loeys-Dietz syndrome; Shprintzen-Goldberg syndrome; TGF-beta; importin 8; knockout mouse model; thoracic aortic aneurysm.

Diabetic kidney disease (DKD) remains the most common cause of kidney failure and the treatment options are insufficient. Here, we used a connectivity mapping approach to first collect 15 gene expression signatures from 11 DKD related published independent studies. Then, by querying the Library of Integrated Network-based Cellular Signatures (LINCS) L1000 dataset, we identified drugs and other bioactive small molecules that are predicted to reverse these gene signatures in the diabetic kidney. Among the top consensus candidates, we selected a PLK1 inhibitor (BI-2536) for further experimental validation. We found that PLK1 expression was increased in the glomeruli of both human and mouse diabetic kidneys and localized largely in mesangial cells. We also found that BI-2536 inhibited mesangial cell proliferation and extracellular matrix in-vitro and ameliorated proteinuria and kidney injury in DKD mice. Further pathway analysis of the genes predicted to be reversed by the PLK1 inhibitor were of members of the TNF-α/NF-κB, JAK/STAT, and TGF-β/Smad3 pathways. In vitro, either BI-2536 treatment or knockdown of PLK1 dampened the NF-κB and Smad3 signal transduction and transcriptional activation. Together, these results suggest that the PLK1 inhibitor BI-2536 should be further investigated as a novel therapy for DKD.

Localized delivery, comparing to systemic drug administration, offers a unique alternative to enhance efficacy, lower dosage, and minimize systemic tissue toxicity by releasing therapeutics locally and specifically to the site of interests. Herein, a localized drug delivery platform ("plum‒pudding" structure) with controlled release and long-acting features is developed through an injectable hydrogel ("pudding") crosslinked <i>via</i> self-assembled triblock polymeric micelles ("plum") to help reduce renal interstitial fibrosis. This strategy achieves controlled and prolonged release of model therapeutics in the kidney for up to three weeks in mice. Following a single injection, local treatments containing either anti-inflammatory small molecule celastrol or anti-<em>TGF</em><i>β</i> antibody effectively minimize inflammation while alleviating fibrosis <i>via</i> inhibiting NF-<i>κ</i>B signaling pathway or neutralizing <em>TGF</em>-<i>β</i>1 locally. Importantly, the micelle-hydrogel hybrid based localized therapy shows enhanced efficacy without local or systemic toxicity, which may represent a clinically relevant delivery platform in the management of renal interstitial fibrosis.

Keywords: Anti-TGFβ antibody; BSA, bovine serum albumin; CLT, celastrol; Celastrol; Controlled release; Cy5.5-NHS, cyanine 5.5-N-hydroxysuccinimide; DAPI, 4′,6-diamidino-2-phenylindole; DEX, dexamethasone; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanineperchlorate; ECM, extracellular matrix; EDCI, carbodiimide hydrochloride; ESR, equilibrium swelling ratio; FITC, fluorescein isothiocyanate; G", the loss modulus; G', storage modulus; HA, hyaluronic acid; HASH, thiolated hyaluronic acid; Hydrogel; IL-1β, interleukin 1β; IL-6, interleukin 6; Inflammation; Localized therapy; MOD, mean optical density; NHS, N-hydroxysuccinimide; PDI, polydispersity index; RIF, renal interstitial fibrosis; RSR, real-time swelling ratio; Renal fibrosis; SD, standard deviation; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TGF-β1, transforming growth factor β1; TNF-α, tumor necrosis factor α; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; UUO, unilateral ureteral obstruction; bis-F127-MA, bis-F127-methacrylate; iNOS, nitric oxide synthase; α-SMA, α-smooth muscle actin; “Plum‒pudding” structure.

1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (tetrahydrocurcumin, THC) is a major bioactive metabolite of curcumin, demonstrating the potential anti-inflammatory, antioxidant and neuroprotective properties, etc. In this study, it was found that Aβ induced decreased cell viability, cell cycle arrest and apoptosis in BV-2 cells, which were ameliorated by THC. In vivo, THC administration rescued learning and memory, and reduced Aβ burden in the hippocampus of APP/PS1 mice. By proteomic analysis of the hippocampus of mice, 157 differentially expressed proteins were identified in APP/PS1 mice treated with THC (comparing with APP/PS1 mice), which also suggested that the effects of THC on the cell cycle and apoptosis were mostly related to the "Ras signaling pathway", etc. In APP/PS1 mice, the down-regulation of Gab2 and K-Ras, and the up-regulation of caspase-3, TGF-β1 and TNF-ɑ were observed; THC attenuated the abnormal expression of Gab2, K-Ras, caspase-3 and TNF-ɑ, and up-regulated TGF-β1 and Bag1 expression. In BV-2 cells, Aβ induced the down-regulation of Gab2, K-Ras and TGF-β1, and the overexpression of caspase-3, PARP1, cleaved-PARP1 and TNF-ɑ, which were restored by THC. Moreover, THC up-regulated Bag1 expression in Aβ-treated BV-2 cells. The decreased transcriptional expression of Ccnd2 and Cdkn1a were also observed in Aβ-treated BV-2 cells, and THC alleviated the down-regulation of Ccnd2. For the first time, we identified that the action of THC in preventing AD was associated with inhibition of cell cycle arrest and apoptosis of microglia via the Ras/ERK signaling pathway, shedding new light on the role of THC in alleviating the progression of AD.

Keywords: Alzheimer’s disease; Cell apoptosis; Cell cycle; Microglia; Ras/ERK signaling pathway; Tetrahydrocurcumin.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) are structurally related growth factors that exert their biological actions by binding to the same cell-surface receptor, EGF receptor. However, in chicken cells, human EGF binds with approximately 100-fold lower affinity than human TGF-alpha. In a previous study, we localized EGF/TGF-alpha receptor immunohistochemically in the granulosa and theca of the developing follicles of laying hens. We have also shown that TGF-alpha binds to cell-surface receptors of the granulosa cells. The present study characterizes the nature of the EGF/TGF-alpha receptor. Immunoprecipitation of receptor proteins from cultured granulosa cells with an anti-EGF receptor antibody (12E) shows the expression of a 170-kDa receptor protein. The expression of the receptor protein decreases with follicular enlargement between the F3 and F1. Incubation of the cells with [125I]TGF-alpha followed by cross-linking with bis(sulphosuccinimidyl)suberate showed that TGF-alpha binds a similar (170 kDa) receptor protein immunoprecipitated with the 12E anti-EGF receptor antibody. The binding of TGF-alpha to granulosa cells caused receptor protein oligomerization, yielding the monomeric (170 kDa) and dimeric (340 kDa) protein forms. Oligomerization seemed to favour the formation of the dimeric rather than the monomeric form. Culturing granulosa cells with luteinizing hormone or follicle-stimulating hormone increased the expression of both monomer and dimer forms of the receptor proteins compared with the control. Western blotting analysis with anti-phosphotyrosine antibody revealed that the lysates of TGF-alpha-stimulated cells express phosphotyrosine-containing receptor proteins of 170 kDa and 340 kDa. The results show that chicken granulosa cells express the 170-kDa EGF/TGF-alpha receptor protein, which dimerizes on binding to TGF-alpha, suggesting that the receptor protein may be involved in the signal transduction of TGF-alpha actions in the chicken granulosa cells.

This study was designed to assess alterations of hormone and cytokine levels associated with growth period during puberty in Honamlı goats which were identified as a new goat breed and had one of the highest meat production potential among the other goat breeds in Turkey. Honamlı goats are originated from native hair goats, so parallel studies of sampling and analyzing were conducted also in native hair goats which have moderate meat production. Blood serum samples of Honamlı (n=90) and native hair goats (n=90) were obtained from the pure herds in Korkuteli and Ka districts of Anatolia. Concentrations of growth hormone (GH), myostatin (MSTN), insulin-like growth factor (IGF), growth hormone releasing hormone (GHRH), growth hormone releasing peptide (GHRP), leptin, transforming growth factor-betal (TGF-β1) and vascular endothelial cell growth factor (VEGF) levels were measured by ELISA in each breed in the age groups of 4, 8 and 12 months. The present results indicate interesting correlations among the age groups and all the examined hormone and cytokine parameters exhibited significant (P<0.05 and P<0.001) differences. The parameters investigated were usually begun to increase after 4 months of age in the both breeds and sexes. Therefore, this paper supported the view that the beginning of hormonal alterations of goats could occur at 4th month of age. The results reported here emphasize the primary role played by GH, MSTN, IGF-1, leptin, GHRH, GHRP, TGF-βi and VEGF in the first year growth period of goats.

Plexiform lesions are characteristic histological changes of pulmonary arteries in human patients with severe pulmonary arterial hypertension (PAH) and are regarded as angiogenic lesions. Meat-type broiler chickens are susceptible to PAH and can develop plexiform lesions spontaneously. Whether the lesion development in broilers is associated with PAH predisposition and lung angiogenic environment remains unclear. Moreover, little is known about the cellular origin of these structures. In this work, plexiform lesions were detected in both layer chickens (a strain known to be resistant to PAH) and broiler chickens aged between 1 and 6 wk with normal pulmonary arterial pressures. Within each of the sampled ages, the lesion density did not differ between strains, with an exception of wk 4 when broiler was higher than layer. In contrast to the trend of age-related decline in layers, lesion densities in broilers demonstrated bi-phasic alterations characterized by a gradual decrease during wk 1 to 3 followed by a sudden increase at wk 4. The mRNA of 6 angiogenic factors in the lung tissue, namely, vascular endothelial growth factor receptor (VEGFR)-2, angiopoietin (Ang)-1, angiopoietin receptor Tie-2, transforming growth factor (TGF)-β1, hepatocyte growth factor (HGF), and interleukin (IL)-8, were differentially expressed between strains. However, none of them was found to be significantly correlated with the lesion density by strain and age-adjusted partial correlation analysis. An in vivo experiment revealed impaired differentiation of endothelial progenitor cells (EPC) into endothelial cells during the producing of plexiform lesions, as evidenced by increased expression of endothelial CD133, a maker of EPC, but reduced expression of CD31, a marker of mature endothelial cells, in the parent vessels of plexiform lesions compared to normal vessels. Collectively, it appears unlikely that the predisposition to PAH or intrapulmonary angiogenic environment contributes to the lesion development in broilers when compared with layers. It is suggested that the lesion development is associated with increased pulmonary arterial pressure, and that local EPC dysfunction may play a role in the process.

Bicarbonate concentration in saliva is controlled by the action of acid-base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor-beta (TGF-β) as a novel regulator of V-ATPase subunits. Using QRT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H(⁺ ) recording with H(⁺ )-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF-β signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF-β1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF-β1 treatment. Following acidosis, a bafilomycin-sensitive and Na⁺ -independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF-βs, inhibition of TGF-β receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis-induced increased V-ATPase activity. The results suggest multiple modes of action of TGF-β1 on V-ATPase subunits in HSG cells: TGF-β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context-dependent manner. Moreover, surface V-ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.

The aim of this study was to evaluate the gene expression of proinflammatory (RANKL, TNF-a and IFN-g) and regulatory (TGF-b and IL-10) cytokines as reaction to experimental infection by mono or bi-association of Fusobacterium nucleatum (ATCC 10953) and Enterococcus faecalis (ATCC 19433). F. nucleatum and E. faecalis, either in mono- or bi-association were inoculated into the root canal system (RCS) of Balb/c mice. Animals were sacrificed at 10 and 20 days after infection and periapical tissues surrounding the root were collected. The mRNA expression of the cytokines RANKL, TNF-a, IFN- g, TGF-b and IL-10 was assessed using real-time PCR. The Kruskal-Wallis test was used for statistical analysis. F. nucleatum mono-infection induced high expression of RANKL and TNF-a, while its modulation was due to IL-10. High expression of IFN-g at day 20 was up-regulated by E. faecalis and RANKL; TNF-a was up-regulated by an independent mechanism via IL-10 and TGF-b. Bi-association (F. nucleatum and E. faecalis) stimulated high expression of RANKL, TNF-a and IFN-g, which seemed to be modulated by TGF-b 20 days later. The gene expression of proinflammatory cytokines was more prominent in the earlier periods of the experimental periapical infection, which concomitantly decreased in the later period. This expression may be regulated by IL-10 and TGF-b in an infection-specific condition.

OBJECTIVE

To study the role of Smad3 in transforming growth factor-beta1 (TGF-beta1)-induced bi-directional effects on skin fibroblast proliferation.

METHODS

The Smad3 small interfering (siRNA) plasmid was constructed using a pSUPER vector. The efficiency of cell transfection was detected by fluorescence microscopy, and the inhibitory effect of the plasmid was assessed by real-time quantitative RT-PCR and immunohistochemistry. The effect of the plasmid on the fibroblast proliferation and Smad3 binding activity was analyzed by BRDU ELISA and EMSA, respectively.

RESULTS

The transfection efficiency of the plasmid into the cells was 41.2%. The Smad3 siRNA plasmid produced efficient and specific inhibition of the expression of Smad3, and promoted the cell proliferation in a dose-dependent manner and abrogated the bi-directional effect of TGF-beta1 on the cell proliferation and Smad3 binding activity.

CONCLUSIONS

The siRNA targeting Smad3 gene can inhibit the protein expression and RNA transcription of Smad3, and TGF-beta1 exerts bi-directional regulation on fibroblast proliferation by modulating Smad3 activity.

To study the regulation of TGF-beta(1) on the development of hemangioblast, embryonic stem cell-derived blast forming cells (BL-CFC) were used as the model of hemangioblast in vitro. TGF-beta(1) or anti-TGF-beta(1) neutralization antibody was added in the medium of embryoid body (EB) generation for observating influence of TGF-B(1) addition in different culture stages on number of BL-CFC and differentiation of BL-CFC to endothelial and hematopoietic cells. The results showed that antagonizing TGF-beta(1) in the course of EB growth could significantly reduce the number of BL-CFC (P<0.01), and the frequency of Flk-1(+) cells was also decreased consistently. Furthermore, the BL-CFC derived from EB pretreated with TGF-beta(1) demonstrated remarkably elevated hematopoietic and endothelial potential, whereas such bi-potential was impaired in the group with neutralizing antibody. It is concluded that TGF-beta(1), a conventional negative regulator in hematopoiesis and angiogenesis exert positive effects on the development and differentiation capacities of BL-CFC in vitro.

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.

Keywords: IL-10; Lactococcus lactis; Leishmania braziliensis; TGF-β; TLR2; heat shock protein 65; oral tolerance.

The current study focused on the regulatory effects of parthenolide (PNL), a bioactive component derived from Chrysanthemum parthenium L., against hepatic fibrosis via regulating the crosstalk of toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) in activated hepatic stellate cells (HSCs). HSCs or Raw 264.7 macrophages were activated by TGF-β or LPS for 1 hr, respectively, and then treated with PNL, CLI-095 (TLR4 inhibitor), or Niclosamide (STAT3 inhibitor) for the indicated time to detect the crosstalk of TLR4 and STAT3. PNL significantly decreased the expressions of α-SMA, collagen I, and the ratio of TIMP1 and MMP13 in TGF-β-activated HSCs. PNL significantly reduced the releases of pro-inflammatory cytokines, including IL-6, IL-1β, IL-1α, IL-18, and regulated signaling P2X7r/NLRP3 axis activation. PNL obviously induced the apoptosis of activated HSCs by regulating bcl-2 and caspases family. PNL significantly inhibited the expressions of TLR4 and STAT3, including their downstream signaling. PNL could regulate the crosstalk of TLR4 and STAT3, which were verified by their inhibitors in activated HSCs or Raw 264.7 cell macrophages. Thus, PNL could decrease the expressions of fibrosis markers, reduce the releases of inflammatory cytokines, and also induce the apoptosis of activated HSCs. In conclusion, PNL could bi-directionally inhibit TLR4 and STAT3 signaling pathway, suggesting that blocking the crosstalk of TLR4 and STAT3 might be the potential mechanism of PNL against hepatic fibrosis.

Keywords: crosstalk of TLR4 and STAT3; hepatic fibrosis; hepatic stellate cells; inflammation; parthenolide.

Periarticular injury usually causes the defects of superficial cartilage and the underlying subchondral bone. Although some efficacious outcomes have been achieved by the existing therapeutic methods both in clinics and research, like symptomatic treatment, microfracture surgery, and tissue engineering technology, they still present specific disadvantages and complications. To improve this situation, we designed a biphasic (bi-) scaffold aiming to repair the structure of cartilage and subchondral bone synchronously. The scaffold consisted of a superior double-network (DN) hydrogel layer and a lower bioactive glass (BG) reinforced hydrogel layer, and the DN hydrogel included glycol chitosan (GC) and dibenzaldhyde functionalized poly(ethylene oxide) network, and sodium alginate (Alg) and calcium chloride (CaCl₂) network. To investigate its effectiveness, we applied this biphasic scaffold to repair osteochondral full-thickness defects in rabbit models. We set up six observation groups in total, including Untreated group, Microfracture group, BG only group, DN gel group, bi-DN gel group, and bi-DN/TGF-β gel group. With a follow-up period of 24 weeks, we evaluated the treatment effects by gross observation, micro-CT scan and histological staining. Besides, we further fulfilled the quantitative analysis of the data from ICRS score, O'Driscoll score and micro-CT parameters. The results revealed that neat GC/Alg DN hydrogel scaffold was only conductive to promoting cartilage regeneration and neat BG scaffold merely showed the excellent ability to reconstruct subchondral bone. While the biphasic scaffold performed better in repairing osteochondral defect synchronously, exhibiting more well-integrated cartilage-like tissue with positive staining of toluidine blue and col II immunohistochemistry, and more dense trabecular bone connecting closely with the surrounding host bone. Therefore, this method possessed the clinical application potential in treating articular injury, osteochondral degeneration, osteochondral necrosis, and sclerosis.

Keywords: biphasic scaffold; double-network hydrogel; dynamic cross linking; experimental research; osteochondral repair.

BACKGROUND

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephaniae tetrandrae, has a long history in Chinese clinical applications as an anti-inflammatory or anti-arrhythmic agent in the treatment of diverse diseases. In our previous study, TET exhibited the synergisitic action on azoles against pathogenic fungi.

OBJECTIVE

In the current study, we examined whether TET can enhance the antifungal activity of FLC against disseminated candidiasis in mice.

METHODS

BALB/c mice were inoculated intravenously with FLC-sensitive or FLC-resistant strains of Candida albicans, randomized and treated intraperitoneally with different doses of TET and/or FLC daily for 7 days. The treatment effectiveness, fungal burdens and the levels of the IFN-γ, IL-10, TGF-β1 and IL-17A are determined in serum by ELISA and in the kidney by Real-time RT-PCR methods.

RESULTS

We found that treatment with 45, 30 and 15 mg/kg of TET, enhanced the antifungal activities of a sub-critical dose (0.4 or 5 mg/kg) and minimal dose (0.8 or 10 mg/kg) of FLC against FLC-sensitive and FLC-resistant (respectively) infected mice. In the resistant strains the resistance mechanisms included MDR1 overexpression-and CDR1/CDR2 overexpression. Furthermore, when animals were treated with a sub-high dose (1.6-3.2 and 20-30 mg/kg) of FLC in the presence of fixed amounts of TET at 45, 30 and 15 mg/kg, the therapeutic doses of FLC could be substantially reduced in all strains tested. The findings in infected animal are consistent with the conclusion that TET exerts a synergistic effect on FLC against C. albicans by fractional inhibitory concentration index (FICI) and time-killing test in vitro.

CONCLUSIONS

In summary, our data indicate that TET will enhance the antifungal activity of FLC against C. albicans infection in disseminated mice model.

Hepatoproliferin (HPF), a liver regeneration factor isolated from rat hepatocytes, was assessed for its mitogenic status in the human hepatoma cell line PLC/PRF-5. HPF was able to enhance hepatoma cell growth on its own without the aid of the established complete mitogens EGF and TGF-alpha or the hepato-priming factor TNF-alpha. HPF therefore acted as a complete hepatomitogen and had no co-mitogenic properties since it did not augment proliferation when combined with EGF or TGF-alpha but showed only an additive effect in the presence of TGF-alpha. Rat HPF was phylogenetically unrestricted, because it was found active in human cells. When each of the established growth factors (GFs) was used alone, the hepatoma cells responded with the same kind of response profile, namely a bi-phasic bell-shaped dose-dependent response due to stimulation at low levels and inhibition at higher levels. However, hepatocyte growth factor (HGF) was an exception since it did not induce a growth response in hepatoma cells. On the contrary HPF, on its own, showed a progressive enhanced linear dose response at the levels used for the GFs (ie 1.0-15 ng/5 x 10(5) cells). The comparative potency (CP) (dpm x 10(3)/microg DNA/ng GF) of HPF (CP = 13) was in the same range as for the complete hepatomitogens EGF (CP = 12) and TGF-alpha (CP = 14), revealing that HPF has indeed the status of a complete mitogen.

OBJECTIVE

The injured anterior cruciate ligament (ACL) is thought to exhibit an impaired healing response, and attempts at surgical repair have not been successful. Connective tissue growth factor (CTGF) is reported to be associated with wound healing, probably through transforming growth factor beta 1 (TGF-β1).

METHODS

A rabbit ACL injury model was used to study the effect of CTGF on ligament recovery. Quantitative real-time PCR (qRT-PCR) was performed for detection of changes in RNA levels of TGF-β1, type 1 collagen (COL1), type 2 collagen (COL2), SRY-related high mobility group-box gene9 (SOX9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase 13 (MMP-13). Expression of related proteins was detected by Western blotting.

RESULTS

The current study showed that CTGF could promote the recovery of an injured anterior cruciate ligament. It can upregulate mRNA and expression of TGF-β1, COL1, COL2, SOX9, and tissue inhibitor of TIMP-1, and downregulate mRNA and expression of MMP-13, suggesting that the curative effect of CTGF on injured rabbit ligaments is through regulation of these cellular factors.

CONCLUSIONS

This finding revealed the healing role of CTGF in injured tissues and provides new possibilities of treating injured tissues and wound healing by using CTGF.Cite this article: X. Sun, W. Liu, G. Cheng, X. Qu, H. Bi, Z. Cao, Q. Yu. The influence of connective tissue growth factor on rabbit ligament injury repair. Bone Joint Res 2017;6:399-404. DOI: 10.1302/2046-3758.67.BJR.2016-0255.R1.

OBJECTIVE

To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model.

METHODS

Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor β (TGF-β) was assessed using real-time PCR. Levene's test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05).

RESULTS

The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-β mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-β mRNA expression during the chronic phase.

CONCLUSIONS

Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-β and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response.

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-β isoforms, TGF-β1 and TGF-β2, and their combination. TGF-β1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, -200b, -200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-β1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.

Coordination of the immune response to injury or disease in the brain is postulated to involve bi-directional discourse between the immune system and the central nervous system. This cross communication involves soluble mediators, including various growth factors, cytokines, and neuropeptides. In this report, we demonstrate that the supernatant from activated T-lymphocytes is able to induce the transcription of a potent cytokine, TGF-beta 2 in glial cells. The activating stimulus invokes signaling mechanisms distinct from known kinase or protease pathways. Activation of TGF-beta 2 transcription correlates with the loss of binding activity for an 80 kDA glial labile repressor protein, GLRP, to a responsive region within the TFG-beta 2 promoter. Although GLRP shares some characteristics with the inducible transcription factor AP-1, it appears to be distinct from known AP-1 family members. These data along with previous observations demonstrating the potent immunosuppressive activity of TGF-beta 2, support a model for a feedback mechanism between the activated T-lymphocytes and astrocytes via TGF-beta 2 to regulate the immune response.

BACKGROUND

The effect of triptolide (TPL) on cardiac fibroblasts (CFbs) and cardiac fibrosis remain unknown till now. This study was conducted to explore the effects of TPL on proliferation and apoptosis of angiotensin II (Ang II)-induced CFbs.

METHODS

Ang II was used to promote proliferation of CFbs. Two dosages of TPL (10ng/ml and 100ng/ml) were chosen. MTT assay was used to detect cell survival rate in vitro. Flow cytometer was performed to analyze apoptosis of CFbs. Hydroxyproline concentration was detected with hydroxyproline assay kit. Quantitative real-time PCR was used to detect the expression of TGF-β1 and Smad3 mRNA.

RESULTS

Ang II promoted CFbs proliferation significantly. Compared to Ang II group, TPL markedly reduced the viability of CFbs and its Hydroxyproline concentration (P<0.05). Besides, TPL can significantly promote apoptosis of CFbs (P<0.05). Furthermore, TPL reduced the expressions of TGF-βΙ and Smad3 mRNA in Ang II-induced CFbs (P<0.05).

CONCLUSIONS

TPL can inhibit the proliferation of CFbs in rats by down-regulating TGF-β1/Smad3 signaling pathway. TPL might be a promising therapeutic drug for myocardial fibrosis.