Galectin 1-A Key Player between Tissue Repair and Fibrosis.

Galectins are ten family members of carbohydrate-binding proteins with a high affinity for ß galactose-containing oligosaccharides. Galectin-1 (Gal-1) is the first protein discovered in the family, expressed in many sites under normal and pathological conditions. In the first part of the review article, we described recent advances in the Gal-1 modulatory role on wound healing, by focusing on the different phases triggered by Gal-1, such as inflammation, proliferation, tissue repair and re-epithelialization. On the contrary, Gal-1 persistent over-expression enhances angiogenesis and extracellular matrix (ECM) production via PI3K/Akt pathway activation and leads to keloid tissue. Therefore, the targeted Gal-1 modulation should be considered a method of choice to treat wound healing and avoid keloid formation. In the second part of the review article, we discuss studies clarifying the role of Gal-1 in the pathogenesis of proliferative diabetic retinopathy, liver, renal, pancreatic and pulmonary fibrosis. This evidence suggests that Gal-1 may become a biomarker for the diagnosis and prognosis of tissue fibrosis and a promising molecular target for the development of new and original therapeutic tools to treat fibrosis in different chronic diseases.

Graphene-Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo.

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

Systemic Beta-Hydroxybutyrate Affects BDNF and Autophagy into the Retina of Diabetic Mice.

Background: Diabetic retinopathy (DR) is a neurovascular disease, characterized by a deficiency of brain-derived neurotrophic factor (BDNF), a regulator of autophagy. Beta-hydroxybutyrate (BHB), previously reported as a protective agent in DR, has been associated with BDNF promotion. Here, we investigated whether systemic BHB affects the retinal levels of BDNF and local autophagy in diabetic mice with retinopathy; Methods: C57BL/6J mice were administered with intraperitoneal (i.p.) streptozotocin (STZ) (75 mg/kg) injection to develop diabetes. After 2 weeks, they received i.p. injections of BHB (25−50−100 mg/kg) twice a week for 10 weeks. Retinal samples were collected in order to perform immunofluorescence, Western blotting, and ELISA analysis; Results: BHB 50 mg/kg and 100 mg/kg significantly improved retinal BDNF levels (p < 0.01) in diabetic mice. This improvement was negatively associated with autophagosome−lysosome formations (marked by LC3B and ATG14) and to higher levels of connexin 43 (p < 0.01), a marker of cell integrity. Moreover, BHB administration significantly reduced M1 microglial activation and autophagy (p < 0.01); Conclusions: The systemic administration of BHB in mice with DR improves the retinal levels of BDNF, with the consequent reduction of the abnormal microglial autophagy. This leads to retinal cell safety through connexin 43 restoration.

Sex and Age-Related Differences in Neuroinflammation and Apoptosis in Balb/c Mice Retina Involve Resolvin D1.

(1) Background: The pro-resolving lipid mediator Resolvin D1 (RvD1) has already shown protective effects in animal models of diabetic retinopathy. This study aimed to investigate the retinal levels of RvD1 in aged (24 months) and younger (3 months) Balb/c mice, along with the activation of macro- and microglia, apoptosis, and neuroinflammation. (2) Methods: Retinas from male and female mice were used for immunohistochemistry, immunofluorescence, transmission electron microscopy, Western blotting, and enzyme-linked immunosorbent assays. (3) Results: Endogenous retinal levels of RvD1 were reduced in aged mice. While RvD1 levels were similar in younger males and females, they were markedly decreased in aged males but less reduced in aged females. Both aged males and females showed a significant increase in retinal microglia activation compared to younger mice, with a more marked reactivity in aged males than in aged females. The same trend was shown by astrocyte activation, neuroinflammation, apoptosis, and nitrosative stress, in line with the microglia and Müller cell hypertrophy evidenced in aged retinas by electron microscopy. (4) Conclusions: Aged mice had sex-related differences in neuroinflammation and apoptosis and low retinal levels of endogenous RvD1.

Paracellular and transcellular migration of metastatic cells through the cerebral endothelium.

Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases. Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood-brain barrier. By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps. We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia-like membrane protrusions towards the tumour cells. Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route. On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells. When co-culturing melanoma cells with cerebral endothelial cells, we observed N-cadherin enrichment at melanoma-melanoma and melanoma-endothelial cell borders. However, for breast cancer cells N-cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo. Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.

Biochemical, Histopathological and Molecular Responses in Gills of Leuciscus cephalus Exposed to Metals.

Gills are major targets for acute metal toxicity in fish, due to their permanent contact with aquatic pollutants. To assess the effects of metals on gills of the Leuciscus cephalus (chub), fish individuals were collected from two sites in the Tur River, Romania, in upstream (site 1) and downstream (site 2) of a metal pollution source. Quantitative and hyperspectral analyses showed that Zn, Sr, and Fe concentrations were significantly higher in gills from site 2 compared with site 1. Malondialdehyde and advanced oxidation protein products levels increased 17 and 28%, respectively, whereas reduced glutathione level diminished significantly in the gills of fish collected from site 2 compared to site 1. The activities of superoxide dismutase, catalase, and glutathione-S-transferase increased significantly at 41, 21, and 28%, respectively. Proliferating cell nuclear antigen (PCNA) protein levels, as well as the amount of DNA damage, were significantly increased for site 2 compared with site 1. The induced oxidative stress generated hyperplasia, hypertrophy, and inflammation in the epithelial cells and apoptosis. Hence, this could suggest that gill cells have tried to counteract the oxidative stress-induced DNA fragmentation by PCNA up-regulation, but the PCNA expression decreased on longer time due to the low level of GSH, resulting in apoptosis.

Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study.

The blood-brain barrier (BBB) is the main interface controlling molecular and cellular traffic between the central nervous system (CNS) and the periphery. It consists of cerebral endothelial cells (CECs) interconnected by continuous tight junctions, and closely associated pericytes and astrocytes. Different parts of the CNS have diverse functions and structures and may be subject of different pathologies, in which the BBB is actively involved. It is largely unknown, however, what are the cellular and molecular differences of the BBB in different regions of the brain. Using in silico, in vitro, and ex vivo techniques we compared the expression of BBB-associated genes and proteins (i.e., markers of CECs, brain pericytes, and astrocytes) in the cortical grey matter and white matter. In silico human database analysis (obtained from recalculated data of the Allen Brain Atlas), qPCR, Western blot, and immunofluorescence studies on porcine and mouse brain tissue indicated an increased expression of glial fibrillary acidic protein in astrocytes in the white matter compared with the grey matter. We have also found increased expression of genes of the junctional complex of CECs (occludin, claudin-5, and α-catenin) in the white matter compared with the cerebral cortex. Accordingly, occludin, claudin-5, and α-catenin proteins showed increased expression in CECs of the white matter compared with endothelial cells of the cortical grey matter. In parallel, barrier properties of white matter CECs were superior as well. These differences might be important in the pathogenesis of diseases differently affecting distinct regions of the brain.

Protective effects of silymarin on epirubicin-induced mucosal barrier injury of the gastrointestinal tract.

Mucositis is a serious disorder of the gastrointestinal tract that results from cancer chemotherapy. We investigated the protective effects of silymarin on epirubicin-induced mucosal barrier injury in CD-1 mice. Immunohistochemical activity of both pro-apoptotic Bax and anti-apoptotic Bcl-2 markers, together with p53, cyt-P450 expression and DNA damage analysis on stomach, small intestine and colon were evaluated. Our results indicated stronger expression for cyt P450 in all analyzed gastrointestinal tissues of Epi group, which demonstrate intense drug detoxification. Bax immunopositivity was intense in the absorptive enterocytes and lamina connective cells of the small intestine, surface epithelial cells of the stomach and also in the colonic epithelium and lamina concomitant with a decreased Bcl-2 expression in all analyzed tissues. Epirubicin-induced gastrointestinal damage was verified by a goblet cell count and morphology analysis on histopathological sections stained for mucins. In all analyzed tissues, Bax immunopositivity has been withdrawn by highest dose of silymarin concomitant with reversal of Bcl-2 intensity at a level comparable with control. p53 expression was found in all analyzed tissues and decreased by high dose of silymarin. Also, DNA internucleosomal fragmentation was observed in the Epi groups for all analyzed tissues was almost suppressed at 100 mg/kg Sy co-treatment. Histological aspect and goblet cell count were restored at a highest dose of Sy for both small and large intestine. In conclusion, our findings suggest that silymarin may prevent cellular damage of epirubicin-induced toxicity and was effective in reducing the severity indicators of gastrointestinal mucositis in mice.

CB2 receptor activation inhibits melanoma cell transmigration through the blood-brain barrier.

During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.

Colloidal and Biological Characterization of Dual Drug-Loaded Smart Micellar Systems.

Smart polymeric micelles (PMs) are of great interest in drug delivery owing to their low critical micellar concentration and sizes. In the present study, two different pH-sensitive poly(2-vinyl pyridine)-b-poly(ethylene oxide) (P2VP-b-PEO) copolymer samples were used for the encapsulation of paclitaxel (PTX), ursolic acid (UA), and dual loading of PTX and UA. Based on the molecular features of copolymers, spherical PMs with sizes of around 35 nm and 140 nm were obtained by dialysis for P2VP55-b-PEO284 and P2VP274-b-PEO1406 samples, respectively. The micellar sizes increased after loading of both drugs. Moreover, drug encapsulation and loading efficiencies varied from 53 to 94% and from 3.2 to 18.7% as a function of the copolymer/drug ratio, molar mass of copolymer sample, and drug type. By FT-IR spectroscopy, it was possible to demonstrate the drug loading and the presence of some interactions between the polymer matrix and loaded drugs. In vitro viability was studied on 4T1 mammary carcinoma mouse cells as a function of time and concentration of drug-loaded PMs. UA-PMs and free PMs alone were not effective in inhibiting the tumor cell growth whereas a viability of 40% was determined for cells treated with both PTX- and PTX/UA-loaded PMs. A synergic effect was noticed for PTX/UA-loaded PMs.

Exploring In Vivo Pulmonary and Splenic Toxicity Profiles of Silicon Quantum Dots in Mice.

Silicon-based quantum dots (SiQDs) represent a special class of nanoparticles due to their low toxicity and easily modifiable surface properties. For this reason, they are used in applications such as bioimaging, fluorescent labeling, drug delivery, protein detection techniques, and tissue engineering despite a serious lack of information on possible in vivo effects. The present study aimed to characterize and evaluate the in vivo toxicity of SiQDs obtained by laser ablation in the lung and spleen of mice. The particles were administered in three different doses (1, 10, and 100 mg QDs/kg of body weight) by intravenous injection into the caudal vein of Swiss mice. After 1, 6, 24, and 72 h, the animals were euthanized, and the lung and spleen tissues were harvested for the evaluation of antioxidant enzyme activity, lipid peroxidation, protein expression, and epigenetic and morphological changes. The obtained results highlighted a low toxicity in pulmonary and splenic tissues for concentrations up to 10 mg SiQDs/kg body, demonstrated by biochemical and histopathological analysis. Therefore, our study brings new experimental evidence on the biocompatibility of this type of QD, suggesting the possibility of expanding research on the biomedical applications of SiQDs.

In Vivo Assessment of Hepatic and Kidney Toxicity Induced by Silicon Quantum Dots in Mice.

In the last decade, silicon-based quantum dots (SiQDs) have attracted the attention of researchers due to their unique properties for which they are used in medical applications and in vivo imaging. Detection of cytotoxic effects in vivo is essential for understanding the mechanisms of toxicity, a mandatory step before their administration to human subjects. In this context, we aimed to evaluate the in vivo hepatic and renal acute toxicity of SiQDs obtained by laser ablation. The nanoparticles were administrated at different doses (0, 1, 10, and 100 mg of QDs/kg of body weight) by intravenous injection into the caudal vein of Swiss mice. After 1, 6, 24, and 72 h, the animals were euthanatized, and liver and kidney tissues were used in further toxicity tests. The time- and dose-dependent effects of SiQDs on the antioxidant defense system of mice liver and kidney were investigated by quantifying the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase) in correlation with the morphological changes and inflammatory status in the liver and kidneys. The results showed a decrease in the activities of antioxidant enzymes and histopathological changes, except for superoxide dismutase, in which no significant changes were registered compared with the control. Furthermore, the immunohistochemical expression of TNF-α was significant at doses over 10 mg of QDs/kg of body weight and were still evident at 72 h after administration. Our results showed that doses under 10 mg of SiQDs/kg of b.w. did not induce hepatic and renal toxicity, providing useful information for further clinical trials.

Dynamic shifts in lung cytokine patterns in post-COVID-19 interstitial lung disease patients: a pilot study.

Introduction: The pathogenesis of post-COVID interstitial lung disease, marked by lung tissue scarring and functional decline, remains largely unknown. Objectives: We aimed to elucidate the temporal cytokine/chemokine changes in bronchoalveolar lavage (BAL) from patients with post-COVID interstitial lung disease to uncover potential immune drivers of pulmonary complications. Design: We evaluated 16 females diagnosed with post-COVID interstitial lung disease, originating from moderate to severe cases during the second epidemic wave in the Autumn of 2020, treated at the Pneumology Department of the Arad County Clinical Hospital, Romania. Their inflammatory response over time was compared to a control group. Methods: A total of 48 BAL samples were collected over three intervals (1, 3, and 6 months) and underwent cytology, gene, and protein expression analyses for pro/anti-inflammatory lung cytokines and chemokines using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Results: One month after infection, there were significant increases in the levels of IL-6 and IL-8. These levels decreased gradually over the course of 6 months but were still higher than those seen in control. Interferon-gamma and tumor necrosis factor alpha exhibited similar patterns. Persistent elevations were found in IL-10, IL-13, and pro-fibrotic M2 macrophages' chemokines (CCL13 and CCL18) for 6 months. Furthermore, pronounced neutrophilia was observed at 1 month post-COVID, highlighting persistent inflammation and lung damage. Neutrophil efferocytosis, aiding inflammation resolution and tissue repair, was evident at the 1-month time interval. A notable time-dependent reduction in CD28 was also noticed. Conclusion: Our research provides insight into the immunological processes that may lead to the fibrotic changes noted in the lungs following COVID-19.

BACKGROUND: Post-COVID lung disease represents a significant health concern that demands comprehensive research. The pathogenesis of post-COVID interstitial lung disease, marked by lung tissue scarring and functional decline, remains largely unknown. METHODS: We evaluated 16 females diagnosed with post-COVID interstitial lung disease, originating from moderate to severe cases during the second epidemic wave in the Autumn 2020, treated at the Pneumology Department of the Arad County Clinical Hospital, Romania. Their inflammatory response over time was compared to a control group. A total of 48 BAL samples were collected over three intervals (1, 3, and 6 months) and underwent cytology, gene, and protein expression analyses for pro/anti-inflammatory lung cytokines and chemokines using RT-PCR and ELISA The interrelationships between the expression levels of various pro-inflammatory and anti-inflammatory cytokines and chemokines by Pearson's correlations was investigated. RESULTS: One month after infection, there were significant increases in the levels of IL-6 and IL-8. These levels decreased gradually over the course of six months but were still higher than those seen in control. IFN-γ and TNF-α exhibited similar patterns. Persistent elevations were found in IL-10, IL-13, and pro-fibrotic M2 macrophages' chemokines (CCL13 and CCL18) for six months. Pronounced neutrophilia was observed at 1 month post-COVID, highlighting persistent inflammation and lung damage. Neutrophils efferocytosis, aiding inflammation resolution and tissue repair, was evident at the 1-month time-interval. A notable time-dependent reduction in CD28 was also noticed. CONCLUSIONS: Our research provides insight into the immunological processes that may lead to the fibrotic changes noted in the lungs following COVID-19.

Dynamic shifts in lung cytokine patterns in post-COVID-19 interstitial lung disease patients: a pilot study The objective of this pilot study was to investigate changes in lung cytokine pro- and anti-inflammatory profiles among patients with interstitial lung disease after COVID-19 infection.

Oral Administration of Vitamin D3 Prevents Corneal Damage in a Knock-Out Mouse Model of Sjögren's Syndrome.

BACKGROUND: Vitamin D deficiency has been associated with dry eye development during Sjögren's syndrome (SS). Here, we investigated whether repeated oral vitamin D3 supplementation could prevent the corneal epithelium damage in an SS mouse model. METHODS: 30 female mouse knock-out for the thrombospondin 1 gene were randomized (six per group) in untreated mice euthanized at 6 weeks as negative control (C-) or at 12 weeks as the positive control for dry eye (C+). Other mice were sacrificed after 6 weeks of oral vitamin D3 supplementation in the drinking water (1000, 8000, and 20,000 IU/kg/week, respectively). RESULTS: The C+ mice showed alterations in their corneal epithelial morphologies and thicknesses (p < 0.01 vs. C-), while the mice receiving 8000 (M) and 20,000 (H) IU/kg/week of vitamin D3 showed preservation of the corneal epithelium morphology and thickness (p < 0.01 vs. C+). Moreover, while the C+ mice exhibited high levels and activity of corneal tumor necrosis factor alpha converting enzyme (TACE), neovascularization and fibrosis markers; these were all reduced in the M and H mice. CONCLUSIONS: Oral vitamin D3 supplementation appeared to counteract the negative effect of TACE on corneal epithelium in a mouse model of SS-associated dry eye.

Biocompatibility and Antimicrobial Profile of Acid Usnic-Loaded Electrospun Recycled Polyethylene Terephthalate (PET)-Magnetite Nanofibers.

The highest amount of the world's polyethylene terephthalate (PET) is designated for fiber production (more than 60%) and food packaging (30%) and it is one of the major polluting polymers. Although there is a great interest in recycling PET-based materials, a large amount of unrecycled material is derived mostly from the food and textile industries. The aim of this study was to obtain and characterize nanostructured membranes with fibrillar consistency based on recycled PET and nanoparticles (Fe3O4@UA) using the electrospinning technique. The obtained fibers limit microbial colonization and the development of biofilms. Such fibers could significantly impact modern food packaging and the design of improved textile fibers with antimicrobial effects and good biocompatibility. In conclusion, this study suggests an alternative for PET recycling and further applies it in the development of antimicrobial biomaterials.

Phytochemical Profiling and Anti-Fibrotic Activities of the Gemmotherapy Bud Extract of Corylus avellana in a Model of Liver Fibrosis on Diabetic Mice.

In this study, we aimed to explore the hepatoprotective effects of the gemmotherapy bud extract of Corylus avellana in a model of liver fibrosis on diabetic mice. An evaluation of total flavonoids and polyphenols contents and LC/MS analyses were performed. Experimental fibrosis was induced with CCl4 (2 mL/kg by i.p. injections twice a week for 7 weeks) in streptozotocin-induced diabetic mice. Our results showed a content of 6-7% flavonoids, while hyperoside and chlorogenic acids were highlighted in the bud extract. Toxic administration of CCl4 increased oxidative stress, mRNA expression of the transforming growth factor-ß1 (TGF-ß1) and Smad 2/3, and reduced Smad 7 expression. Furthermore, up-regulation of α-smooth muscle actin (α-SMA) revealed an activation of hepatic stellate cells (HSCs), while collagen I (Col I) up-regulation and matrix metalloproteinases (MMPs) unbalance led to an altered extracellular matrix enriched in collagen, confirmed as well by a trichrome stain and electron microscopy analysis. Treatment with gemmotherapy extract significantly restored the liver architecture and the antioxidant balance, and significantly decreased collagen deposits in the liver and improved the liver function. Our results suggest that Corylus avellana gemmotherapy extract may have anti-fibrotic effects and could be useful in the prevention and treatment of liver fibrosis. The hepatoprotective mechanism is based on HSC inhibition, a reduction in oxidative stress and liver damage, a downregulation of the TGF-ß1/Smad signaling pathway and a MMPs/TIMP rebalance.

Chrysin-based supramolecular cyclodextrin-calixarene drug delivery system: a novel approach for attenuating cardiac fibrosis in chronic diabetes.

Introduction: Cardiac fibrosis is strongly induced by diabetic conditions. Both chrysin (CHR) and calixarene OTX008, a specific inhibitor of galectin 1 (Gal-1), seem able to reduce transforming growth factor beta (TGF-ß)/SMAD pro-fibrotic pathways, but their use is limited to their low solubility. Therefore, we formulated a dual-action supramolecular system, combining CHR with sulfobutylated ß-cyclodextrin (SBECD) and OTX008 (SBECD + OTX + CHR). Here we aimed to test the anti-fibrotic effects of SBECD + OTX + CHR in hyperglycemic H9c2 cardiomyocytes and in a mouse model of chronic diabetes. Methods: H9c2 cardiomyocytes were exposed to normal (NG, 5.5 mM) or high glucose (HG, 33 mM) for 48 h, then treated with SBECD + OTX + CHR (containing OTX008 0.75-1.25-2.5 µM) or the single compounds for 6 days. TGF-ß/SMAD pathways, Mitogen-Activated Protein Kinases (MAPKs) and Gal-1 levels were assayed by Enzyme-Linked Immunosorbent Assays (ELISAs) or Real-Time Quantitative Reverse Transcription Polymerase chain reaction (qRT-PCR). Adult CD1 male mice received a single intraperitoneal (i.p.) administration of streptozotocin (STZ) at a dosage of 102 mg/kg body weight. From the second week of diabetes, mice received 2 times/week the following i.p. treatments: OTX (5 mg/kg)-SBECD; OTX (5 mg/kg)-SBECD-CHR, SBECD-CHR, SBECD. After a 22-week period of diabetes, mice were euthanized and cardiac tissue used for tissue staining, ELISA, qRT-PCR aimed to analyse TGF-ß/SMAD, extracellular matrix (ECM) components and Gal-1. Results: In H9c2 cells exposed to HG, SBECD + OTX + CHR significantly ameliorated the damaged morphology and reduced TGF-ß1, its receptors (TGFßR1 and TGFßR2), SMAD2/4, MAPKs and Gal-1. Accordingly, these markers were reduced also in cardiac tissue from chronic diabetes, in which an amelioration of cardiac remodeling and ECM was evident. In both settings, SBECD + OTX + CHR was the most effective treatment compared to the other ones. Conclusion: The CHR-based supramolecular SBECD-calixarene drug delivery system, by enhancing the solubility and the bioavailability of both CHR and calixarene OTX008, and by combining their effects, showed a strong anti-fibrotic activity in rat cardiomyocytes and in cardiac tissue from mice with chronic diabetes. Also an improved cardiac tissue remodeling was evident. Therefore, new drug delivery system, which could be considered as a novel putative therapeutic strategy for the treatment of diabetes-induced cardiac fibrosis.

Adipose-Derived Stem Cells (ADSCs) Supplemented with Hepatocyte Growth Factor (HGF) Attenuate Hepatic Stellate Cell Activation and Liver Fibrosis by Inhibiting the TGF-ß/Smad Signaling Pathway in Chemical-Induced Liver Fibrosis Associated with Diabetes.

Liver fibrosis can develop on the background of hyperglycemia in diabetes mellitus. However, xenobiotic-related factors may accelerate diabetes-associated liver fibrosis. In this study, we aimed to assess the antfibrotic effect of ADSC and HGF therapy and to establish the cellular and molecular mechanisms through in vitro and in vivo experiments. In vitro, TGF-ß1-activated hepatic stellate cells (HSCs) were cocultured with ADSCs or HGF, and the expression of several fibrosis markers was investigated. The antifibrotic effect of the ADSCs, HGF, and ADSCs supplemented with HGF was further assessed in vivo on diabetic mice with liver fibrosis experimentally induced. In vitro results showed the inhibition of HSC proliferation and decrease in fibrogenesis markers. Coadministration of ADSCs and HGF on diabetic mice with liver fibrosis enhanced antifibrotic effects confirmed by the downregulation of Col I, α-SMA, TGF-ß1, and Smad2, while Smad7 was upregulated. Moreover, stem cell therapy supplemented with HGF considerably attenuated inflammation and microvesicular steatosis, decreased collagen deposits, and alleviated liver fibrosis. In conclusion, the HGF-based ADSC therapy might be of interest for the treatment of liver fibrosis in diabetic patients, consecutive aggression exerts by different environmental factors.

Silk ProteinsEnriched Nanocomposite Hydrogels Based on Modified MMT Clay and Poly(2-hydroxyethyl methacrylate-co-2-acrylamido-2-methylpropane Sulfonic Acid) Display Favorable Properties for Soft Tissue Engineering.

Due to their remarkable structures and properties, three-dimensional hydrogels and nanostructured clay particles have been extensively studied and have shown a high potential for tissue engineering as solutions for tissue defects. In this study, four types of 2-hydroxyethyl methacrylate/2-acrylamido-2-methylpropane sulfonic acid/montmorillonite (HEMA/AMPSA/MMT) hydrogels enriched with sericin, and fibroin were prepared and studied in the context of regenerative medicine for soft tissue regenerative medicine. Our aim was to obtain crosslinked hydrogel structures using modified montmorillonite clay as a crosslinking agent. In order to improve the in vitro and in vivo biocompatibility, silk proteins were further incorporated within the hydrogel matrix. Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) were performed to prove the chemical structures of the modified MMT and nanocomposite hydrogels. Swelling and rheological measurements showed the good elastic behavior of the hydrogels due to this unique network structure in which modified MMT acts as a crosslinking agent. Hydrogel biocompatibility was assessed by MTT, LDH and LIVE/DEAD assays. The hydrogels were evaluated for their potential to support adipogenesis in vitro and human stem cells isolated from adipose tissue were seeded in them and induced to differentiate. The progress was assessed by evaluation of expression of adipogenic markers (ppar-γ2, perilipin) evaluated by qPCR. The potential of the materials to support tissue regeneration was further evaluated on animal models in vivo. All materials proved to be biocompatible, with better results on the 95% HEMA 5% AMPSA enriched with sericin and fibroin material. This composition promoted a better development of adipogenesis compared to the other compositions studied, due the addition of sericin and fibroin. The results were confirmed in vivo as well, with a better progress of soft tissue regeneration after implantation in mice. Therefore, hydrogel 95% HEMA 5% AMPSA enriched with sericin as well as fibroin showed the best results that recommend it for future soft tissue engineering application.

New Insights into the Cell Death Signaling Pathways Triggered by Long-Term Exposure to Silicon-Based Quantum Dots in Human Lung Fibroblasts.

This report is the first research study that aims to explore the molecular mechanisms involved in the in vitro pulmonary cytotoxicity triggered by long-term exposure to silicon-based quantum dots (QDs). Human lung fibroblasts (MRC-5 cell line) were exposed to 5 µg/mL silicon-based QDs for 5 weeks and the concentration was increased up to 40 µg/mL QDs during the next 4 weeks. Cell viability and population doubling level were calculated based on Trypan blue staining. The expression levels of proteins were established by Western blotting and the telomeres' length was determined through Southern blotting. Prolonged exposure of lung fibroblasts to QDs reduced the cell viability by 10% compared to untreated cells. The level of p53 and apoptosis-inducing factor (AIF) expression increased during the exposure, the peak intensity being registered after the seventh week. The expressions of autophagy-related proteins, Beclin-1 and LC-3, were higher compared to untreated cells. Regarding the protein expression of Nrf-2, a progressive decrease was noticed, suggesting the downregulation of a cytoprotective response to oxidative stress. In contrast, the heat shock proteins' (HSPs) expression was increased or maintained near the control level during QDs exposure in order to promote cell survival. Furthermore, the telomeres' length was not reduced during this exposure, indicating that QDs did not induce cellular senescence. In conclusion, our study shows that silicon-based QDs triggered the activation of apoptotic and autophagy pathways and downregulation of survival signaling molecules as an adaptive response to cellular stress which was not associated with telomeres shortening.