Multifunctional alginate/polydeoxyribonucleotide hydrogels for promoting diabetic wound healing.

A multifunctional alginate/PDRN hydrogel system by ionic crosslinking and the Schiff base reaction between oxidized alginate (OA) and PDRN was developed in the present study. Biocompatibility assessment of the PDRN-loaded OA hydrogels showed a significant enhancement in cell viability in human dermal fibroblast (HDF) cells. In addition, hydrogels showed migratory, anti-inflammatory, intracellular reactive oxygen species scavenging, and anti-apoptotic activities. In vivo studies using a streptozotocin-induced diabetic Wister rat model indicated that OA-4PDRN had the highest percentage of wound closure (96.1 ± 2.6 %) at day 14 compared to the control (79.0 ± 2.3 %) group. This was accompanied by up-regulation of vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-ß) accompanied by down-regulation of pro-inflammatory markers (IL-6, IL-1ß). Following histopathological observations, PDRN-loaded OA hydrogel ensured tissue safety and induced wound healing with granular tissue formation, collagen deposition, re-epithelialization, and regeneration of blood vessels and hair follicles. The downregulation of inflammatory cytokines (CD68) and expression of angiogenesis-related cytokines (CD31) in wound sites revealed the suppression of inflammation and increased angiogenesis, ensuring skin tissue regeneration in diabetic wound healing. In conclusion, the findings suggest that PDRN-loaded OA hydrogel has enormous therapeutic potential as a diabetic wound dressing.

Radiation therapy attenuates lymphatic vessel repair by reducing VEGFR-3 signalling.

Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood. Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema. Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury. Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema.

Key signaling networks are dysregulated in patients with the adipose tissue disorder, lipedema.

OBJECTIVES: Lipedema, a poorly understood chronic disease of adipose hyper-deposition, is often mistaken for obesity and causes significant impairment to mobility and quality-of-life. To identify molecular mechanisms underpinning lipedema, we employed comprehensive omics-based comparative analyses of whole tissue, adipocyte precursors (adipose-derived stem cells (ADSCs)), and adipocytes from patients with or without lipedema. METHODS: We compared whole-tissues, ADSCs, and adipocytes from body mass index-matched lipedema (n = 14) and unaffected (n = 10) patients using comprehensive global lipidomic and metabolomic analyses, transcriptional profiling, and functional assays. RESULTS: Transcriptional profiling revealed >4400 significant differences in lipedema tissue, with altered levels of mRNAs involved in critical signaling and cell function-regulating pathways (e.g., lipid metabolism and cell-cycle/proliferation). Functional assays showed accelerated ADSC proliferation and differentiation in lipedema. Profiling lipedema adipocytes revealed >900 changes in lipid composition and >600 differentially altered metabolites. Transcriptional profiling of lipedema ADSCs and non-lipedema ADSCs revealed significant differential expression of >3400 genes including some involved in extracellular matrix and cell-cycle/proliferation signaling pathways. One upregulated gene in lipedema ADSCs, Bub1, encodes a cell-cycle regulator, central to the kinetochore complex, which regulates several histone proteins involved in cell proliferation. Downstream signaling analysis of lipedema ADSCs demonstrated enhanced activation of histone H2A, a key cell proliferation driver and Bub1 target. Critically, hyperproliferation exhibited by lipedema ADSCs was inhibited by the small molecule Bub1 inhibitor 2OH-BNPP1 and by CRISPR/Cas9-mediated Bub1 gene depletion. CONCLUSION: We found significant differences in gene expression, and lipid and metabolite profiles, in tissue, ADSCs, and adipocytes from lipedema patients compared to non-affected controls. Functional assays demonstrated that dysregulated Bub1 signaling drives increased proliferation of lipedema ADSCs, suggesting a potential mechanism for enhanced adipogenesis in lipedema. Importantly, our characterization of signaling networks driving lipedema identifies potential molecular targets, including Bub1, for novel lipedema therapeutics.

Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment.

Enhancing differentiation of mesenchymal stem cells (MSCs) to endothelial cells may improve their ability to vascularize tissue and promote wound healing. This study describes a novel role for nitric oxide (NO) in reprogramming MSCs towards an endothelial lineage and highlights the role of Wnt signaling and epigenetic modification by NO. Rat MSCs were transduced with lentiviral vectors expressing endothelial nitric oxide synthase (pLV-eNOS) and a mutated caveolin gene (pLV-CAV-1F92A ) to enhance NO generation resulting in increased in vitro capillary tubule formation and endothelial marker gene expression. An exogenous source of NO could also stimulate CD31 expression in MSCs. NO was associated with an arterial-specific endothelial gene expression profile of Notch1, Dll4, and Hey2 and significantly reduced expression of venous markers. Wnt signaling associated with NO was evident through increased gene expression of Wnt3a and ß-catenin protein, and expression of the endothelial marker Pecam-1 could be significantly reduced by treatment with the Wnt signaling inhibitor Dkk-1. The role of NO as an epigenetic modifier was evident with reduced gene expression of the methyltransferase, DNMT1, and bisulfite sequencing of the endothelial Flt1 promoter region in NO-producing MSCs showed significant demethylation compared to control cells. Finally, subcutaneous implantation of NO-producing MSCs seeded in a biomaterial scaffold (NovoSorb®) resulted in survival of transplanted cells and the formation of blood vessels. In summary, this study describes, NO as a potent endothelial programming factor which acts as an epigenetic modifier in MSCs and may provide a novel platform for vascular regenerative therapy.

Lentiviral vector expression of Klf4 enhances chondrogenesis and reduces hypertrophy in equine chondrocytes.

Monolayer expansion of chondrocytes in culture results in the dedifferentiation of chondrocytes with inferior cartilage specific extracellular matrix synthesis and proliferation when compared with its native counterpart. We aimed to enhance chondrocyte proliferation and articular cartilage specific gene expression through ectopic expression of the major pluripotency transcription factors (Oct4, Sox2, Klf4 and c-Myc). We also aimed to provide insights to the modulation of TGFß receptor mRNA with Klf4 overexpression. Equine chondrocytes pooled from three donors were transduced with lentiviral vectors expressing the induced pluripotency factors, Oct4, Sox2. Klf4 and c-Myc (OSKM), singly, or in combination or together with green fluorescent protein (GFP) as a control. Klf4 and c-Myc overexpressing chondrocytes showed a significant increase in mitosis when compared to the control (P < 0.01 and P < 0.0001 respectively). Furthermore, overexpression of Klf4 or OSKM in three dimensional (3D) culture of equine chondrocytes resulted in a significant increase in Col2a1 mRNA levels relative to the controls (P < 0.05 and P < 0.01 respectively) while all transcription factors significantly lowered the mRNA of the fibrocartilage marker Col1a1. We also employed a Col2a1 promoter driven GFP reporter for real time monitoring of Col2a1 gene activation in 3D micromass culture, which showed significantly higher promoter activity when cultures were treated with the growth factor TGFß3 (P < 0.05). The chondrogenic properties of Klf4 transduced chondrocytes at a lower passage (P4) showed significant increases in Sox9 (P < 0.001), Col2a1 (P < 0.05) and TGFß receptor I (P < 0.05) and II (P < 0.001) expression relative to the DS-Red expressing control. The chondrocyte dedifferentiation marker Col1a1 and hypertrophic marker Col10a1 were significantly downregulated with the inclusion of Klf4 (P < 0.01 and P < 0.05 respectively). In Conclusion, chondrogenic re-differentiation and proliferation of equine chondrocytes is promoted through ectopic expression of Klf4 while suppressing chondrocyte dedifferentiation.

Mitochondrial fission protein Drp1 inhibition promotes cardiac mesodermal differentiation of human pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Biologically active constituents of the secretome of human W8B2+ cardiac stem cells.

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

Generation of Immortalized Equine Chondrocytes With Inducible Sox9 Expression Allows Control of Hypertrophic Differentiation.

Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P < 0.0001) and up regulation of the hypertrophic marker collagen X (P < 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFß-3 (P < 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over-expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P < 0.05), while up-regulating collagen II and Aggrecan mRNA (P < 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 118: 1201-1215, 2017. © 2016 Wiley Periodicals, Inc.

Molecular control of nitric oxide synthesis through eNOS and caveolin-1 interaction regulates osteogenic differentiation of adipose-derived stem cells by modulation of Wnt/ß-catenin signaling.

BACKGROUND: Nitric oxide (NO) plays a role in a number of physiological processes including stem cell differentiation and osteogenesis. Endothelial nitric oxide synthase (eNOS), one of three NO-producing enzymes, is located in a close conformation with the caveolin-1 (CAV-1WT) membrane protein which is inhibitory to NO production. Modification of this interaction through mutation of the caveolin scaffold domain can increase NO release. In this study, we genetically modified equine adipose-derived stem cells (eASCs) with eNOS, CAV-1WT, and a CAV-1F92A (CAV-1WT mutant) and assessed NO-mediated osteogenic differentiation and the relationship with the Wnt signaling pathway. METHODS: NO production was enhanced by lentiviral vector co-delivery of eNOS and CAV-1F92A to eASCs, and osteogenesis and Wnt signaling was assessed by gene expression analysis and activity of a novel Runx2-GFP reporter. Cells were also exposed to a NO donor (NONOate) and the eNOS inhibitor, L-NAME. RESULTS: NO production as measured by nitrite was significantly increased in eNOS and CAV-1F92A transduced eASCs +(5.59 ± 0.22 µM) compared to eNOS alone (4.81 ± 0.59 µM) and un-transduced control cells (0.91 ± 0.23 µM) (p < 0.05). During osteogenic differentiation, higher NO correlated with increased calcium deposition, Runx2, and alkaline phosphatase (ALP) gene expression and the activity of a Runx2-eGFP reporter. Co-expression of eNOS and CAV-1WT transgenes resulted in lower NO production. Canonical Wnt signaling pathway-associated Wnt3a and Wnt8a gene expressions were increased in eNOS-CAV-1F92A cells undergoing osteogenesis whilst non-canonical Wnt5a was decreased and similar results were seen with NONOate treatment. Treatment of osteogenic cultures with 2 mM L-NAME resulted in reduced Runx2, ALP, and Wnt3a expressions, whilst Wnt5a expression was increased in eNOS-delivered cells. Co-transduction of eASCs with a Wnt pathway responsive lenti-TCF/LEF-dGFP reporter only showed activity in osteogenic cultures co-transduced with a doxycycline inducible eNOS. Lentiviral vector expression of canonical Wnt3a and non-canonical Wnt5a in eASCs was associated with induced and suppressed osteogenic differentiation, respectively, whilst treatment of eNOS-osteogenic cells with the Wnt inhibitor Dkk-1 significantly reduced expressions of Runx2 and ALP. CONCLUSIONS: This study identifies NO as a regulator of canonical Wnt/ß-catenin signaling to promote osteogenesis in eASCs which may contribute to novel bone regeneration strategies.

Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells.

BACKGROUND: Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses. METHODS: Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction. RESULTS: Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFRα and FGFR2, respectively. CONCLUSIONS: A novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.

Complete genome sequence analysis and identification of putative metallo-beta-lactamase and SpoIIIE homologs in Bacillus cereus group phage BCP8-2, a new member of the proposed Bastille-like group.

Bacillus cereus group-specific bacteriophage BCP8-2 exhibits a broad lysis spectrum among food and human isolates (330/364) of B. cereus while not infecting B. subtilis (50) or B. licheniformis (12) strains. Its genome is 159,071 bp long with 220 open reading frames, including genes for putative methyltransferases, metallo-beta-lactamase, and a sporulation-related SpoIIIE homolog, as wells as 18 tRNAs. Comparative genome analysis showed that BCP8-2 is related to the recently proposed Bastille-like phages, but not with either SPO1-like or Twort-like phages of the subfamily Spounavirinae.

Bacteriophages BCP1-1 and BCP8-2 require divalent cations for efficient control of Bacillus cereus in fermented foods.

Bacillus cereus is a foodborne bacterial pathogen that causes diarrhea and vomiting. In this study, the usefulness of bacteriophages to eradicate B. cereus from fermented foods was investigated. A total of 13 phages were isolated from Korean fermented food products, and 2 (BCP1-1 and BCP8-2) were further characterized. Transmission electron microscopy (TEM), restriction enzyme digestion pattern analysis, and SDS-PAGE of the structural proteins suggest that both phages belong to the family Myoviridae, containing approximately 150 kbp-long genomes. The host ranges of both phages were limited to B. cereus group species (12/13), as they were not able to lyse other Gram-positive or negative strains including Bacillus subtilis. Purified phages were used to inhibit B. cereus growth in a model fermented food system, cheonggukjang, a fast-fermented soybean paste product. BCP1-1 and BCP8-2 were able to effectively eradicate B. cereus from the food only if divalent cations (Ca²âº, Mg²âº, or Mn²âº) were added to the medium. Further studies reveal that divalent cations are essential for phage adsorption, while a monovalent cation (Na⁺) is required for the post-adsorption phase of phage infection. Taken together, our findings imply that a phage could be an ideal anti-bacterial agent for use in fermented food products that require the presence of beneficial microflora and, during phage application, optimization of phage reaction conditions is critical for the successful utilization of phage biocontrol.

Prevalence of Bacillus cereus bacteriophages in fermented foods and characterization of phage JBP901.

Bacillus cereus is widely distributed in the environment and is responsible for foodborne illnesses, causing diarrhea and vomiting. In this study, the prevalence of B. cereus bacteriophages in Korean fermented foods was studied using 6 reference strains, and JBP901 was further characterized. In a total of 47 samples, 19 (40.4%) contained more than one kind of B. cereus phage. Fourteen phages were purified and determined for host lysis profile. Among them, JBP901 was specific to B. cereus group strains (forming plaques in 13 of 14 isolates) and not able to lyse other tested Gram-positive and Gram-negative strains including Bacillus subtilis. Transmission electron microscopy analysis suggested that JBP901 is a member of the family Myoviridae. Restriction enzyme digestion pattern analysis and one-step growth study indicated a genome size of approximately 150 kbp and a burst size of 100. Furthermore, bacterial lysis studies revealed that JBP901 could be used to control the growth of B. cereus in liquid culture and in food. This is the first report to examine the prevalence of phages in fermented foods in Korea, and suggests that fermented foods are useful sources of B. cereus phage isolation; moreover, B. cereus phages could provide an additional tool to tackle problems associated with B. cereus contamination.