Association of gut microbiota characteristics and metabolites reveals the regulation mechanisms under cadmium consumption circumstance.

BACKGROUND: Cadmium is a non-biodegradable heavy metal with a long biological half-life. Although its negative impact on human health has been previously reported, the association of cadmium consumption overdose with changes in the gut microbiota and its corresponding metabolites has not been fully elucidated so far. RESULTS: Cadmium consumption overdose led to a reduced body weight gain accompanied by an enhanced level of the proinflammatory cytokine tumor necrosis factor-α, interleukin-6, and histamine in the serum of the rats in comparison with normal rats. Furthermore, hepatotoxicity was also observed to be induced by cadmium, which was consistent with abnormal hepatic activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase and oxidative stress. In contrast, Lactobacillus rhamnosus-fermented Ganoderma lucidum (FGL) slice supplementation improved the aforementioned physiological properties. More importantly, microbiome and metabolites analysis indicated cadmium exposure significantly reduced the generation of short-chain fatty acids in the gut, particularly butyrate. However, rats in the FGL group had the highest level of butyrate in the feces, characterized with significantly enriched probiotics (Lactobacillus, Bifidobacterium) and butyrate-producing bacteria (Roseburia). CONCLUSION: The targeted regulation of the gut microbial community and its metabolites might be the essential association for attenuating body dysfunction induced by cadmium. The supplementation of FGL, as evidenced in this study, might highlight a novel approach to this field. © 2022 Society of Chemical Industry.

Phenolic Compounds with Antioxidant Properties from Canola Meal Extracts Inhibit Adipogenesis.

The extraction of phenolic compounds from canola meal produces functional health products and renders the canola meal a more digestible animal feed. The extracted phenolics may have novel bioactivity worth investigation. In this study, several solvents were evaluated for their ability to extract phenolic compounds from canola meal: water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The in vitro antioxidant and anti-obesity properties of various extracts were investigated. Anti-obesity properties were studied using adipogenic differentiation inhibition of a murine mesenchymal stem cell line (C3H10T1/2) and a pancreatic lipase inhibition assay. AE, ME, and BE showed significant (p < 0.05) adipogenesis and pancreatic lipase inhibitory activities and may have more pharmacological properties. AE down-regulated the gene expression of the major adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), correlating to phenolic content in a dose-dependent manner. The chemical characterization of AE revealed the presence of sinapic acid, ferulic acid, and kaempferol derivatives as main bioactive phenols.

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.

Peptides derived from lupin proteins confer potent protection against oxidative stress.

BACKGROUND: Lupin seeds are rich in proteins, which are utilized in the food industry. There is an increased interest in lupin research due to its association with health-related benefits, such as reduction of hypertension and hyperglycemia. However, studies on the peptides derived from lupin proteins are rare. RESULTS: Lupin protein hydrolysates (LPHs) were prepared by proteolysis using alcalase, trypsin and pepsin, respectively. All the hydrolysates demonstrated higher antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities compared to lupin proteins. The hydrolysates were fractionated into three fractions based on molecular weight (MW), and the peptides with MW < 3 kDa (LPH3) had the highest antioxidant and ACE inhibitory activities compared to other fractions. Cell model study revealed that LPH3 fraction had the highest protection against the generation of reactive oxygen species in HepG2 cells, which was associated with increased activities of superoxide dismutase and glutathione peroxidase through upregulation of SOD1, GPX1, GCLM, SLC7A11 and SRXN1 expression. CONCLUSIONS: The analysis of amino acid composition indicated that the peptides were characterized with high content of hydrophobic amino acids, which may be responsible for the greatest antioxidant activity. This study highlights the promising potential of lupin peptides as a functional ingredient in healthy foods. © 2018 Society of Chemical Industry.

Characterization of endogenous antioxidant attributes and its influence on thermal stability of canola oil.

Difference in thermal stability of two commercially available canola oils prepared by either expeller-extraction (EE) or solvent-extraction (SE) method was investigated. After 5 days consecutive deep-fry, content of oxidized-triacylglycerols (oxTAGs) in SE oil increased by 250.0% compared to its original status. However, 62.5% increase of oxTAGs in EE oil occurred, indicating that EE oil exhibits superior thermal stability to SE oil. Antioxidant capacity of EE oil was highly retained and loss rate of tocopherols in EE oil was much slower than in SE oil during deep-fry. Lipidomics showed that although there was no significant difference in molecular profile of either triacylglycerols or diacylglycerols between two oils, EE oil was characterized with 19 times higher phosphatidylcholine contents than SE oil. Considering no difference in antioxidant capacity between the two oils in their original status, it is proposed that synergetic mechanism is simultaneously initiated by antioxidant compounds and phosphatidylcholines, which plays key roles for maintaining better thermo-stability of vegetable oil during deep-fry.

Mesenchymal Stem Cells Expressing eNOS and a Cav1 Mutant Inhibit Vascular Smooth Muscle Cell Proliferation in a Rat Model of Pulmonary Hypertension.

BACKGROUND: This study aimed to investigate the effect of bone marrow derived mesenchymal stem cells (rBMSCs) transduced with lentiviral vectors expressing endothelial nitric oxide synthase (eNOS) and/or a mutant caveolin-1(F92A-Cav1), on the pulmonary haemodynamics and structure in a rat model of pulmonary arterial hypertension (PAH). METHODS: Pulmonary arterial hypertension was induced with monocrotaline (MCT) in 60 adult male Wistar rats prior to delivery of lentiviral vector transduced rBMSCs expressing Cav1, eNOS and/or F92A-Cav1. Changes in pulmonary haemodynamics, right ventricular hypertrophy index (RVHI), and serum nitric oxide (NO) were evaluated. Ultrastructure changes in lung tissues were observed by transmission electron microscopy. Expression of Kruppel-like factor 4 (KLF4), p53, P21, eNOS, and alpha-smooth muscle actin were evaluated by real time PCR, western blotting or immunohistochemistry. RESULTS: Treatment of PAH rats with gene modified rBMSCs (eNOS +/- Cav1 F92A) decreased right ventricular systolic pressure and improved pulmonary haemodynamics. The protein of alpha-smooth muscle actin expression was decreased whilst KLF4, p53, P21, eNOS expression, and serum NO concentration was elevated. The survival rate of rats in the treatment groups was also improved, after 35 days of observation. CONCLUSION: Intravenous delivery of rBMSCs expressing eNOS/F92A-Cav1 to PAH rats inhibits pulmonary vascular smooth muscle cell proliferation, and improves pulmonary haemodynamics, vascular remodelling and short-term survival. Activation of KLF4-p53 signalling pathway may be involved in these beneficial effects.

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.

Construction of local gene network for revealing different liver function of rats fed deep-fried oil with or without resistant starch.

To study the mechanism underlying the liver damage induced by deep-fried oil (DO) consumption and the beneficial effects from resistant starch (RS) supplement, differential gene expression and pathway network were analyzed based on RNA sequencing data from rats. The up/down regulated genes and corresponding signaling pathways were used to construct a novel local gene network (LGN). The topology of the network showed characteristics of small-world network, with some pathways demonstrating a high degree. Some changes in genes led to a larger probability occurrence of disease or infection with DO intake. More importantly, the main pathways were found to be almost the same between the two LGNs (30 pathways overlapped in total 48) with gene expression profile. This finding may indicate that RS supplement in DO-containing diet may mainly regulate the genes that related to DO damage, and RS in the diet may provide direct signals to the liver cells and modulate its effect through a network involving complex gene regulatory events. It is the first attempt to reveal the mechanism of the attenuation of liver dysfunction from RS supplement in the DO-containing diet using differential gene expression and pathway network.

Gene expression profiling of common signal transduction pathways affected by rBMSCs/F92A-Cav1 in the lungs of rat with pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with sustained vasoconstriction, inflammation and suppressed apoptosis of smooth muscle cells. Our previous studies have found that rat bone marrow-derived mesenchymal stem cells (rBMSCs) transduced with a mutant caveolin-1(F92A-Cav1) could enhance endothelial nitric oxide synthase (eNOS) activity and improve pulmonary vascular remodeling, but the potential mechanism is not yet fully explored. The present study was to investigate the gene expression profile upon rBMSCs/F92A-Cav1delivered to PAH rat to evaluate the role of F92A-Cav1 in its regulation. METHODS: PAH was induced with monocrotaline (MCT, 60mg/kg) prior to delivery of lentiviral vector transduced rBMSCs expressing Cav1 or F92A-Cav1. Gene expression profiling was performed using Rat Signal Transduction PathwayFinder array. The expression changes of 84 key genes representing 10 signal transduction pathways in rat following rBMSCs/F92A-Cav1 treatment was examined. RESULTS: Screening with the Rat Signal Transduction PathwayFinder R2 PCR Array system and subsequent western blot, immunohistochemistry or real time PCR analysis revealed that F92A-Cav1 modified rBMSCs can inhibit the inflammation factors (TNF-alpha, Icam1 and C/EBPdelta), pro-proliferation genes (c-Myc, Bcl2a1d, Notch1and Hey2), oxidative stress gene (Hmox1) and activate cell cycle arrested gene Cdkn1a, ameliorating inflammation and inhibiting cell proliferation in PAH rat. CONCLUSION: rBMSCs/F92A-Cav1 inhibits inflammation and cell proliferation by regulating signaling pathways that related to inflammation, proliferation, cell cycle and oxidative stress.

Epigallocatechin Gallate (EGCG) Decorating Soybean Seed Ferritin as a Rutin Nanocarrier with Prolonged Release Property in the Gastrointestinal Tract.

The instability and low bioavailability of polyphenols limit their applications in food industries. In this study, epigallocatechin gallate (EGCG) and soybean seed ferritin deprived of iron (apoSSF) were fabricated as a combined double shell material to encapsulate rutin flavonoid molecules. Firstly, due to the reversible assembly characteristics of phytoferritin, rutin was successfully encapsulated within apoSSF to form a ferritin-rutin complex (FR) with an average molar ratio of 28.2: 1 (rutin/ferritin). The encapsulation efficiency and loading capacity of rutin were 18.80 and 2.98 %, respectively. EGCG was then bound to FR to form FR-EGCG composites (FRE), and the binding number of EGCG was 27.30 ± 0.68 with a binding constant K of (2.65 ± 0.11) × 10(4) M(-1). Furthermore, FRE exhibited improved rutin stability, and displayed prolonged release of rutin in simulated gastrointestinal tract fluid, which may be attributed to the external attachment of EGCG to the ferritin cage potentially reducing enzymolysis in GI fluid. In summary, this work demonstrates a novel nanocarrier for stabilization and sustained release of bioactive polyphenols.

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.

Deep-fried oil consumption in rats impairs glycerolipid metabolism, gut histology and microbiota structure.

BACKGROUND: Deep frying in oil is a popular cooking method around the world. However, the safety of deep-fried edible oil, which is ingested with fried food, is a concern, because the oil is exposed continuously to be re-used at a high temperature, leading to a number of well-known chemical reactions. Thus, this study investigates the changes in energy metabolism, colon histology and gut microbiota in rats following deep-fried oil consumption and explores the mechanisms involved in above alterations. METHODS: Deep-fried oil was prepared following a published method. Adult male Wistar rats were randomly divided into three groups (n = 8/group). Group 1: basal diet without extra oil consumption (control group); Group 2: basal diet supplemented with non-heated canola oil (NEO group); Group 3: basal diet supplemented with deep-fried canola oil (DFEO group). One point five milliliters (1.5 mL) of non-heated or heated oil were fed by oral gavage using a feeding needle once daily for 6 consecutive weeks. Effect of DFEO on rats body weight, KEGG pathway regarding lipids metabolism, gut histology and gut microbiota were analyzed using techniques of RNA sequencing, HiSeq Illumina sequencing platform, etc. RESULTS: Among the three groups, DFEO diet resulted in a lowest rat body weight. Metabolic pathway analysis showed 13 significantly enriched KEGG pathways in Control versus NEO group, and the majority of these were linked to carbohydrate, lipid and amino acid metabolisms. Comparison of NEO group versus DFEO group, highlighted significantly enriched functional pathways were mainly associated with chronic diseases. Among them, only one metabolism pathway (i.e. glycerolipid metabolism pathway) was found to be significantly enriched, indicating that inhibition of this metabolism pathway (glycerolipid metabolism) may be a response to the reduction in energy metabolism in the rats of DFEO group. Related gene analysis indicated that the down-regulation of Lpin1 seems to be highly associated with the inhibition of glycerolipid metabolism pathway. Histological analysis of gastrointestinal tract demonstrated several changes induced by DFEO on intestinal mucosa with associated destruction of endocrine tissue and the evidence of inflammation. Microbiota data showed that rats in DFEO group had the lowest proportion of Prevotella and the highest proportion of Bacteroides among the three groups. In particular, rats in DFEO group were characterized with higher presence of Allobaculum (Firmicutes), but not in control and NEO groups. CONCLUSION: This study investigated the negative effect of DFEO on health, in which DFEO could impair glycerolipid metabolism, destroy gut histological structure and unbalance microbiota profile. More importantly, this is the first attempt to reveal the mechanism involved in these changes, which may provide the guideline for designing health diet.

The stimulatory effects of eNOS/F92A-Cav1 on NO production and angiogenesis in BMSCs.

BACKGROUND: Nitric oxide (NO) is generated in endothelial cells by endothelial nitric oxide synthase (eNOS). Caveolin-1 (Cav1) inhibits eNOS function and NO production. Modifying Cav1 scaffold domain, in particular Phenylalanine at position 92 (F92) is critical for the inhibitory actions of Cav1 toward eNOS. The aims of this study were to investigate the effect of enhanced NO production in term of in vitro angiogenesis on rat bone marrow derived mesenchymal stem cells (BMSCs) transduced with a novel bicistronic lentiviral vector co-expressing eNOS and mutant Cav1 (F92A). METHODS: A bicistronic eNOS/F92-Cav1 lentiviral vector was constructed, and used to transduce rat BMSCs. The expression of eNOS and VEGF protein were confirmed by western-blot. NO production was detected by the greiss assay and in vitro angiogenesis was assessed by matrigel assisted capillary tube formation. The cell viability was evaluated using a Cell Counting Kit (CCK)-8. RESULTS: The bicistronic eNOS/F92A-Cav1 lentiviral vector increased eNOS and VEGF protein expression, NO production compared to controls. In vitro capillary formation was increased in eNOS-F92A transduced cells and cell viability was not affected by transduction. CONCLUSION: Transduction of rat BMSCs with an eNOS-F92A-Cav1 lentiviral vector can increase NO production by enhancing eNOS protein expression. The increased NO production did not reduce cell viability. This study demonstrates that genetic modification of BMSCs to enhance NO producton could be applied in stem cell based therapeutic approaches to treat diseases such as pulmonary arterial hypertension (PAH) which is characterized by decreased endothelial NO release.

Effects of canola proteins and hydrolysates on adipogenic differentiation of C3H10T/2 mesenchymal stem cells.

This study assessed the ability of canola protein isolate (CPI) and enzymatic hydrolysates (CPHs) to inhibit adipogenic differentiation of C3H10T1/2 murine mesenchymal stem cells in vitro. Cell viability was maintained at concentrations of 60 µg/ml of sample. Cells treated with Alcalase hydrolysate demonstrated a higher reduction in anti-adipogenic differentiation through quantitation by oil-red O staining. qPCR analysis showed that CPI and CPH-treated cells significantly inhibited PPARγ expression, a key transcription factor involved in adipocyte differentiation, as evident in an ∼ 60-80% fold reduction of PPARγ mRNA. Immunofluorescence staining for PPARγ protein also showed a reduced expression in some treated cells when compared to differentiated untreated cells. The 50% inhibition concentration (IC50) of CPI, CPHs and their membrane ultrafiltration fractions on pancreatic lipase (PL) activity ranged between 0.75 and 2.5 mg/ml, (p < 0.05) for the hydrolysed and unhydrolysed samples. These findings demonstrate that CPI and CPHs contain bioactive components which can modulate in vitro adipocyte differentiation.

Efficient transduction of equine adipose-derived mesenchymal stem cells by VSV-G pseudotyped lentiviral vectors.

Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomatitis virus (V-GFP) or, for the first time, the baculovirus gp64 envelope protein (G-GFP). In this study, we produced similarly high titre V-GFP and G-GFP lentiviral vectors. Flow cytometric analysis showed efficient transduction using V-GFP; however G-GFP exhibited a poor ability to transduce EADMSC. Transduction resulted in sustained GFP expression over four passages, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential.

Gene modification of mesenchymal stem cells and articular chondrocytes to enhance chondrogenesis.

Current cell based treatment for articular cartilage and osteochondral defects are hampered by issues such as cellular dedifferentiation and hypertrophy of the resident or transplanted cells. The reduced expression of chondrogenic signalling molecules and transcription factors is a major contributing factor to changes in cell phenotype. Gene modification of chondrocytes may be one approach to redirect cells to their primary phenotype and recent advances in nonviral and viral gene delivery technologies have enabled the expression of these lost factors at high efficiency and specificity to regain chondrocyte function. This review focuses on the various candidate genes that encode signalling molecules and transcription factors that are specific for the enhancement of the chondrogenic phenotype and also how epigenetic regulators of chondrogenesis in the form of microRNA may also play an important role.

Endothelial progenitor cells and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterised by lung endothelial cell dysfunction and vascular remodelling. A number of studies now suggest that endothelial progenitor cells (EPCs) may induce neovascularisation and could be a promising approach for cell based therapy for PAH. On the contrary EPCs may contribute to pulmonary vascular remodelling, particularly in end-stage pulmonary disease. This review article will provide a brief summary of the relationship between PAH and EPCs, the application of the EPCs to PAH and highlight the potential clinical application of the EPCs cell therapy to PAH.

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia.

INTRODUCTION: A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia. METHODS: Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated. RESULTS: The second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression. CONCLUSIONS: Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.