Stem cell enriched lipotransfer reverses the effects of fibrosis in systemic sclerosis.

Oro-facial fibrosis in systemic sclerosis (Scleroderma;SSc) has a major impact on mouth function, facial appearance, and patient quality of life. Lipotransfer is a method of reconstruction that can be used in the treatment of oro-facial fibrosis. The effect of this treatment not only restores oro-facial volume but has also been found to reverse the effects of oro-facial fibrosis. Adipose derived stem cells (ADSCs) within the engrafted adipose tissue have been shown to be anti-fibrotic in SSc and are proposed as the mechanism of the anti-fibrotic effect of lipotransfer. A cohort of 62 SSc patients with oro-facial fibrosis were assessed before and after stem cell enriched lipotransfer treatment. Clinical evaluation included assessment of mouth function using a validated assessment tool (Mouth Handicap in Systemic Sclerosis Scale-MHISS), validated psychological measurements and pre and post-operative volumetric assessment. In addition, to understand the mechanism by which the anti-fibrotic effect of ADSCs occur, SSc derived fibroblasts and ADSCs from this cohort of patients were co-cultured in direct and indirect culture systems and compared to monoculture controls. Cell viability, DNA content, protein secretion of known fibrotic mediators including growth factor- ß1 (TGF ß-1) and connective tissue growth factor (CTGF) using ELISA analysis and fibrosis gene expression using a fibrosis pathway specific qPCR array were evaluated. Mouth function (MHISS) was significantly improved (6.85±5.07) (p<0.0001) after treatment. All psychological measures were significantly improved: DAS 24 (12.1±9.5) (p<0.0001); HADS-anxiety (2.8±3.2) (p<0.0001), HADS-depression (2.0±3.1) (p<0.0001); BFNE (2.9 ± 4.3) (p<0.0001); VAS (3.56±4.1) (p<0.0001). Multiple treatments further improved mouth function (p<0.05), DAS (p<0.0001) and VAS (p = 0.01) scores. SSc fibroblast viability and proliferation was significantly reduced in co-culture compared to monoculture via a paracrine effect over 14 days (p < 0.0001). Protein secretion of transforming growth factor (TGF-ß1) and connective tissue growth factor (CTGF) was significantly reduced in co-culture compared to monoculture (p < 0.0001). Multiple fibrosis associated genes were down regulated in SSc co-culture compared to monoculture after 14 days including Matrix metalloproteinase-8 (MMMP-8), Platelet derived growth factor-ß (PDGF-ß) and Integrin Subunit Beta 6 (ITG-ß6). Autologous stem cell enriched lipotransfer significantly improved the effects of oro-facial fibrosis in SSc in this open cohort study. Lipotransfer may reduce dermal fibrosis through the suppression of fibroblast proliferation and key regulators of fibrogenesis including TG-ß1 and CTGF. Our findings warrant further investigation in a randomised controlled trial.

Characteristics of human adipose derived stem cells in scleroderma in comparison to sex and age matched normal controls: implications for regenerative medicine.

BACKGROUND: Adipose-derived stem cells (ADSCs) are emerging as an alternative stem cell source for cell-based therapies. Recent data suggest that autologous ADSC-enriched micrografting improves the effects of facial involvement in systemic sclerosis (SSc). We have extensively characterised ADSCs from SSc patients and compared their phenotype and function to healthy age- and sex-matched control ADSCs. METHODS: ADSCs were isolated and characterised from a cohort of six SSc patients (ADSC-SSc) and were compared to six healthy age- and sex-matched controls (ADSC-N). Cell surface phenotype lineage commitment was explored by flow cytometric analysis of mesenchymal and hematopoietic markers and by the capacity to differentiate to chondrogenic, osteogenic, and adipogenic lineages. Functional activities of ADSCs were assessed by biochemical and cellular assays for proliferation, metabolism, adhesion, morphology, migration, and invasion. RESULTS: Upon characterization of ADSC-SSc, we found that there was no alteration in the phenotype or surface antigen expression compared to healthy matched control ADSCs. We found that the differentiation capacity of ADSC-SSc was equivalent to that of ADSC-N, and that ADSC-SSc did not display any morphological or adhesive abnormalities. We found that the proliferation rate and metabolic activity of ADSC-SSc was reduced (p < 0.01). We found that the migration and invasion capacity of ADSC-SSc was reduced (p < 0.01) compared to healthy matched control ADSCs. CONCLUSIONS: This study provides important findings that can differentially characterise ADSCs from SSc patients. Results indicate that the surface phenotype and differentiation capacity of ADSCs from SSc patients are identical to healthy matched ADSCs. While the findings indicate that the proliferation and migration capacity of ADSC-SSc is reduced, ADSC-SSc are capable of ex-vivo culture and expansion. These findings encourage further investigation into the understanding by which ADSCs can impact upon tissue fibrosis.

Flavanol concentrations do not predict dipeptidyl peptidase-IV inhibitory activities of four cocoas with different processing histories.

Cocoa and its constituent bioactives (particularly flavanols) have reported anti-diabetic and anti-obesity activities. One potential mechanism of action is inhibition of dipeptidyl peptidase-IV (DPP4), the enzyme that inactivates incretin hormones such as glucagon-like peptide-1 and gastric inhibitory peptide. The objective of this study was to determine the DPP4 inhibitory activities of cocoas with different processing histories, and identify processing factors and bioactive compounds that predict DPP4 inhibition. IC25 values (µg mL-1) were 4.82 for Diprotin A (positive control), 2135 for fermented bean extract, 1585 for unfermented bean extract, 2871 for unfermented liquor extract, and 1076 for fermented liquor extract This suggests mild inhibitory activity. Surprisingly, protein binding activity, total polyphenol, total flavanol, individual flavanol and complex fermentation/roasting product levels were all positively correlated to IC25 concentrations (greater levels correspond to less potent inhibition). For the representative samples studied, fermentation appeared to improve inhibition. This study suggests that cocoa may possess mild DPP4 inhibitory activity, and that processing steps such as fermentation may actually enhance activity. Furthermore, this activity and the variation between samples were not easily explainable by traditional putative bioactives in cocoa. The compounds driving this activity, and the associated mechanism(s) by which this inhibition occurs, remain to be elucidated.

Loss of Native Flavanols during Fermentation and Roasting Does Not Necessarily Reduce Digestive Enzyme-Inhibiting Bioactivities of Cocoa.

Polyphenol profiles and in vitro digestive enzyme inhibitory activities were compared between cocoa extracts from unfermented beans (UB), fermented beans (FB), unfermented liquor (UL), and fermented liquor (FL). Total polyphenols, total flavanols, and individual flavanols were significantly different between UB/FB and UL/FL. All extracts effectively inhibited α-glucosidase (lowest IC50 = 90.0 µg/mL, UL) and moderately inhibited α-amylase (lowest IC50 = 183 µg/mL, FL) and lipase (lowest IC25 = 65.5 µg/mL, FB). Our data suggest that fermentation does not reduce α-glucosidase inhibition, while roasting may enhance inhibition. For α-amylase, both fermentation and roasting improved inhibition. Finally, for lipase, both fermentation and roasting attenuated inhibition. Conclusive correlations between inhibition and mDP, total polyphenol, and flavanol contents were not found. Our data suggest that enzyme inhibition activities of cocoa are not uniformly reduced by polyphenol/flavanol losses during fermentation and roasting. This paradigm-challenging finding suggests other cocoa constituents, potentially formed during processing, contribute to digestive enzyme inhibition.

ROCK activity and the Gßγ complex mediate chemotactic migration of mouse bone marrow-derived stromal cells.

INTRODUCTION: Bone marrow-derived stromal cells (BMSCs), also known as mesenchymal stem cells, are the focus of intensive efforts worldwide to elucidate their function and biology. Despite the importance of BMSC migration for their potential therapeutic uses, the mechanisms and signalling governing stem cell migration are still not fully elucidated. METHODS: We investigated and detailed the effects of MCP-1 activation on BMSCs by using inhibitors of G protein-coupled receptor alpha beta (GPCR αß), ROCK (Rho-associated, coiled-coil containing protein kinase), and PI3 kinase (PI3K). The effects of MCP-1 stimulation on intracellular signalling cascades were characterised by using immunoblotting and immunofluorescence. The effectors of MCP-1-mediated migration were investigated by using migration assays (both two-dimensional and three-dimensional) in combination with inhibitors. RESULTS: We established the kinetics of the MCP-1-activated signalling cascade and show that this cascade correlates with cell surface re-localisation of chemokine (C motif) receptor 2 (CCR2) (the MCP-1 receptor) to the cell periphery following MCP-1 stimulation. We show that MCP-1-initiated signalling is dependent on the activation of ßγ subunits from the GPCR αßγ complex. In addition, we characterise a novel role for PI3Kγ signalling for the activation of both PAK and ERK following MCP-1 stimulation. We present evidence that the Gßγ complex is responsible for PI3K/Akt, PAK, and ERK signalling induced by MCP-1 in BMSCs. Importantly, we found that, in BMSCs, inhibition of ROCK significantly inhibits MCP-1-induced chemotactic migration, in contrast to previous reports in other systems. CONCLUSIONS: Our results indicate differential chemotactic signalling in mouse BMSCs, which has important implications for the translation of in vivo mouse model findings into human trials. We identified novel components and interactions activated by MCP-1-mediated signalling, which are important for stem cell migration. This work has identified additional potential therapeutic targets that could be manipulated to improve BMSC delivery and homing.