Molecular‐ and microarray‐based analysis of diversity among resting and osteogenically induced porcine mesenchymal stromal cells of several tissue origin

Mesenchymal stromal cells (MSCs) play a pivotal role in modern therapeutic approaches in bone‐healing disorders. Although bone marrow‐derived MSCs are most frequently used, the knowledge that many other adult tissues represent promising sources for potent MSCs has gained acceptance. In the present study, the osteogenic differentiation potential of porcine skin fibroblasts (FBs), as well as bone marrow‐ (BMSCs), adipose tissue‐ (ASCs) and dental pulp‐derived stromal cells (DSCs) were evaluated. However, additional application of BMP‐2 significantly elevated the delayed osteogenic differentiation capacity of ASC and FB cultures, and in DSC cultures the supplementation of platelet‐rich plasma increased osteogenic differentiation potential to a comparable level of the good differentiable BMSCs. Furthermore, microarray gene expression performed in an exemplary manner for ASCs and BMSCs revealed that ASCs and BMSCs use different gene expression patterns for osteogenic differentiation under standard media conditions, as diverse MSCs are imprinted dependent from their tissue niche. However, after increasing the differentiation potential of ASCs to a comparable level as shown in BMSCs, a small subset of identical key molecules was used to differentiate in the osteogenic lineage. Until now, the importance of identified genes seems to be underestimated for osteogenic differentiation. Apparently, the regulation of transmembrane protein 229A, interleukin‐33 and the fibroblast growth factor receptor‐2 in the early phase of osteogenic differentiation is needed for optimum results. Based on these results, bone regeneration strategies of MSCs have to be adjusted, and in vivo studies on the osteogenic capacities of the different types of MCSs are warranted. Copyright © 2016 The Authors Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

Failure of collateral blood flow is associated with infarct growth in ischemic stroke

Changes in collateral blood flow, which sustains brain viability distal to arterial occlusion, may impact infarct evolution but have not previously been demonstrated in humans. We correlated leptomeningeal collateral flow, assessed using novel perfusion magnetic resonance imaging (MRI) processing at baseline and 3 to 5 days, with simultaneous assessment of perfusion parameters. Perfusion raw data were averaged across three consecutive slices to increase leptomeningeal collateral vessel continuity after subtraction of baseline signal analogous to digital subtraction angiography. Changes in collateral quality, Tmax hypoperfusion severity, and infarct growth were assessed between baseline and days 3 to 5 perfusion–diffusion MRI. Acute MRI was analysed for 88 patients imaged 3 to 6 hours after ischemic stroke onset. Better collateral flow at baseline was associated with larger perfusion–diffusion mismatch (Spearman''s Rho 0.51, P<0.001) and smaller baseline diffusion lesion volume (Rho −0.70, P<0.001). In 30 patients without reperfusion at day 3 to 5, deterioration in collateral quality between baseline and subacute imaging was strongly associated with absolute (P=0.02) and relative (P<0.001) infarct growth. The deterioration in collateral grade correlated with increased mean Tmax hypoperfusion severity (Rho −0.68, P<0.001). Deterioration in Tmax hypoperfusion severity was also significantly associated with absolute (P=0.003) and relative (P=0.002) infarct growth. Collateral flow is dynamic and failure is associated with infarct growth.     

Arginase 1 overexpression in psoriasis: limitation of inducible nitric oxide synthase activity as a molecular mechanism for keratinocyte hyperproliferation

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.     

Comparative analysis of Shwachman‐Diamond syndrome to other inherited bone marrow failure syndromes and genotype–phenotype correlation

Hashmi SK, Allen C, Klaassen R, Fernandez CV, Yanofsky R, Shereck E, Champagne J, Silva M, Lipton JH, Brossard J, Samson Y, Abish S, Steele M, Ali K, Dower N, Athale U, Jardine L, Hand JP, Beyene J, Dror Y. Comparative analysis of Shwachman‐Diamond syndrome to other inherited bone marrow failure syndromes and genotype–phenotype correlation. Our knowledge of the phenotypes of inherited bone marrow failure syndromes (IBMFSs) derives from case reports or case series in which only one IBMFS was studied. However, the substantial phenotypic overlap necessitates comparative analysis between the IBMFSs. Shwachman–Diamond syndrome (SDS) is an IBMFS that the appreciation of what comprises its clinical phenotype is still evolving. In this analysis we used data on 125 patients from the Canadian Inherited Marrow Failure Study (CIMFS), which is a prospective multicenter population‐based study. Thirty‐four cases of SDS patients were analyzed and compared to other patients with the four most common IBMFSs on the CIMFS: Diamond Blackfan anemia, Fanconi anemia (FA), Kostmann/severe congenital neutropenia and dyskeratosis congenita (DC). The diagnosis of SDS, FA and DC was often delayed relative to symptoms onset; indicating a major need for improving tools to establish a rapid diagnosis. We identified multiple phenotypic differences between SDS and other IBMFSs, including several novel differences. SBDS biallelic mutations were less frequent than in previous reports (81%). Importantly, compared to patients with biallelic mutations, patients with wild type SBDS had more severe hematological disease but milder pancreatic disease. In conclusion, comprehensive study of the IBMFSs can provide useful comparative data between the disorders. SBDS‐negative SDS patients may have more severe hematological failure and milder pancreatic disease.     

Initial combination therapy with saxagliptin and metformin provides sustained glycaemic control and is well tolerated for up to 76 weeks

Aim: To assess the efficacy and safety of saxagliptin + metformin initial combination therapy compared with saxagliptin or metformin alone over 76 weeks (24‐week short‐term + 52‐week long‐term extension) in treatment‐naÏve type 2 diabetes mellitus patients with inadequate glycaemic control. Methods: In this phase 3, parallel‐group, double‐blind, active‐controlled study, 1306 patients 18–77 years of age (HbA1c 8.0–12.0%) were randomized to saxagliptin 5 mg + 500 mg metformin, saxagliptin 10 mg + 500 mg metformin, saxagliptin 10 mg + placebo or 500 mg metformin + placebo. Blinded metformin was titrated during weeks 1–5 of the short‐term treatment period in 500 mg/day increments to 2000 mg/day maximum in the metformin‐based treatment groups. No titration of metformin was permitted during the long‐term treatment period. A total of 888 patients completed the study (76 weeks), 613 without being rescued. Changes in HbA1c, fasting plasma glucose, 120‐min postprandial glucose (PPG) and PPG‐area under the curve (AUC) from baseline to week 76 were analysed using a repeated‐measures model. Results: At 76 weeks, adjusted mean changes from baseline HbA1c (95% CI) for saxagliptin 5 mg + metformin, saxagliptin 10 mg + metformin, saxagliptin 10 mg and metformin were ?2.31 (?2.44, ?2.18), ?2.33 (?2.46, ?2.20), ?1.55 (?1.70, ?1.40) and ?1.79% (?1.93, ?1.65), respectively (post hoc and nominal p < 0.0001 vs. metformin and saxagliptin monotherapies for saxagliptin 5 mg + metformin and saxagliptin 10 mg + metformin). The proportions of patients requiring rescue or discontinuation for insufficient glycaemic control were lower for saxagliptin + metformin than for either monotherapy. Little or no attenuation in PPG‐AUC or 120‐min PPG was observed between weeks 24 and 76 for saxagliptin + metformin, indicating persistent efficacy. Adverse event rates were similar across groups; hypoglycaemic events occurred at a low frequency. Conclusion: Saxagliptin + metformin initial combination therapy was well tolerated and produced sustained glycaemic control for up to 76 weeks, with greater improvements in glycaemic parameters compared with either drug alone.