Integrin α10ß1-selected mesenchymal stem cells reduced hypercoagulopathy in a porcine model of acute respiratory distress syndrome.

Mesenchymal stem cells (MSCs) have been studied for their potential benefits in treating acute respiratory distress syndrome (ARDS) and have reported mild effects when trialed within human clinical trials. MSCs have been investigated in preclinical models with efficacy when administered at the time of lung injury. Human integrin α10ß1-selected adipose tissue-derived MSCs (integrin α10ß1-MSCs) have shown immunomodulatory and regenerative effects in various disease models. We hypothesized that integrin α10ß1 selected-MSCs can be used to treat a sepsis-induced ARDS in a porcine model when administering cells after established injury rather than simultaneously. This was hypothesized to reflect a clinical picture of treatment with MSCs in human ARDS. 12 pigs were randomized to the treated or placebo-controlled group prior to the induction of mild to moderate ARDS via lipopolysaccharide administration. The treated group received 5 × 106 cells/kg integrin α10ß1-selected MSCs and both groups were followed for 12 h. ARDS was confirmed with blood gases and retrospectively with histological changes. After intervention, the treated group showed decreased need for inotropic support, fewer signs of histopathological lung injury including less alveolar wall thickening and reduction of the hypercoagulative disease state. The MSC treatment was not associated with adverse events over the monitoring period. This provides new opportunities to investigate integrin α10ß1-selected MSCs as a treatment for a disease which does not yet have any definitive therapeutic options.

Global genomic and transcriptomic analysis of human pancreatic islets reveals novel genes influencing glucose metabolism.

Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

Guthrie card methylomics identifies temporally stable epialleles that are present at birth in humans.

A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time consuming. Here, we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in-utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify interindividual DNA methylation variation that is present both at birth and 3 yr later. These findings suggest that disease-relevant epigenetic variation could be detected at birth, i.e., before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of interindividual epigenomic variation in a range of common human diseases.

Integrin α10ß1-Selected Mesenchymal Stem Cells Reduce Pain and Cartilage Degradation and Increase Immunomodulation in an Equine Osteoarthritis Model.

OBJECTIVE: Integrin α10ß1-selected mesenchymal stem cells (integrin α10-MSCs) have previously shown potential in treating cartilage damage and osteoarthritis (OA) in vitro and in animal models in vivo. The aim of this study was to further investigate disease-modifying effects of integrin α10-MSCs. DESIGN: OA was surgically induced in 17 horses. Eighteen days after surgery, horses received 2 × 107 integrin α10-MSCs intra-articularly or were left untreated. Lameness and response to carpal flexion was assessed weekly along with synovial fluid (SF) analysis. On day 52 after treatment, horses were euthanized, and carpi were evaluated by computed tomography (CT), MRI, histology, and for macroscopic pathology and integrin α10-MSCs were traced in the joint tissues. RESULTS: Lameness and response to carpal flexion significantly improved over time following integrin α10-MSC treatment. Treated horses had milder macroscopic cartilage pathology and lower cartilage histology scores than the untreated group. Prostaglandin E2 and interleukin-10 increased in the SF after integrin α10-MSC injection. Integrin α10-MSCs were found in SF from treated horses up to day 17 after treatment, and in the articular cartilage and subchondral bone from 5 of 8 treated horses after euthanasia at 52 days after treatment. The integrin α10-MSC injection did not cause joint flare. CONCLUSION: This study demonstrates that intra-articular (IA) injection of integrin α10-MSCs appears to be safe, alleviate pathological changes in the joint, and improve joint function in an equine post-traumatic osteoarthritis (PTOA) model. The results suggest that integrin α10-MSCs hold promise as a disease-modifying osteoarthritis drug (DMOAD).

Human integrin α10ß1-selected mesenchymal stem cells home to cartilage defects in the rabbit knee and assume a chondrocyte-like phenotype.

BACKGROUND: Mesenchymal stem cells (MSCs) have shown promising results in stimulating cartilage repair and in the treatment of osteoarthritis (OA). However, the fate of the MSCs after intra-articular injection and their role in cartilage regeneration is not clear. To address these questions, this study investigated (1) homing of labeled human adipose tissue derived integrin α10ß1-selected MSCs (integrin α10-MSCs) to a cartilage defect in a rabbit model and (2) the ability of the integrin α10-MSCs to differentiate to chondrocytes and to produce cartilage matrix molecules in vivo. DESIGN: Integrin α10-MSCs were labeled with superparamagnetic iron oxide nanoparticles (SPIONs) co-conjugated with Rhodamine B to allow visualization by both MRI and fluorescence microscopy. A cartilage defect was created in the articular cartilage of the intertrochlear groove of the femur of rabbits. Seven days post-surgery, labeled integrin α10-MSCs or vehicle were injected into the joint. Migration and distribution of the SPION-labeled integrin α10-MSCs was evaluated by high-field 9.4 T MRI up to 10 days after injection. Tissue sections from the repair tissue in the defects were examined by fluorescence microscopy. RESULTS: In vitro characterization of the labeled integrin α10-MSCs demonstrated maintained viability, proliferation rate and trilineage differentiation capacity compared to unlabeled MSCs. In vivo MRI analysis detected the labeled integrin α10-MSCs in the cartilage defects at all time points from 12 h after injection until day 10 with a peak concentration between day 1 and 4 after injection. The labeled MSCs were also detected lining the synovial membrane at the early time points. Fluorescence analysis confirmed the presence of the labeled integrin α10-MSCs in all layers of the cartilage repair tissue and showed co-localization between the labeled cells and the specific cartilage molecules aggrecan and collagen type II indicating in vivo differentiation of the MSCs to chondrocyte-like cells. No adverse effects of the α10-MSC treatment were detected during the study period. CONCLUSION: Our results demonstrated migration and homing of human integrin α10ß1-selected MSCs to cartilage defects in the rabbit knee after intra-articular administration as well as chondrogenic differentiation of the MSCs in the regenerated cartilage tissue.

Temporal extracellular vesicle protein changes following intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells in equine osteoarthritis.

Introduction: Equine osteoarthritis (OA) is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterized by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilizes their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) treatment in an experimental model of equine osteoarthritis with longitudinal sampling. Methods: Adipose tissue derived, integrin α10-MSCs were injected intraarticularly in six horses 18 days after experimental induction of OA. Synovial fluid samples were collected at day 0, 18, 21, 28, 35, and 70. Synovial fluid was processed and extracellular vesicles were isolated and characterized. Extracellular vesicle cargo was then analyzed using data independent acquisition mass spectrometry proteomics. Results: A total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR ≤ 0.05) between sham-operated control joint without MSC treatment and OA joint treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p = 0.023), complement activation (classical pathway) (p = 0.023), and collagen containing extracellular matrix (p = 0.034). Due to the lack of an OA group without MSC treatment, findings cannot be directly correlated to only MSCs. Discussion: To date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis. This is an important step toward understanding the potential therapeutic mechanisms of MSC therapy, ultimately enabling the improvement of therapeutic efficacy.

Discovery of selective small-molecule CD80 inhibitors.

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs.

Identification of protein-protein interfaces implicated in CD80-CD28 costimulatory signaling.

The B7 ligands CD80 and CD86 on APCs deliver either costimulatory or inhibitory signals to the T cell when interacting with their counter-receptors CD28 and CD152 (CTLA-4) on the T cell surface. Although crucial for lymphocyte regulation, the structural basis of these interactions is still not completely understood. Using multivalent presentation and conditions mimicking clustering, believed to be essential for signaling through these receptors, and by applying a combined differential mass spectrometry and structural mapping approach to these conditions, we were able to identify a putative contact area involving hydrophilic regions on both CD28 and CD80 as well as a putative CD28 oligomerization interface induced by B7 ligation. Analysis of the CD80-CD28 interaction site reveals a well-defined interface structurally distinct from that of CD80 and CD152 and thus provides valuable information for therapeutic intervention targeted at this pathway, suggesting a general approach for other receptors.