Characterization and therapeutic application of canine adipose mesenchymal stem cells to treat elbow osteoarthritis

Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia. © 2017, Canadian Veterinary Medical Association. All rights reserved

Licensing by Inflammatory Cytokines Abolishes Heterogeneity of Immunosuppressive Function of Mesenchymal Stem Cell Population

When mesenchymal stem cells (MSCs) are used for therapy of immunological pathologies, they get into an inflammatory environment, altering the effectiveness of the treatment. To establish the impact of environmental inflammatory factors on MSCs' immunofunction in the mirror of intrinsic heterogeneity of mouse MSC population, individual MSC clones were generated and characterized. Adipogenic but not osteogenic differentiation and pro-angiogenic activity of five independent MSC cell lines were similar. Regarding osteogenic differentiation, clones MSC3 and MSC6 exhibited poorer capacity than MSC2, MSC4, and MSC5. To study the immunosuppressive heterogeneity, in vitro and in vivo experiments have been carried out using T-cell proliferation assay and delayed-type hypersensitivity (DTH) response, respectively. A remarkable difference was found between the clones in their ability to inhibit T-cell proliferation in the following order: MSC2MSC5>MSC4>MSC3>>MSC6. Nevertheless, the differences between the immunosuppressive activities of the individual clones disappeared on pretreatment of the cells with pro-inflammatory cytokines, a procedure called licensing. Stimulation of all clones with IFN- and TNF- resulted in elevation of their inhibitory capability to a similar level. Nitric oxide (NO) and prostaglandin E2 (PGE2) were identified as major mediators of immunofunction of the MSC clones. The earlier findings were also supported by in vivo results. Without licensing, MSC2 inhibited DTH response, while MSC6 did not affect DTH response. In contrast, prestimulation of MSC6 with inflammatory cytokines resulted in strong suppression by this clone as well. Here, we have showed that MSC population is functionally heterogeneous in terms of immunosuppressive function; however, this variability is largely reduced under pro-inflammatory conditions

Novel role for galectin-1 in T-cells under physiological and pathological conditions

Secreted, extracellular galectin-1 (exGal-1) but not intracellular Gal-1 (inGal-1) has been described as a strong immunosuppressive protein due to its major activity of inducing apoptosis of activated T-cells. It has previously been reported that T-cells express Gal-1 upon activation, however its participation in T-cell functions has remained largely elusive. To determine function of Gal-1 expressed by activated T-cells we have carried out a series of experiments. We have shown that Gal-1, expressed in Gal-1-transgenic Jurkat cells or in activated T-cells, remained intracellularly indicating that Gal-1-induced T-cell death was not a result of an autocrine effect of the de novo expressed Gal-1. Rather, a particular consequence of the inGal-1 expression was that T-cells became more sensitive to exGal-1 added either as a soluble protein or bound to the surface of a Gal-1-secreting effector cell. This was also verified when the susceptibility of activated T-cells from wild type or Gal-1 knockout mice to Gal-1-induced apoptosis were compared. Murine T-cells expressing Gal-1 were more sensitive to the cytotoxicity of the exGal-1 than their Gal-1 knockout counterparts. We also conducted a study with activated T-cells from patients with systemic lupus erythematosus (SLE), a disease in which dysregulated T-cell apoptosis has been well described. SLE T-cells expressed lower amounts of Gal-1 than healthy T-cells and were less sensitive to exGal-1. These results suggested a novel role of inGal-1 in T-cells as a regulator of T-cell response to exGal-1, and its likely contribution to the mechanism in T-cell apoptosis deficiency in lupus

Characterization and therapeutic application of canine adipose mesenchymal stem cells to treat elbow osteoarthritis

Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia. © 2017, Canadian Veterinary Medical Association. All rights reserved

Identification of Galectin-1 as a Critical Factor in Function of Mouse Mesenchymal Stromal Cell-Mediated Tumor Promotion

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development. These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression

A mezenhimális őssejt-eredetű galektin-1 meghatározó faktor a felnőtt szöveti őssejtek pro-angiogén funkciójában

A tumorigenezis során az egészséges sejtek növekedést szabályozó génjeiben mutációkat halmoznak fel, melynek következtében kikerülnek a szigorú kontroll alól, és korlátlan osztódásnak indulnak. A tumorsejtek önmagukban azonban nem lennének képesek az egész szervezetet érintő megbetegedés kialakítására, ehhez szükség van arra, hogy a fennmaradásukat támogató kötőszövettel, tumor-asszociált sztrómával rendelkezzen, mely extracelluláris mátrixból, valamint különféle sejttípusokból, például fibroblasztokból, endotél sejtekből, pericitákból/ér simaizom sejtekből, immunsejtekből tevődik össze. A tumorsejtek és a sztróma komponensek egymásra gyakorolt hatásának eredményeként alakulnak ki a sokszor leküzdhetetlennek bizonyuló daganatos megbetegedések. A daganat kötőszöveti sejtjeinek eredete nem teljesen ismert. Egyik forrása a csontvelői mezenhimális őssejtek (MSC-k) lehetnek. A mezenhimális őssejtek - más néven felnőtt szöveti őssejtek – olyan multipotens sejtek, melyek kis százalékban valamennyi szövetben megtalálhatók, fiziológiás feladatuk a szervek kötőszövetének megújítása. Az MSC-k a tumorsejtekből felszabaduló faktorok hatására a tumorba vándorolnak. A daganatszövethez érve heterotípusos kapcsolataik, szekretált molekuláik vagy fibroblaszttá és ereket támogató pericitákká/ér simaizom sejtekké történő differenciáció révén támogatják a tumor fejlődését. Az MSC-k nagy mennyiségben termelik a galektin-1 (Gal-1) szénhidrát kötő fehérjét, melynek funkciója a daganatfejlődés szepomtjából kevéssé ismert. Munkánk során ezért arra a kérdésre kerestük a választ, hogy az MSC-eredetű Gal-1 milyen szerepet játszik a mezenhimális őssejtek tumornövekedést befolyásoló hatásában

Foldameric α/β-Peptide Analogs of the β‑Sheet-Forming Antiangiogenic Anginex: structure and Bioactivity

The principles of beta-sheet folding and design for alpha-peptidic sequences are well established, while those for sheet mimetics containing homologated amino acid building blocks are still under investigation. To reveal the structure-function relations of beta-amino-acid-containing foldamers, we followed a top-down approach to study a series of alpha/beta-peptidic analogs of anginex, a beta-sheet-forming antiangiogenic peptide. Eight anginex analogs were developed by systematic alpha --> beta(3) substitutions and analyzed by using NMR and CD spectroscopy. The foldamers retained the beta-sheet tendency, though with a decreased folding propensity. beta-Sheet formation could be induced by a micellar environment, similarly to that of the parent peptide. The destructuring effect was higher when the alpha --> beta(3) exchange was located in the beta-sheet core. Analysis of the beta-sheet stability versus substitution pattern and the local conformational bias of the bulky beta(3)V and beta(3)I residues revealed that a mismatch between the H-bonding preferences of the alpha- and beta-residues played a minor role in the structure-breaking effect. Temperature-dependent CD and NMR measurements showed that the hydrophobic stabilization was scaled-down for the alpha/beta-peptides. Analysis of the biological activity of the foldamer peptides showed that four anginex derivatives dose-dependently inhibited the proliferation of a mouse endothelial cell line. The alpha --> beta(3) substitution strategy applied in this work can be a useful approach to the construction of bioactive beta-sheet mimetics with a reduced aggregation tendency and improved pharmacokinetic properties

GM1 controlled lateral segregation of tyrosine kinase Lck predispose T-cells to cell-derived galectin-1-induced apoptosis

One prominent immunoregulatory function of galectin-1 (Gal-1), a beta-galactoside binding mammalian lectin, is induction of apoptosis in activated T-cells by a process depending on the activity of Src family tyrosine kinase, Lck. Although the requirement for Lck in Gal-1 induced T-cell death and the ability of Gal-1 to affect the membrane localization of extracellular Gal-1-binding proteins have been well documented, the consequence of the complex and related reorganization of extra- and intracellular signaling components upon Gal-1 treatment of T-cells has not yet been revealed. Therefore, we have analyzed the plasma membrane movement of Lck upon Gal-1 triggered signaling, and the significance of this event in Gal-1 induced T-cell death. Non-receptor tyrosine kinase, Lck primarily localized in the synapse of tumor cell-T-cell during 15min of the established direct cell contact. Later, after 30min, a lateral segregation of Lck from the cell synapse was observed. The migration of Lck to the opposite of the cell contact apparently depended on the expression and cell surface presentation of Gal-1 on the effector (tumor) cells and was accompanied by phosphorylation on the negative regulatory tyrosine residue, Tyr505. Receptor tyrosine phosphatase, CD45 played crucial role in this event since CD45 deficiency or inhibition of its phosphatase activity resulted in the failure of Lck membrane movement. Level of the Gal-1-binding glycolipid GM1 ganglioside also essentially regulated Lck localization. Segregation of Lck and Gal-1 induced apoptosis was diminished in T-cells with low GM1 expression compared to T-cells with high GM1. Our results show that spatial regulation of Lck by CD45 and GM1 ganglioside determines the outcome of apoptotic response to Gal-1 and this local regulation may occur only upon intimate effector (Gal-1 expressing) cell-T-cell attachment

Two-stage interaction of the tumor nursing galectin-1 with the antiangiogenic peptide anginex

The 33mer peptide anginex is a potent inhibitor of angiogenesis and tumor growth. Its biological activity is dependent on the beta-galactoside-binding protein galectin-1 (gal-1), which has been reported to be the main receptor for anginex. The gal-1-anginex interaction has been observed using surface plasmon resonance and mass spectrometric methods, but the stoichiometry and affinity in the solution remain elusive. Our aim was to characterize the gal-1-anginex interaction via isothermal titration calorimetry. In order to ensure protein purity and integrity, native gel electrophoresis, Western blot analysis, mass spectrometric measurements, and ultracentrifugation were carried out for the recombinant wild-type human gal-1 and V5D gal-1 expressed in E. coli. Two stages were identified in the titration curves: (i) formation of a 4:1 galectin-1-anginex complex with low nM affinity, and (ii) a complex with 1:1 stoichiometry exhibiting K (D) > 200 nM. The 4:1 complex was robust at different concentrations, and neither the oxidation state nor the V5D mutation (a monomeric gal-1 mutant) of gal-1 affected this stoichiometry. The presence of the high-affinity 4:1 interaction may have implications for the biological applications of anginex