alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Ischemia

Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

Topics: Animals; Biocompatible Materials; Cadherins; Cell Line; Collagen; Embryonic Stem Cells; Extracellular Matrix; Female; Gene Expression; Homeodomain Proteins; Insulin-Like Growth Factor I; Ischemia; Major Histocompatibility Complex; Mice; Mice, Inbred C57BL; Muscle Development; Muscle, Skeletal; Myogenic Regulatory Factor 5; Myogenin; Oligosaccharides; Paired Box Transcription Factors; PAX3 Transcription Factor; Regeneration; Sialyl Lewis X Antigen

Circulating progenitor cells home to and engraft to sites of ischemia, mediated in part by the adhesion molecule L-selectin; however, accumulation in tissues such as the heart is low. In this study, an acellular collagen-based matrix containing sialyl Lewis(X) (sLe(X)), which binds L-selectin, was developed in order to enhance the endogenous progenitor cell therapeutic response. Its effect on progenitor cells and angiogenesis were assessed in vitro and using a hindlimb ischemia model with rats. In culture, the sLe(X)-collagen matrix recruited more CD133(+)CD34(+)L-selectin(+) cells than collagen-only matrix, with adhesion mediated by L-selectin binding. Increased angiogenic/chemotactic cytokine production and improved resistance to apoptosis appeared in cells cultured on sLe(X)-collagen matrix. In vivo, mobilization of endogenous circulating progenitor cells was increased, and greater recruitment of these and systemically injected human peripheral blood CXCR4(+)L-selectin(+) cells to sLe(X)-collagen treated limbs was observed compared to collagen-only. This condition was associated with differences in angiogenic/chemotactic cytokine levels, with greater arteriole density and increased perfusion in sLe(X)-collagen treated hindlimbs. With these factors taken together, we demonstrated that an acellular matrix-bound ligand approach can enhance the mobilization, recruitment, and therapeutic effects of endogenous and/or transplanted progenitor cells, possibly through paracrine and antiapoptotic mechanisms, and could be used to improve cell-based regenerative therapies.

Topics: AC133 Antigen; Adult; Animals; Antigens, CD; Antigens, CD34; Cell Adhesion; Cell- and Tissue-Based Therapy; Collagen; Cytokines; Glycoproteins; Hindlimb; Humans; Intercellular Signaling Peptides and Proteins; Ischemia; L-Selectin; Leukocytes, Mononuclear; Neovascularization, Physiologic; Oligosaccharides; Peptides; Rats; Rats, Sprague-Dawley; Sialyl Lewis X Antigen; Stem Cells

The aim of this study was to look at the role of alpha 1-acid glycoprotein as a natural anti-inflammatory agent with particular respect to its antineutrophil and anticomplement activity. A recombinantly engineered form of sialyl Lewisx (sLe(x))-bearing alpha 1-acid glycoprotein (sAGP) was administered intravenously to pentobarbital-anesthetized rats after 50 min of intestinal ischemia just before 4 h of reperfusion. A non-sLe(x)-bearing form of AGP (nsAGP) was used as control. sAGP-treated animals had a 62% reduction (P < 0.05) in remote lung injury, assessed by 125I-albumin permeability, compared with those treated with nsAGP (permeability index of 3.61 +/- 0.15 x 10(-3) and 5.18 +/- 0.67 x 10(-3), respectively). There was a reduction in pulmonary myeloperoxidase levels in sAGP-treated rats compared with nsAGP-treated rats. Complement-dependent intestinal injury, assessed by 125I-albumin permeability was reduced by 28% (P < 0.05) in animals treated with sAGP (7.58 +/- 0.63) compared with those treated with nsAGP (10.4 +/- 0.54). We conclude that sAGP ameliorates both complement- and neutrophil-mediated injuries.

Topics: Animals; Cell Membrane Permeability; Complement Pathway, Alternative; Humans; Intestines; Iodine Radioisotopes; Ischemia; Lung; Male; Mesenteric Vascular Occlusion; Oligosaccharides; Orosomucoid; Peroxidase; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion; Sialyl Lewis X Antigen

Neutrophil adhesion and migration is associated with hepatic ischemia and reperfusion. The role of a Sialyl Lewis(x) (SLe)(x) oligosaccharide, a ligand for selections, was studied in hepatic ischemia and reperfusion injury.. Total hepatic ischemia was produced in rats for 90 minutes using an extracorporeal portosystemic shunt. To assess the role of SLe(x) in hepatic ischemia and reperfusion injury, 25 mg/kg of an SLe(x) analog, CY-1503, was given five minutes before reperfusion or at reperfusion. Biochemical tests of hepatic injury, myeloperoxidase activity in hepatic tissue, and histologic studies, including neutrophil infiltration determined by the naphthol esterase technique, were analyzed six hours after reperfusion.. Significantly improved protection in biochemical hepatic injury tests (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase) was noted between the ischemic and the SLe(x) treated groups. Myeloperoxidase activity and polymorphonuclear cell infiltration in hepatic tissue were decreased in the SLe(x) groups. Histologic protection from hepatic damage was observed in the treated groups.. The SLe(x) oligosaccharide analog, CY-1503, had an important protective role in hepatic ischemia and reperfusion injury. Modulation of SLe(x) in the neutrophil decreased the adhesion of polymorphonuclear cells and their subsequent migration after hepatic ischemia and reperfusion.

Topics: Animals; Drug Evaluation, Preclinical; Endothelium, Vascular; Ischemia; Lewis Blood Group Antigens; Ligands; Liver; Male; Neutrophils; Oligosaccharides; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Selectins; Sialyl Lewis X Antigen

We investigated the role of adhesion molecules in the early phase of reperfusion following cold ischemia. Livers of male Lewis rats were preserved for 0 h (group A) or 24 h in University of Wisconsin (UW) solution without additives (group B) or in UW solution with anti-ICAM-1 antibody (group C) or anti-E-selectin-1, SLe(x) and SLe(a) antibodies (group D). The livers were then reperfused with diluted rat whole blood (DWB; groups A and B). DWB containing anti-ICAM-1 and LFA-1 antibodies (group C) or DWB containing anti-L-selectin, SLe(x) and SLe(a) antibodies (group D). The reperfusion was performed at 37 degrees C for 1 h at 5 cm H2O of perfusion pressure. During reperfusion, hepatic microcirculation was assessed by monitoring portal and peripheral tissue blood flow. Bile production was significantly reduced in group B livers compared with those in group A. Anti-ICAM-1 and LFA-1 antibodies failed to improve hepatic microcirculation, whereas anti-LECAM-1, SLe(x) and SLe(a) antibodies significantly improved the microcirculation. Bile production in group C and D livers was comparable to that in group B livers. Preservation for 24 h significantly increased the release of TNF-alpha from 0.207 to 43.7 pg/g per hour during reperfusion. Monoclonal antibodies to the adhesion molecules did not suppress the release of TNF-alpha in groups C and D. Histological examination demonstrated a lack of leukocyte infiltration or thrombus in hetapic microvessels. The extent of hepatocyte necrosis did not differ among groups B, C, and D. We conclude that the microcirculatory disturbance in the early phase of reperfusion occurs as a result of the tethering of leukocytes through the interaction of the selectin family and their ligands, and that the ICAM-1-LFA-1 pathway is not involved in this step. The lack of improvement in bile production with antibodies to the selectin family and their ligands strongly suggests that other mechanisms participate in the deterioration of hepatic function.

Topics: Adenosine; Allopurinol; Animals; Antibodies, Monoclonal; Antibody Specificity; Bile; CA-19-9 Antigen; Cell Adhesion; Cold Temperature; E-Selectin; Glutathione; Insulin; Intercellular Adhesion Molecule-1; Ischemia; L-Lactate Dehydrogenase; L-Selectin; Leukocytes; Liver; Lymphocyte Function-Associated Antigen-1; Male; Microcirculation; Necrosis; Oligosaccharides; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Sialyl Lewis X Antigen; Tumor Necrosis Factor-alpha