Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.

Intravenous immunoglobulin (IVIg) therapy is associated with a broad range of immunomodulatory activities. Several of the postulated mechanisms of IVIg action relate to the presence of antibodies to molecules relevant for regulation of the immune response. This article reports that IVIg contains antibodies to the Arg-Gly-Asp (RGD) sequence, and the attachment site of a number of adhesive extracellular matrix proteins, including ligands for β1, β3, and β5 integrins. Anti-RGD antibodies were identified in IVIg by enzyme-linked immunosorbent assay and by using the BIAcore (BIAcore, Uppsala, Sweden) technology. The affinity of anti-RGD antibodies to a synthetic RGD-containing peptide and to fibronectin (Fn) was found to be in the micromolar range. F(ab′)2 fragments specific for RGD were purified from IVIg by affinity chromatography. Anti-RGD F(ab′)2 antibodies inhibited adenosine diphosphate induced IIb/β3 integrin-mediated platelet aggregation and the adhesion of activated 4β1 integrin-expressing B cells to Fn. Adhesion of unstimulated platelets to fibrinogen (Fg) involving both the γ-chain dodecapeptide sequence and the RGD sequence was inhibited by anti-RGD antibodies. In addition, adhesion of thrombin-stimulated platelets to von Willebrand factor or Fg was completely inhibited by affinity-purified anti-RGD antibodies. Our results suggest that the presence of natural IgG antibodies to the RGD motif may contribute to the immunomodulatory and anti-inflammatory effects of therapeutic preparations of normal IgG.