Mesenchymal stem cells pretreated with delivered Hph-1-Hsp70 protein are protected from hypoxia-mediated cell death and rescue heart functions from myocardial injury.

Mesenchymal stem cell (MSC) therapy for myocardial injury has inherent limitations due to the poor viability of MSCs after cell transplantation. In this study, we directly delivered Hsp70, a protein with protective functions against stress, into MSCs, using the Hph-1 protein transduction domain ex vivo for high transfection efficiency and low cytotoxicity. Compared to control MSCs in in vitro hypoxic conditions, MSCs delivered with Hph-1-Hsp70 (Hph-1-Hsp70-MSCs) displayed higher viability and anti-apoptotic properties, including Bcl2 increase, reduction of Bax, JNK phosphorylation and caspase-3 activity. Hsp70 delivery also attenuated cellular ATP-depleting stress. Eight animals per group were used for in vivo experiments after occlusion of the left coronary artery. Transplantation of Hph-1-Hsp70-MSCs led to a decrease in the fibrotic heart area, and significantly reduced the apoptotic positive index by 19.5 +/- 2%, compared to no-treatment controls. Hph-1-Hsp70-MSCs were well-integrated into the infarcted host myocardium. The mean microvessel count per field in the infarcted myocardium of the Hph-1-Hsp70-MSC-treated group (122.1 +/- 13.5) increased relative to the MSC-treated group (75.9 +/- 10.4). By echocardiography, transplantation of Hph-1-Hsp70-MSCs resulted in additional increases in heart function, compared to the MSCs-transplanted group. Our results may help formulate better clinical strategies for in vivo MSC cell therapy for myocardial damage.

Role of disulfide bonds in the structure and activity of human insulin.

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness. Deletion of the A6-A11 disulfide bond caused the least perturbation to the structure. In addition, different refolding efficiencies between the three des mutants suggest that the disulfide bonds are formed sequentially in the order A20-B19, A7-B7 and A6-A11 in the folding pathway of proinsulin.