BACKGROUND:Fibrosis around cardiac cell injections represents an obstacle to graft integration in cell-based cardiac repair. Thrombospondin-2 (TSP-2) is a pro-fibrotic, anti-angiogenic matricellular protein and an attractive target for therapeutic knockdown to improve cardiac graft integration and survival. METHODS:We used a TSP-2 knockout (KO) mouse in conjunction with a fetal murine cardiomyocyte grafting model to evaluate the effects of a lack of TSP-2 on fibrosis, vascular density, and graft size in the heart. RESULTS:Two weeks after grafting in the uninjured heart, fibrosis area was reduced 4.5-fold in TSP-2 KO mice, and the thickness of the peri-graft scar capsule was reduced sevenfold compared to wild-type (WT). Endothelial cell density in the peri-graft region increased 2.5-fold in the absence of TSP-2, and cardiomyocyte graft size increased by 46% in TSP-2 KO hearts. CONCLUSIONS:TSP-2 is a key regulator of fibrosis and angiogenesis following cell grafting in the heart, and its absence promotes better graft integration, vascularization, and survival. SUMMARY:Fibrosis around cardiac cell injections impairs graft integration in cell-based cardiac repair. TSP-2 is a pro-fibrotic, anti-angiogenic matricellular protein. Using a TSP-2-knockout mouse model and cardiac cell transplantation, we found significantly reduced fibrosis and increased endothelial cell density in the peri-graft region. Thus, TSP-2 is an attractive target for therapeutic knockdown to improve cardiac graft integration and survival.

Stem cell transplantation may repair the injured heart, but tissue regeneration is limited by death of transplanted cells. Most cell death occurs in the first few days post-transplantation, likely from a combination of ischemia, anoikis and inflammation. Interventions known to enhance transplanted cell survival include heat shock, over-expressing anti-apoptotic proteins, free radical scavengers, anti-inflammatory therapy and co-delivery of extracellular matrix molecules. Combinatorial use of such interventions markedly enhances graft cell survival, but death still remains a significant problem. We review these challenges to cardiac cell transplantation and present an approach to systematically address them. Most anti-death studies use histology to assess engraftment, which is time- and labor-intensive. To increase throughput, we developed two biochemical approaches to follow graft viability in the mouse heart. The first relies on LacZ enzymatic activity to track genetically modified cells, and the second quantifies human genomic DNA content using repetitive Alu sequences. Both show linear relationships between input cell number and biochemical signal, but require correction for the time lag between cell death and loss of signal. Once optimized, they permit detection of as few as 1 graft cell in 40,000 host cells. Pro-survival effects measured biochemically at three days predict long-term histological engraftment benefits. These methods permitted identification of carbamylated erythropoietin (CEPO) as a pro-survival factor for human embryonic stem cell-derived cardiomyocyte grafts. CEPO's effects were additive to heat shock, implying independent survival pathways. This system should permit combinatorial approaches to enhance graft viability in a fraction of the time required for conventional histology.

Intramyocardial injection of therapeutic agents may enhance heart repair after infarction. Incomplete retention of intramyocardial injections has been reported, but modes of loss are undefined. We determined the fate of neutron-activated microspheres injected into acutely ischemic rat myocardium using saline, Pluronic F127, or Matrigel as vehicle. Twenty minutes after injection in saline, 63% +/- 12% of 10-mum microspheres was retained in the heart. Similar retention was observed after 6 days. Injection site leakage accounted for 14% +/- 5% of the microspheres, whereas exit via coronary veins resulted in 11.2% +/- 9.5% collecting in the lungs. Microspheres distribution to other organs was minimal. Retention of 40-mum microspheres was similar to that observed with the 10-mum microspheres. Pluronic F127 and Matrigel reduced immediate leakage to 4% +/- 1% and 2% +/- 1%, respectively. Surprisingly, microsphere retention in the heart was not improved at 20 min using either gelling vehicle, suggesting that leakage occurs over a prolonged period. Thus, most injected particles are retained in the ischemic rat heart following direct injection, but significant fractions are lost from the injection site and through coronary veins. Gelling agents reduced short-term leakage, but failed to enhance longer-term retention. Hydrogels with stiffer mechanical properties might enhance retention and reduce variability.

BACKGROUND:Myocardial infarcts in mammals heal by scar formation rather than formation of new muscle tissue. The MRL/MpJ [Murphy Roths large (MRL) derived by the Murphy group of the Jackson Laboratory (MpJ)] mouse, however, has been reported to exhibit minimal scarring and subsequent cardiac regeneration after cryoinjury of the right ventricle. Other groups have reported that permanent and temporary ligation of the coronary artery resulted in scarring without regeneration. METHODS:To clarify these contradictory results, we studied the temporal evolution of infarcts in MRL/MpJ and C57BL/6 control mice from 1 to 90 days post injury and the effects of intrathoracic cryoinjury to 28 days. RESULTS:After infarction, the conversion from necrotic myocardium to granulation tissue and then to scar proceeded identically in the two groups. Infarct DNA synthesis, measured by incorporation of a 5-bromo-2-deoxyuridine pulse, peaked at 4 days in both strains and did not differ between strains at any time point. Endothelial cell and total vascular density in the both the infarcted and noninfarcted cardiac tissue did not differ between groups at any time. Histological analysis of directly cryoinjured right and left ventricular myocardium showed indistinguishable wound healing in both strains, and final scar size was identical in each group. CONCLUSIONS:These studies demonstrate that both myocardial infarcts and cryoinjuries in MRL/MpJ mice heal by typical scar formation rather than muscle regeneration, in a manner very similar to C57BL/6 controls. We conclude that the MRL mouse is not a model for myocardial regeneration.

Capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated as alternatives to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis for peptide mapping with Staphylococcus aureus protease (V8) of a hydrophobic recombinant hepatitis C virus antigen, HC-31, which required 0.1% SDS for solubility. Controls (V8 only) or HC-31 digests were extracted with chloroform-methanol-water (1:4:3) to remove SDS, which interferes with MALDI-TOF, and high salt content, which affects CE. In two different runs by CE, the elution times of each of 11 peptide peaks were very reproducible (R.S.D.<0.016). 25 fragments were resolved by MALDI-TOF-MS, including six smaller peptides (M(r)<13 000) resulting from V8 autodigestion. MALDI-TOF-MS indicated that partial cleavages occurred, primarily at sites where there are paired glutamic and/or aspartic acid residues.