Microvessel networks [corrected] pre-formed in artificial clinical grade dermal substitutes in vitro using cells from haematopoietic tissues.

Forming a microcirculation is critical for vascularisation of artificial skin substitutes. One strategy to improve speed of grafting is to pre-form microvascular networks in the substitute before applying to a wound. For clinical application, this requires sufficient functional endothelial cell numbers. In vitro endothelial colony forming cells (ECFCs) derived cells were expanded from cord and adult blood donations and co-cultured with human dermal fibroblasts or bone marrow mesenchymal stem/stromal cells to form microvascular networks in the presence or absence of dermal substitutes which are in clinical use. The number of endothelial cells generated ranged from 1.03×10(9) to 2.18×10(11) from 10 adult blood donations and 1×10(12) to 1.76×10(13) from 6 cord blood units after 50 days in culture. Two adult donations failed to generate ECFCs. Both cord and adult blood cells formed 2D microvascular networks in vitro, although there was a significant difference in the functional capacity of adult and cord blood ECFCs. While co-culture of the latter within dermal substitutes Matriderm or Integra demonstrated the formation of 3D microvascular networks penetrating 100µm, enhanced expansion, while maintaining functional capacity, of adult blood cells is required for fully pre-vascularising the clinical grade acellular dermal substitutes used here prior to applying these to burns.

Effects of obstetric factors and storage temperatures on the yield of endothelial colony forming cells from umbilical cord blood.

As umbilical cord blood (UCB) is a rich source of endothelial colony-forming cells (ECFC), our aim was twofold: (1) to examine potential obstetric selection criteria for achieving the highest ECFC yields from UCB units, and (2) to determine whether transient storage temperatures of fresh UCB and cryopreservation of UCB units affected ECFC yield and function. ECFC quality was assessed before and after cryopreservation by their clonogenic proliferative potential. Of the 20 factors examined, placental weight was the only statistically significant obstetric factor that predicted ECFC frequency in UCB. Studies on the effects of storage revealed that transient storage of fresh UCB at 4°C reduced ECFC yield compared with storage at 22°C, while cryopreservation of UCB MNCs significantly reduced ECFC recoveries. To our knowledge, this is the first demonstration that placental weight and temperature of storage prior to processing or culture have significant effects on ECFC frequency in UCB. Our studies further support the evidence that cryopreservation of UCB MNCs compromises ECFC recovery.

Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner.

Here, we show how targeting protein phosphatase 2A (PP2A), a key regulator of cellular protein phosphorylation, can either induce or prevent apoptosis depending on what other signals the cell is receiving. The oncoprotein polyoma small T interacts with PP2A to regulate survival. In the presence of growth factors, small T induces apoptosis. Akt activity, which usually promotes survival, is required for this death response, because inhibitors of Akt or PI3 kinase protect cells from death. The activation of Akt under these conditions is partial, characterized by T308 phosphorylation but not S473 phosphorylation. In the absence of growth factors, small T protects from cell death. Here, small T uses PP2A to promote phosphorylation of Akt on both T308 and S473. This effect results in a different pattern of phosphorylation of Akt substrates and shifts Akt from a proapoptotic (presence of growth factors) to an antiapoptotic mode (absence of growth factors). An intriguing possibility is that Akt phosphorylation could be therapeutically disregulated to decrease the survival of cancer cells.

Genetic analysis of the polyomavirus DnaJ domain.

Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60(c-src) and in transformation.

Activation of CREB/ATF sites by polyomavirus large T antigen.

Polyomavirus large T antigen (LT) has a direct role in viral replication and a profound effect on cell phenotype. It promotes cell cycle progression, immortalizes primary cells, blocks differentiation, and causes apoptosis. While much of large T function is related to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family, we have previously shown that activation of the cyclin A promoter can occur through a non-Rb-dependent mechanism. Here we show that activation occurs via an ATF/CREB site. Investigation of the mechanism indicates that large T can synergize with CREB family members to activate transcription. Experiments with Gal4-CREB constructs show that synergy is independent of CREB phosphorylation by protein kinase A. Examination of synergy with Gal4-CREB deletion constructs indicates that large T acts on the constitutive activation domain of CREB. Large T can bind to CREB in vivo. Genetic analysis shows that the DNA-binding domain (residues 264 to 420) is sufficient to activate transcription when it is localized to the nucleus. Further analysis of the DNA-binding domain shows that while site-specific DNA binding is not required, non-site-specific DNA binding is important for the activation. Thus, CREB binding and DNA binding are both important for large T activation of CREB/ATF sites. In contrast to previous models where large T transactivation occurred indirectly, these results also suggest that large T can act directly at promoters to activate transcription.