In-vivo Murine Model Research Articles

Differential promotion of angiogenesis by distinct bone marrow cell subpopulations

Abstract Introduction: Bone marrow mononuclear cells (BMMC) have the capacity to differentiate into endothelial cells and promote neovascularization in hindlimb ischemia. However, the specific bone marrow cell population that promotes revascularization is unknown. Therefore, we determined the ability of unfractionated BMMC, CD34+ and CD34− from bone marrow to promote angiogenesis in an in-vivo murine model. Methods: BMMC were obtained from total bone marrow harvested from C57Bl/6 mice. CD34+ and CD34− subpopulations were isolated using magnetic bead separation. 1cc Matrigel was admixed with either unfractionated BMMC or CD34+ or CD34− cells was injected subcutaneously. Matrigel +/− VEGF (500ng/cc) was injected as positive and negative controls. Matrigel implants were harvested 14 days later and were analyzed by immunohistochemistry with determination of CD31+ and smooth-muscle actin (SMA) positive areas by morphometry. Results: No angiogenesis was observed in plain Matrigel. All implants seeded with bone-marrow-derived cells promoted neovessel formation in a dose-dependent manner. Unfractionated BMMC promoted formation of neovessels containing both CD31+ and SMA + cells. CD31+ area was greatest in implants seeded with CD34+ cells. In contrast, SMA+ area was greatest in implants containing CD34− cells, which promoted development of larger SMA-containing vessels. Conclusions: BMMC are capable of promoting angiogenesis in an in-vivo Matrigel model in a dose-dependent manner. CD34+ bone marrow cells are most capable of promoting endothelial development. In contrast, the CD34− subpopulation promotes development of larger arteries containing vascular SMA. These results have important implications for the development of cell-based therapies for limb ischemia.

Stress proteins are proinflammatory in vivo

Fibrosis of biosensors, placed in vivo, represents a major hurdle to the wide-spread application of real-time monitoring of biochemical and metabolic processes in animals and humans. The fibrotic reaction is the end result of the acute inflammatory response caused by significant tissue trauma at the time of biosensor placement. This response may be associated with increased expression and release from cells of cellular stress proteins. We tested the hypothesis that subcutaneous injection of stress proteins will cause acute inflammatory swelling in an in-vivo murine model. Comparisons between groups were performed using the Dunnet T3 multiple comparison test and significance was accepted when p < 0.05. Subcutaneous injection of stress proteins (HSP70 or GRP78) was associated with significantly increased acute swelling in comparison to injections of saline or autologous serum (AS). The histological analysis demonstrated increased inflammatory cell infiltrate in the tissues that received the stress protein injections. Stress proteins, within the acute wound environment, are proinflammatory and may set the stage for peribiosensor fibrosis and eventual failure. Stress proteins may be future targets for therapeutic manipulation wherever acute inflammation is followed by problematic fibrosis.