The stem cell fate of pericytes in tissue regeneration

Pericytes are perivascular cells that wrap endothelial cells in capillaries and form by that the structural support of small blood vessels, necessary to maintain blood pressure. It is now clear that pericytes are also involved in diseases that are closely linked with the formation of new blood vessels, such as diabetic retinopathy or cancer. Previous research into pericyte function has been limited as the isolation of pure cells proved difficult. But now this obstacle has been overcome by the development of sophisticated equipment and by the genetic engineering of mice in which mutations were found to affect pericyte function. Isolated pericytes were shown to be capable to differentiate into various cell types, and it was therefore suggested that they represent stem cell-like progenitor cells. Furthermore, pericytes that were isolated from skeletal muscle and injected into injured muscle were shown to participate in muscle regeneration. This has created big hope for gene therapy approaches using these cells. However, in order to safely use and to maximise the efficiency of pericytes in human applications it is necessary to fully characterise the potential of the endogenous pericytes, the degree to which they spontaneously contribute to tissue remodelling, and potential undesired side-effects,
We have previously generated a mouse model in which pericytes can be specifically identified by a colour-based staining procedure in isolated tissues. We have now crossed these mice with mdx mice, the mouse model for the human muscle wasting disease Duchenne muscular dystrophy, to determine by colour reaction whether or not endogenous pericytes contribute to new muscle formation. This has shown that there is massive remodelling of these cells in injured skeletal muscle. However, we are limited in the further identification of cells that originated from pericytes. We therefore propose to generate a more versatile mouse model, which marks the cells genetically with a fluorescent protein and enables us to follow the fate of pericytes in muscle regeneration by using imaging techniques that allow a high penetrance in the intact tissue. Moreover, this mouse model will be invaluable in the future to characterise pericyte progenitor cells from a variety of tissues for their capacity in tissue remodelling.

Perivascular cells/pericytes are adjacent to capillaries and have been named due to their anatomical location. Pericytes are in intimate contact with endothelial cells in capillaries and share a common basement membrane. They are thought to stabilise the vascular wall and control blood pressure and vascular permeability. In recent years there has been increasing evidence that pericytes represent a cell population with mesenchymal stem cell-like properties as they retain the capacity to differentiate into distinct mesenchymal lineages in vitro. However, there is little information available with respect to their differentiation potential in vivo. Two recent reports demonstrated that pericytes, isolated from skeletal muscle, can participate in muscle regeneration. This has raised the question as to whether endogenous pericytes contribute to regeneration of skeletal muscle and other tissues after injury or in disease.
Previously, we have generated a mouse model in which the LacZ gene was targeted into the annexin V (Anxa5) locus. The expression of the Anxa5-LacZ fusion gene defined a specific marker for perivascular cells during development and in the adult, and allowed the isolation of pericytes from embryos and adult meninges. The purified cells express pericyte-specific markers as well as markers characteristic for stem cell populations and are capable to differentiate in vitro into mesenchymal lineages. We have now crossed Anxa5-LacZ mice with mdx mice, the mouse model for human Duchenne muscular dystrophy, to determine the endogenous contribution of pericytes to muscle regeneration by LacZ staining. The intriguing results of these experiments suggest that Anxa5-LacZ-positive precursor cells actively participate in remodelling of the tissue. However, further interpretation of these results is limited due to the static histological procedures, necessary for LacZ staining, and the uncertainty of a regulated Anxa5 promoter activity in pericyte descendents. We therefore propose to generate a pericyte reporter mouse strain with which pericytes and all of its descendents can be permanently genetically labelled by targeting Cre recombinase into the Anxa5 locus (Anxa5-Cre). Using skeletal muscle as model tissue, this will allow us to trace and characterise pericytes and its descendents in muscle regeneration after induced injury by a myotoxic agent using ex vivo 2-photon laser scanning microscopy. Furthermore, Anxa5-Cre mice will be a versatile model to study the role of pericytes associated with other diseases (cancer, diabetes) in future studies and help to develop and test novel therapeutic strategies.