RESEARCH PROPOSAL: Scientific Merit:
The aims of this proposal are intriguing, focused on mechanisms, and clinically relevant. Why exercise leads to angiogenesis is a profoundly important biological questions, and understanding how this process is influenced by aging is, as America's population ages, likely to lead to important therapeutic approaches as well. But buried in the proposal are far too many questions too be answered short of a Program Project Grant, and, as a consequence, a crisp, doable set of experiments is lacking. The candidate and her mentors ought to decide on a focused project rather than on a bundled group of somewhat loosely related ideas ranging from the effect of hypoxia-ischemia on induction of angiogenic growth factors to the influence of exercise training on these same variables. As a result of putting so many ideas into the proposal, none are really dealt with in sufficient depth. For example, hypoxia and ischemia are lumped together in a set of experiments. But the consequences of reduced oxygenation on local angiogenesis may differ if the cause of the hypoxia is ischemia as opposed to a reduction in PO2 in an otherwise normally perfused system. Ischemia leads to reduced removal of metabolic products, any one of which might influence cytokines or growth factors, while hypoxia with perfusion is profoundly different. Although these issues are referred to by the candidate, the experimental protocols do not reflect these important concerns. This reviewer has a great deal of concern with the experimental design. Training and interventions are to be compared usually among three groups of rats with n = 8 in each group. Only 4 rats per group will be used for subsequent analysis of the effects of the interventions on a variety of growth factors. Northern analysis will be used to determine gene expression and protein levels in the 4 remaining rats will also be determined. The noise-to-signal relationship that exists at all levels of the proposed experiments is so large that one must question whether or not any information so obtained may be reliable. The candidate did not address some critical issues. For example, of 8 rats beginning an eight week training regimen, one can expect that between 40-70 percent may actually complete the training in a reliable manner. The percentage of drop-outs increases in males compared with females and in older versus younger animals. Densitometric analysis of gel autoradiographs is fraught with error. Far too much emphasis is placed on the single observation of Breen et al., who used, in this reviewers opinion, far too few animals to make a convincing case (n = 4). The data presented in the Breen article are impressive, but in terms of a training environment for a new scientist, one can be pretty sure about the Journal Club Discussion of an n = 4 northern blot result from rat studies presented as a ground breaking study. It is unfair for the candidate to believe that when doing multiple comparisons, so small an n is acceptable. Statistical consultation is needed and should have been provided by the mentors. Finally, the techniques proposed, ranging from basic molecular biological techniques to microsphere studies of capillarity, are enormous in scope.
