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Principal Investigator:
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Peter Davies, Ph.D.
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Organization:
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University of Pennsylvania
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Project Ended:
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2004
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The body's cardiovascular system adapts to different gravitational environments. Dr. Peter Davies is studying vascular adaptation by looking at blood vessel cells and tissues exposed to simulated weightlessness, normal gravity and simulated hypergravity (gravity greater than Earth's). He is comparing the different environments' effects on gene expression levels in vascular cells and tissues, which will lead to an understanding of which genes are involved in the vascular system's adaptation to various gravitational environments.
|
Principal Investigator:
|
Peter Davies, Ph.D.
|
|
Organization:
|
University of Pennsylvania
|
|
Project Ended:
|
2004
|
Aims When changes in the biomechanical environment of the circulation occur, blood vessels undergo well-orchestrated structural and metabolic remodeling to restore optimal function. We propose that this remarkable adaptive ability lies at the center of orthostatic intolerance exhibited by most astronauts on return to Earths gravitational field after modest-to-long periods in microgravity. We are therefore mapping gene expression (transcription profiling) of the different vascular steady states exhibited in vivo (mouse) in simulated hypergravity and microgravity, and the transitions between them, in order to design better countermeasures for undesired vascular consequences in long-term space flight. In particular, the transition to hypergravity will simulate the effects experienced by astronauts upon return to a significant gravitational field (Earth, Mars) following adaptation to long periods of microgravity. The studies will generate a reference genomics database identifying gene expression changes in the arteries, heart and lungs, induced by gravitational shifts. Mining of such databases will provide a guide to potential countermeasures to offset deleterious effects.
Key Findings
During the first year, we refined the antisense RNA techniques necessary to amplify RNA from small numbers of cells with high fidelity. This became necessary when it was apparent that no literature existed for a rigorous test of the protocols required in the mouse experiments. In a model experiment, vascular cells were stimulated with the cytokine TNF for which a small number of genes are known (through conventional Northern analyses) to change. RNA from the same pool was analyzed by microarray with and without amplification. Sophisticated bioinformatics analysis of 13,800 genes was performed. The data from unamplified and amplified RNA were analyzed for fidelity, sensitivity and utility. The expected prominent changes in known genes were detected in both groups with high retention of accuracy, an essential requirement for the proposed in vivo gravity experiments.
An interesting additional and unexpected finding is that RNA amplification increased the detection rate of genes whose differential expression was just below a significance threshold in the unamplified assay, i.e., greater sensitivity of detection of differential gene expression conferred by the linear amplification techniques employed. Most important, these differences were confirmed by real-time quantitative PCR of unamplified RNA. This work was published in the journal Physiological Genomics in April 2003. The studies were a prerequisite for the gravitational experiments because no such analysis existed that rigorously evaluated the accuracy of the transcription profiles arising from amplification of small amounts of blood vessel.
In extending the RNA-amplification techniques, we next addressed differential, vascular-cell gene expression in two sites in the aorta of the normal adult pig. Endothelial cell mRNA was isolated from two regions of the aortic arch, characteristic of disturbed flow (pro-atherogenic) and undisturbed flow respectively. RNA from paired sites in individual aortas (n = eight) was isolated, linearly amplified, reverse-transcribed, and cDNA was hybridized to microarrays custom-prepared from the University of Toronto human cDNA cardiovascular database (approximately 8,000 genes) plus several thousand proprietary Incyte clones. Bioinformatics analyses identified expression patterns in the disturbed flow region, indicative of an antioxidant endothelial profile that may be protective of a pro-inflammatory state.
Some genes associated with major mechanisms believed to initiate atherogenesis, e.g., pro-inflammation, were elevated but the critical adhesion molecules necessary to initiate inflammation were not differentially expressed in this region, consistent with the absence of any pathology by histological assessment. This is an intriguing result that demonstrates the power of this approach in identifying the interactions of multiple genes need to be considered in defining atheroprotective or susceptible situations. As far as we are aware, these are the first high-throughput array analyses of arterial endothelial gene expression directly obtained from discrete regions of blood vessel.
When compared with several studies that have profiled the effects of different flow treatments on cultured (as opposed to in vivo) cells, many differences of gene pathways were noted. This work has been submitted to the journal Proceedings National Academy of Sciences USA. While these studies were performed with larger blood vessels (porcine) in order to obtain enough lining cells (endothelium), the cell numbers used are comparable to, in fact less than, those we will obtain from whole mouse aorta for the gravity studies. Techniques for the dissection of mouse blood vessels, RNA isolation and amplification has been verified under normal gravitational conditions. These evaluative experiments demonstrate that we can successfully perform the entire sets of protocols - from tissue isolation to bioinformatics and gene annotation - prior to the gravitational shift experiments at NASA-Ames Research Center.
Hypergravity and transitional procedures on 96 mice at NASA Ames, using the 24-foot centrifuge in close collaboration with Ames staff members, is under way. Arterial, heart and lung tissues harvested will then be analyzed at Penn. The molecular biology is demanding and lengthy, and the bioinformatics is complex. As in the case of our publications to date, we are taking steps to ensure that the data are openly available to the widest scientific community.
Impact
New techniques addressing vascular genomics have been developed, tested and have withstood critical peer-review in leading journals in the field of genomics and biology. We are now implementing them in carefully designed experiments in which gravitational shift is the variable.
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