The response to mechanical stimulus' gene set was ranked at number two in our absolute enrichment analysis and it was ranked at number one in our upregulated enrichment analysis. This gives us a strong measure of confidence that our t paired statistic is capturing relevant themes in our rat ear stretch system.
In the absolute enrichment framework, Permutation testing [5
] gave the hypoxia gene set a highly significant P value of 0.0033 at the 0.05 level. The two prototypical behaviors seen in the time trajectories (seen in Figures through ) tell us that the genes in our hypoxia pathway show either a sharp rise or a sharp fall at earlier time points and then recover. This behavior was then compared with what has been reported in the literature. Specifically, we note that SOD3 shows a decline at earlier time points and then a recovery while NOS2, Slc2a8, and Hmox1 all show sharp rises at earlier time points. Maiti et al [6
] have stated that "under hypoxic stress, the cellular defence systems such as antioxidant enzymes (GPx, GR, SOD, etc.) get disturbed and their activity decreases." Further, they report that in the rat brain, hypoxia leads to an increase in nitric oxide. In our system, NOS2 is sharply rising at earlier time points while SOD3 is falling at earlier time points.
It has also been reported in the literature that NO production upregulates heme oxygenase (Hmox-1) production [7
]. This may explain why we see a sharp rise in Hmox in the early time points. It also may explain why on our co-expression network graph, NOS2 and Hmox show a positive correlation of 0.69. At later time points, however, NOS2 and Hmox-1 do not move together (for example NOS2 shows downregulation while Hmox shows upregulation). Nitric oxide isn't the only mediator of Hmox-1 upregulation. For example, it has been reported that Hmox-1 is the major stress protein induced by UVA, hydrogen peroxide and arsenite [9
]. Further, it is known that Hmox-1 expression is linked to tissue stretch. Many pathways lead to heme oxygenase I expression through renal injury [7
]. Mechanical stress has been shown to cause oxidant stress [10
] and Hmox-1 levels are increased when cells are exposed to oxidative stress [11
]. Hmox-1 prevents oxidant-induced microvascular leukocyte adhesion [12
]. Hmox-1 has cytoprotective roles [14
], and is anti-inflammatory [15
Hmox-1 itself is a negative regulator of NO [16
]. Thus, if Hmox-1 is upregulated independently of NO (as may be happening at the later time points), then NO is strongly inhibited. For example, hemin can upregulate Hmox-1 (independently of NO). This induction has been shown to strongly inhibit NO production of LPS-activated macrophages [18
]. Loike et al.
] have reported that hypoxia leads to glucose transporter expression in endothelial cells. Thus, it may be that we see a sharp spike in slc2a8 (which is a glucose transporter [21
]) as a consequence of hypoxia at earlier time points in our system.
Thus, our system may be increasing glucose intake to make up for lack of oxygen, and it seems that our system may be undergoing hypoxia [23
] at least at earlier time points. According to [23
], "...the molecular mechanisms by which muscle contraction/hypoxia increase glucose uptake are less well defined, although they appear to be independent of the PI3K pathway. Most intriguing is the observation that the recently identified hormone adiponectin also stimulates skeletal muscle glucose uptake in a PI3K-independent manner." The adiponectin gene is not part of the hypoxia gene set. We went to our dataset to see if seemed to show a trend similar to the genes in our hypoxia pathway. The adiponectin gene showed an upregulation or a rise at earlier time points dropping to a downregulation at later time points (plot not shown).
To our knowledge this is the first evidence that tissue stretch may lead to hypoxia. However, we should stress that we do not have replicates at each time point. Thus, the conclusions on the time trajectories follow from the results of the enrichment analysis and not the other way round. Because we feel that the hypoxia pathway is important, we then study its time trajectory in more detail for further insights.