A set of genes previously implicated in the hypoxia response might be an important modulator in the rat ear tissue response to mechanical stretch

Clinical context

Worldwide, wounds pose a major health issue. Lower extremity ulcers alone cost the US Medicare system $1.5 billion. Unless wound therapies see a large improvement, we will see escalating treatment costs and morbidity as the population ages and as the incidence of diabetes and obesity increases. An understanding of the mechanisms underlying wound healing will shed light on how normal physiology adapts to changes in the normal homeostatic environment. The vacuum assisted closure (VAC) device (KCI, San Antonio Texas) is a relatively new modality in wound healing. Although the device has been shown to accelerate wound healing, its mode of action remains to be proven convincingly. Figure 1 shows the application of the device. This involves packing a polyurethane sponge into the wound bed and then sealing the wound including the sponge with an occlusive dressing that has one outlet tube going to a vacuum (a vacuum of about 115 mmHg is applied through the tube). Theories about how this device obtains its efficacy range from a reduction in bacterial load [1] to a reduction in edema. The blowup in Figure 1 shows that with the application of the vacuum the skin is pulled into the intra-strut spacings of the sponge thereby stretching the skin.

In an earlier publication, we have used a numerical model to show how the imposition of the vacuum forces the tissue to stretch by small amounts (micromechanically) as the tissue is 'pulled' into the empty space defined by regular holes in the sponge causing a tension to be applied to the tissue substrate and through it to the cells embedded within the substrate [2]. Previous work [3] by Donald Ingber, one of the co-authors on our microstreatch paper [2] has shown that cells stretched in vitro proliferate, whereas cells that are not stretched are cell cycle arrested. We thus, in that paper, hypothesized that through a similar mechanism the cells in the wound bed subjected to stretch by the application of negative suction pressure are also proliferating and thus helping the wound heal faster [2]. If we are able to understand the molecular mechanisms that underlie tissue response to forces, then we could substitute forces with interventions that mimic these molecular events. The effects of tissue expansion mechanisms [4] are little studied in the literature. A rat ear was chosen as the system in which to study the effects of forces on tissues in a perfused system (Figure 2). The rat ear is thin and the blood vessels can be easily visualized under a light microscope. Further, it is highly amenable to force experiments in vivo.

Enrichment analyses

It should be noted that in gene set enrichment analysis we typically do not calculate the P values for each gene separately. Rather we perform the permutation test (as we explain in the methods section) and look for the P values of the gene set. This is why we have not listed the P values of the genes in the hypoxia pathway. In the upregulated enrichment analysis, 'response to mechanical stimulus' achieves the top position. Interestingly, the c26 cluster which is enriched for Reproduction genes from EASE and which was derived from Mootha's original gene sets has obtained second rank in the up regulated analysis. There are a total of 36 c clusters derived by Mootha through the use of self organizing maps over the GNF mouse expression atlas which itself is derived from expression profiling of different mouse tissue [5]. There were no gene sets in the down regulated analysis that had an ambiguous interpretation and thus none had to be analyzed through EASE. The Hypoxia gene set obtains rank 6 in the down regulated enrichment analysis. To see how members of the hypoxia gene set may be co-regulated, we ran a co-expression network analysis.

Co-expression network analysis

Pairwise correlations between the gene expression trajectories provide the correlation network weights shown in Table 5. The correlation network structure based on a threshold of 0.5 is shown in Figure 3. To obtain the connectivity values (Table 5), absolute values of the correlations were summed for each gene.

Gene symbol (or probeset when symbol unavailable)	Connectivity values (in descending order)
Nos2	5.6852
Casp12	5.365
Casp4	5.3197
Brd4	5.038
Brd2	4.8786
Ep300_1369307_at	4.7816
Casp1	4.7395
Hmox1	4.6694
Slc2a8	4.5987
Sod3	4.5886
RGD1562168_predicted	4.3523
Epas1	4.1956
1371289_at	4.1945
Ep300_1373916_at	3.8843
Camk2g	2.888

Expression ratio plot over time

The hypoxia gene set members were further visualized by plotting the stretch to control expression values in three groups. The first group was made up of the positively upregulated members of the hypoxia pathway (positive given by the sign of the paired t score). The second and third groups were obtained by taking the remaining members of the hypoxia pathway (these all had negative paired t scores) and then splitting them by inspection of their time trajectories into two groups. These results are presented in Figures 4 through 6.

The time trajectories of the members of the hypoxia gene set show two prototypical behaviors. They show either a sharp spike at earlier time points that is then dampened or they show a sharp dip at earlier time points that then recovers at later time points. These two are displayed on the co-expression network graph (Figure 3). We note that the members shown in light green shading show the first prototypical behavior while members negatively correlated with this set show the dip behavior (Casp12 for example). Ep300_1373916_at and RGD_1561628_predicted are negatively correlated with Casp12 and thus show a rise at earlier time points but we haven't shaded these because their rise isn't as pronounced.

Applying forces to rat ears and obtaining the ear RNA

Male wistar rats weighing approximately 180 g were chosen for this study. All animal studies were conducted according to institutional guidelines. Strips of latex were fastened to the ears and, using appropriately calibrated rubber bands, forces measuring 50 g were applied to the ears at three points (Figure 2). Eight time points were sampled at 1 h, 4 h, 9 h, 13 h, 19 h, 48 h, 72 h, 96 h. Three rats were used per time point. One ear of each rat (chosen randomly) was stretched while the other served as control. At the time points listed above, the ears were excised and immediately frozen in liquid nitrogen and then stored at -80°C. The three stretched ears at each time point and the three unstretched ears at the same point were separately pooled (70 to 80% of the whole ear was used) and the RNA extractions carried out. The RNA was then hybridized to RAE 230 2.0 Affymetrix rat gene chips. MAS5 was used to obtain the expression values from the images. Expression datasets will be deposited at geodatasets.

Analysis scheme

To gain insights into the molecular mechanisms and pathways underlying the cellular response to forces and tissue stretch, a gene expression analysis was conducted by stretching rat ears (see Figure 2) over a period of five days and then obtaining their expression profiles using Affymetrix arrays. Traditional techniques that analyze gene expression datasets rely on clustering techniques that do not take advantage of pre-existing information about pathways thus not taking account of biological context. This is also true of other analyses that rank genes by fold change or other measures of significance. Enrichment techniques which include The Absolute Enrichment (AE), The Gene Set Enrichment Analysis (GSEA), and what we call The Down Regulated Analysis (DRE) are powerful analysis techniques that not only take advantage of pre-existing pathway knowledge but also in their implementation increase the signal to noise ratio thereby giving us a higher chance of capturing subtle signals in the expression set. Enrichment techniques start by using a ranking metric to reorder the expression dataset.

Next, a priori created groups of genes are scored on this reordered dataset and ranked from most highly scored at the top of the reordered dataset to the least highly scored or 'enriched' groups or gene sets. Thus, rather than looking at significance of each gene within the expression dataset, we look at significance of groups of genes. Since the gene set enrichment analysis is not limited by the type of statistic used, we should use a statistic that will be best at elucidating the changes between the groups that we are studying. We thus conducted our analysis by reordering our dataset using the paired t-statistic as our ranking metric (as our dataset consists of paired data points and thus this statistic will be best for capturing differences). Enrichment techniques can be powerful tools in analyzing data. However, within the enrichment analysis framework, people have focused solely on upregulation. They have perhaps neglected to look at down regulation. Downregulation itself can be just as important as upregulation [5]. However, both downregulated and upregulated (enrichment) analyses may miss differential effects that show some balance between up and downregulation (what essentially we have termed "homeostatic" systems) [5].

Let's look at a (metabolic) pathway (with feedback) to make this more clear. Let's say a pathway has 3 members A through C, and let's say A leads to upregulation of B which leads to upregulation of C (Figure 7). Further, if B can be upregulated independently of A, then B will downregulate A and if C can be upregulated independently of B, then its upregulation will downregulate both A and B (Figure 8). And further, if C itself feeds into another pathway which itself (the pathway) may be upregulated independently of C, then that pathway when independently upregulated will downregulate A, B, and C (Figure 9). We can essentially perturb a pathway at any point from start to finish (and not just at the start or at the end). If we perturb it in between such that there is a good mixture of both upregulation (coming from the downstream parts of the pathway) and downregulation (coming from the upstream parts of the pathway through feedback mechanisms), then this pathway may be captured by the absolute enrichment analysis (see Figure 10). For more details on absolute enrichment see below as well as ref [5]. (It must be noted that pathways don't have to have this serial relationship as in our conceptualized pathway. There can be bifurcations and so on.)

In essence, we are really looking for a change between an "affected" condition and a "nonaffected" or control condition. A change can be either an upward change (upregulation) or a downward change (downregulation). Thus, we're really looking for the highest differential regulation [5]. Sometimes, the absolute enrichment technique captures gene sets that are seen (at the very top) in either the up or down regulated analyses. Many times, however, it captures other gene sets. Because we run an exhaustive search through enrichment techniques that encompasses both up (or down) regulated and absolute enrichment techniques, we test whether the absolute enrichment technique captures anything extra at the top (of the list of ranked gene sets) compared with either of the up or downregulated techniques.

When what the absolute enrichment captures is found at the top of the up or downregulated analyses, then this analysis doesn't add new information (although a few of the rest of the top ranked gene sets in all the enrichment analyses can still be useful to study). However, when it does capture something new, we focus on it (or at least look at its result) because what it has captured is the most differentially expressed [5]. In our analysis, we captured a new gene set that was higher ranked than even the highest ranked upregulated and downregulated gene sets. Thus, we studied this gene set in more detail. This included running a co-expression network analysis (the fact that we could even run this analysis was driven by the small size of the top ranked hypoxia gene set).

Thus, to recap, our analysis scheme consisted of choosing the appropriate ranking metric (or statistic), running the three enrichment techniques, and then focusing on the gene set obtained from the absolute enrichment analysis (since it captured the most differentially regulated gene set). Our results show that the response to hypoxia pathway was the most differentially regulated pathway and that this pathway showed a homeostatic (comprising components of upregulation and downregulation) response. We believe that this pathway is being most differentially expressed through a homeostatic perturbation.

The rationale for the AE is that in a time series analysis it may be important to see how the system responds to the (mechanical stretch) perturbations that we have imposed on the system by regulating itself. Pathways are essentially composed of elements that respond in tandem to perturbations. Often these responses are self controlled through feedback mechanisms and thus homeostatic response analyses can often give us powerful indicators of pathways that are important in gene expression datasets.

Enrichment analyses

Our data analysis uses enrichment techniques to understand important pathways in our system. Enrichment techniques start out by reordering the dataset using an appropriate statistic (please see Figure 11 for an overview of these techniques using absolute enrichment as an example). Consider the dataset to be a rectangular two dimensional array with the different conditions (control or stretch) represented by columns and the different genes represented along the rows. A suitable statistic is chosen that best helps us differentiate between stretch and control for each gene. Mootha's implementation of the gene set enrichment analysis (GSEA) [25] used the signal to noise ratio statistic to differentiate between the affected condition in his dataset and the control condition. Our dataset has paired samples (in the sense that one ear of the same rat was subjected to stretch while the other ear from the same rat at the same time point served as control). Thus, a better statistic to use in our case is the paired t-statistic since it takes better account of pair-wise differences. Enrichment techniques are not limited to the type of statistic used as the ranking metric. Any statistic that best lets us differentiate between the two classes (control versus stretch) can be used [5] (please see the Appendix for a description of the paired t-statistic).

if the probeset ID is part of the gene set, where G is the number of genes in the gene set and N is the total number of genes (or rather probeset IDs in the whole dataset – we had 31099 probeset IDs).

We next compute a running sum of this statistic on the reordered dataset. Where the running sum reaches a maximum score is our enrichment score for the gene set in question [25, 5]. (Figure 11 shows the overall steps in running enrichment techniques). The gene sets are then ranked from highest enrichment score to the lowest. To test whether the results of the analysis are significant, we perform permutation testing of the dataset. To obtain the permutes, we swapped random stretch samples with random control samples. One thousand permuted datasets were constructed. All three enrichment analyses were run on each of these newly constructed datasets, giving us one thousand rankings of gene sets based on their enrichment scores (for each enrichment analysis). We then counted the number of times each gene set achieved top rank in the one thousand permutes. This number divided by 1000 gave us the permutation test P value. If this number was lower than 0.05, then the gene set was significant. For more details on the permutation test, please see Mootha et al.[25]. We didn't run technical replicates at each time point because our analysis is looking at an aggregate change between the stretched ear and control ear conditions across all time points. By doing this we look for overall changes between the eight time points versus their corresponding paired controls. We next describe how the various gene sets that were used in the enrichment techniques were constructed.

Construction of gene sets

Many gene sets were compiled by the use of rat orthologs of previously published genesets [25] (according to [26] "it is estimated that 90% of rat genes have orthologs in the mouse and human genomes that have persisted since they shared a common ancestor"). Gene sets are essentially collections of genes. To derive additional gene sets that may show relevance, we searched the literature for different analyses that have been performed on cell excitation such as response to serum. When these analyses listed groups of genes obtained through various clustering or classifying techniques, we created gene sets for the various clusters listed in the papers. Other gene sets were created by looking up all possible relevant keywords at the geneontology.org website (angiogenesis, response to mechanical stimulus and so on). The gene sets obtained through the various mechanisms outlined above when they comprised genes not in Affymetrix Probeset ID format were then converted to collections of Probeset IDs through various platforms (The Affymetrix website, matchminer, Onto tools from the Intelligent systems Bioinformatics Laboratory). Some of the gene sets that were obtained were derived from clusters obtained through classifying gene expression datasets. Sometimes, the clusters were annotated by the publications as being enriched for certain pathways or GO categories. When they were not (as for example the 'c' clusters in Mootha's published gene set list [25]), we used EASE (a tool for obtaining enrichment information on groups of genes) as we explain next. A total of 173 gene sets were constructed.

EASE

For calculation of GO category enrichment, we used EASE. EASE is a program that determines the over-represented GO categories in a group of genes (specified either by probeset ID, accession number or other identifier) by calculating the hypergeometric ratio of the GO annotations of the genes found in an analysis versus the background distribution of each of the GO annotations. It should be noted that EASE is distinct from the various enrichment tests that are used in this paper. Enrichment techniques (such as AE, GSEA up or down regulated enrichment) measure differential expression of a set or group of genes. These groups of genes are pre-defined by us independently of the data set. EASE is independent of the enrichment techniques that we use. We can run EASE on any (possibly random) group of genes. EASE will then find what annotations are over-represented in these groups. Enrichment techniques try to see which pre-defined gene sets are most important in a re-ordered data set (thus the gene sets are previously defined and then compared to the data set), while EASE takes any group of genes and tries to find which annotations are over-represented in that group. Through the use of the various enrichment techniques, we found the hypoxia gene set to be the most significantly differentially expressed. We next ran a co-expression network analysis on this gene set.

Co-expression network analysis

To understand connection hubs in the hypoxia pathway, a co-expression network was constructed similar to the methodology presented by Zhang and Horvath [27]. The stretch to control expression value ratios were obtained at each time point. Correlations were then obtained for all possible pairs of genes across all the time point ratios. Next we took a threshold of 0.5 of the absolute values of the correlations leaving us with correlations between genes that were higher than 0.5. The genes were then 'connected' to other genes when their pairwise correlations were above this threshold.

Paired t-statistic

The 'Y' variable here stands for the stretch condition while the 'X' variable stands for the control condition. We calculate the paired t-statistic for each gene ('n' is eight, the number of time points).