Regulatory networks in retinal ischemia-reperfusion injury
© Andreeva et al.; licensee BioMed Central. 2015
Received: 23 January 2015
Accepted: 14 April 2015
Published: 24 April 2015
Retinal function is ordered by interactions between transcriptional and posttranscriptional regulators at the molecular level. These regulators include transcription factors (TFs) and posttranscriptional factors such as microRNAs (miRs). Some studies propose that miRs predominantly target the TFs rather than other types of protein coding genes and such studies suggest a possible interconnection of these two regulators in co-regulatory networks.
Our lab has generated mRNA and miRNA microarray expression data to investigate time-dependent changes in gene expression, following induction of ischemia-reperfusion (IR) injury in the rat retina. Data from different reperfusion time points following retinal IR-injury were analyzed. Paired expression data for miRNA-target gene (TG), TF-TG, miRNA-TF were used to identify regulatory loop motifs whose expressions were altered by the IR injury paradigm. These loops were subsequently integrated into larger regulatory networks and biological functions were assayed. Systematic analyses of the networks have provided new insights into retinal gene regulation in the early and late periods of IR. We found both overlapping and unique patterns of molecular expression at the two time points. These patterns can be defined by their characteristic molecular motifs as well as their associated biological processes. We highlighted the regulatory elements of miRs and TFs associated with biological processes in the early and late phases of ischemia-reperfusion injury.
The etiology of retinal ischemia-reperfusion injury is orchestrated by complex and still not well understood gene networks. This work represents the first large network analysis to integrate miRNA and mRNA expression profiles in context of retinal ischemia. It is likely that an appreciation of such regulatory networks will have prognostic potential. In addition, the computational framework described in this study can be used to construct miRNA-TF interactive systems networks for various diseases/disorders of the retina and other tissues.
Retinal ischemia is a consequence of restrained blood flow that causes severe imbalance between the supply and the demand of nutrients and oxygen resulting in neuronal damage and impaired retinal function . Immediate reperfusion attenuates the retinal damage, however, it is accompanied by mechanisms such as excessive reactive oxygen species (ROS) generation, low nitric oxide, and inflammation, and might accelerate neuronal cell death [2-4]. Retinal ischemia-reperfusion (IR) injury is associated with a wide range of conditions [5-9] that can culminate in blindness due to relatively ineffective treatment . Detailed understanding of the molecular events following ischemia-reperfusion induced retinal damage would facilitate development of relevant treatments.
It is widely acknowledged that complex diseases and/or disorders, including those resulting in altered vision, are more likely linked to groups of genes, gene modules or gene pathways than to any single gene [11,12]. The transcriptional regulation of genes is mediated in part by transcription factors (TFs), while their post-transcriptional regulation is mediated in part by small non coding RNAs, a prominent class of which are microRNAs (miRs) . Despite the different levels of regulation, both transcriptional and post-transcriptional regulatory interactions are not isolated from each other, but interact to execute complex regulatory programs which, in turn, modulate cellular functions [14,15]. Cellular and tissue functions rely on well-coordinated molecular interactions between genes, TFs and miRs, all integrated within regulatory networks [16,17]. The networks are fairly complex, and consist of a variety of patterns of interaction. For example, one possible pattern consists of a miRNA and a TF that coordinate one another and which also co-regulate a common gene  or gene-transcript. Since there is no general nomenclature to name this pattern, at this time, we have used the term “closed loop-motif” throughout this manuscript. The “closed loop” infers an interaction between all 3 elements of the loop. The “motif” part of the terminology infers a special relationship of the closed loop to some context driven activity within gene networks. In these closed loop-motifs, the TF, miRs and genes/transcripts can be viewed as nodes whereas the regulatory influences between them are seen as connecting lines or edges [18-20]. Since the loop motifs are highly interconnected within a regulatory network, the altered expressions of context-driven genes might also influence the expression of genes from neighboring loop-motifs. Emerging evidence indicates that loop-motifs which contain disease-driven differentially expressed molecular components (genes, miRs or TFs) are linked to different aspects of the etiology and/or expression of diseases and/or disorders [21,22].
In recent years extensive efforts have been focused on modeling of regulatory networks combining TFs and miRs [15,23-25]. The majority of these early studies focused on the development of algorithms or tools but did not address the biological context of the networks [25-28]. Furthermore, the construction of regulatory networks related to particular disorders is still in the very early stages of development. However, advances in the construction of such networks is essential and will eventually contribute to the identification of better drug targets and biomarkers for monitoring and controlling the progression of more complex disorders, such as glaucoma, ophthalmic artery occlusion and other retinopathies associated with retinal ischemia.
The goal of this study is to construct regulatory networks associated with early and late reperfusion time points following retinal ischemia and to capture transient changes in the regulatory networks.
Ischemia-Reperfusion injury (IR-injury) related mRNAs, TFs and miRNA
Microarray data were obtained and analyzed for miRNA and mRNA transcript levels for reperfusion times of 0 h, 24 h and 7d after an initial 1 h period of ischemia as previously described . A total of 36 animals were used for the mRNA microarray study. Sham control and IR injured animal groups contained 18 rats per group. Each of the sham and IR injury related groups were divided into 3 sub-groups of 6 animals based on the 3 time points used for this study (0 h, 24 h, 7d). A total of 60 animals were used for the miRNA microarray study. Sham control and IR injured animal groups contained 30 rats per group. Each of the sham and IR injury related groups were divided into 5 sub-groups of 6 animals based on the 5 time points used for this study (0 h, 2 h, 24 h, 48 h, 7d). The treatment and care of all animals used in this study were approved by the University of Louisville Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with the ARVO Statement for the Use and Care of Animals in Ophthalmic and Vision Research. The mRNA and miRNA datasets are deposited into the Gene Expression Omnibus (GEO) data repository (GSE43671 and GSE61072), where the information about the data normalization is available. In brief, the raw data files for the mRNA array (.txt) were imported into GeneSpring (GX 11.1) for normalization and analyses. GeneSpring generates an average value from the six animal/samples for each gene. Data were transformed to bring any negative values or values less than 0.01 to 0.01 and then log2-transformed. Normalization was performed using a per-chip 75 percentile method that normalizes each chip on its 75 percentile, allowing comparison among chips. Then a per-gene on median normalization was performed, which normalized the expression of every gene on its median among the samples. We retained a total of 23897 transcripts for further statistical analysis.
The raw data files with total 350 miRNAs extracted from Agilent Feature extraction software were further processed and analyzed by GeneSpring GX10.0 software. The raw data were at first normalized with the following conditions and then filtered by the flag using GeneSpring GX10.0 software. The normalization included log2 transformation, per chip normalization to 75% quantile and dropped per gene normalization to median. We retained the 219 normalized miRNAs for the further statistical analysis.
Differentially expressed mRNAs, TFs and miRs at 0 h, 24 h, and 7d post-IR periods, filtered by fold change ≥ 2 and p-Value ≤ 0.05
Differentially expressed molecular components
Inference of closed loop-motifs
To identify TFs in the rat genome as well as TF-target gene pairs, we used three publicly available databases (ITFP , PAZAR [38,39] and TRED [40,41]) as well as the commercial database TRANSFAC  (professional release 2014). Additionally, the Match Analysis tool  associated with TRANSFAC was used to investigate the promoter regions of genes (5 kb upstream) to identify predicted TF-target gene pairs. To minimize false positive as well as false negative relationships, only pairs of transcription factors and genes with the highest matrix score (0.8) were collected. Genes unknown to TRANSFAC were re-analyzed with the aid of Match using either different aliases (gene symbol or RefSeq ID) or through use of the promoter sequence of the gene as found with the UCSC table browser .). We added connecting edges to the 3 types of pairs; TF-Gene; miR-TF and miR-Gene without regard to direction of interaction (Figure 1.II).
Subsequently, we constructed putative tripartite loops by attaching edges between the interactions previously paired. These tripartite loop-motifs contain 3 different molecular entities, mRNA, miR, TF (Figure 1.III). The loop-motifs are building blocks and these are then combined to form the larger regulatory networks (Figure 1.IV). Due to the complex nature of the different relationships that might exist in a regulatory network, we restricted our inference to loop-motifs where the miR targets a TF and both co-regulate the expression of a co-targeted gene. Other combinations were not considered. We obtained a total of 4218 loop-motifs for the 24 h post-IR period and 957 loop-motifs for the 7d time point. These data were further reduced since only loops with three significantly correlated edges were considered (see below).
Functional analysis of mRNAs within the miRNA-TF-TG closed loop-motifs at 24h and 7d
Early (24 h)
Total loop-motifs 24h
Immune response *
Total loop-motifs 7d
Evaluation of regulatory loops
Although the prevailing approach when inferring regulation relationships is to assume linear dependencies between bio-elements, it is possible for some elements to have nonlinear dependency. DC is a novel method for evaluating nonlinear dependency that has many appealing features when compared to Pearson. Unlike Pearson, DC scores zero if and only if variables are independent (a Pearson correlation of zero does not imply independence between variables). Since our miRNA array data were generated for 5 time points (0 h, 2 h, 24 h, 48 h and 7d) and mRNA for only 3 time points (0 h, 24 h and 7d) post-IR injury, we imputed mRNA expression data for two additional time points (2 h and 48 h) using the simple least square method [52-54]. This approach allowed us to calculate both, linear and nonlinear dependencies for all predicted miRNA-mRNA pairs at matching time points. Detailed results from both correlation methods could be found in Additional file 1.
Number of closed loop-motifs and their molecular components for each of the three reperfusion time points (0 h, 24 h, and 7d) following 1 h of ischemia
Closed loop motifs
Construction of regulatory networks
Significantly correlated closed loop-motifs identified in the previous step were integrated into regulatory networks associated with 24 h and 7d time points following retinal IR-injury. The Gephi open graph visualization platform  was used to develop graphic representations of the regulatory networks containing nodes, each consisting of either miR, TF, or TG and their interconnecting edges representing interactions between the nodes. We analyzed the topological structure of the networks to identify regulators (TFs and miRs) with major regulatory roles in 24 h and 7d post-IR-injury based on node degree. The node degree is defined as the number of directly connected neighbors of a node in a particular network. Nodes that have a high number of directly connected neighbors are thought to be important regulatory hubs within the regulatory network.
Analysis of regulatory closed loop-motifs associated with IR-Injury
Initial analyses indicated that different numbers of mRNAs, TFs and miRs were present at the three different time points following the initial ischemic condition (Table 1). The lowest number of changes occurred at 0 h whereas the largest number occurred at 24 h. These changes were reflected in the number of closed loop motifs observed for each time point (Table 3). Thus, there were no closed loop motifs at the 0 h time point, and the maximum number was observed at 24 h. The absence of closed loop motifs at 0 h may indicate a lack of sensitivity or a lack of data at this time point. This is an area that may need further investigation.
Properties of time point specific regulatory networks associated with IR-Injury
Top ten transcription factors, mRNAs and miRs ranked by the number of their connections at the 2 time points 24 h and 7d post-IR injury
miR (24 h)
mRNA (24 h)
TF (24 h)
Reportedly, the nodes that have a high degree of connectivity are known as hub nodes (or hubs) and play major roles in the regulatory networks. The top three rno-miRs at 24 h were rno-miR-495* (degree of 172), followed by rno-miR-214 (degree of 170) and rno-miR-207(degree of 143). In contrast at 7d, rno-miR-873 (degree of 55), rno-miR-223 (degree of 48) and miR-410 (degree of 45) had the highest degrees of connectivity. The top three TFs were Maf (degree of 389), Stat1 (degree of 165) and Creb1 (degree of 104) in the network associated with the 24 h time point and Lef1 (degree of 124), Stat1 (degree of 106) and Bcl6 (degree of 98) in the regulatory network associated with the 7d post-IR. Of particular note, at both time points, with the exception of the top 3 TFs, most TFs had relatively low levels of connectivity (Table 4). We didn’t distinguish between in- and out-degree and we ranked the molecular components based on connectivity (the sum of in- and out-degree). However, further analyses showed that the ranking of the top 3 TFs would not change if we consider the out-degree only. The top 10 gene-transcripts, distinct for each of the 24 h and 7d time points, were moderately well connected at between 12 and 22 connections each.
The gene-transcripts in the regulatory loops in these networks were evaluated for their biological relevance with the aid of the Database for Annotation, Visualization and Integrated Discovery (DAVID) [45,46] and pathway analysis (Ingenuity Pathway Analysis, IPA®,QIAGEN Redwood City, CA) and the most enriched biological processes were listed (Table 2). The networks associated with the 24 h time point were significantly enriched for genes participating in cell death, apoptosis, caspase-activation, ion transport and synaptic activities. The networks associated with the 7d time point were significantly enriched for genes participating in inflammatory responses, immune responses, antigen presentation, ion transport and also cell death. Similarities and differences between the processes in each time point are discussed below.
Sub-networks at 24 h post-IR period
Sub-networks at 7d post-IR period
Cell death sub-networks at 24 h vs. 7d post IR-injury
Ion transport sub-networks at 24 h vs. 7d post IR-injury
We analyzed mRNA and miRNA arrays for ischemic-reperfusion injury in the rat retina for 0 h, 24 h and 7 days following a 1 h ischemic period. We developed a protocol to look at the correlated expressions between 3 nodes, miRs, mRNAs and TFs, connected by edges, in what we have termed closed loop-motifs. All three molecular elements are required to be related source/targets and have correlated expressions in order to be part of a closed loop. A context dependent and regulatory relationship between the 3 members of these loop-motifs is inferred. The edges in a closed loop-motif show significantly correlated regulation between the 3 nodes. Because of this particular requirement, our loops happened to contain only positive correlations. In this analysis, we have not given any weight to any particular direction of interaction. However, we made every attempt to show that the members of the closed regulatory loops are related, such that they have significantly correlated expressions and that the TF and the miR are known to interact with the target gene or its transcript in the closed regulatory loop. The 0 h time point showed few changes in correlated expression, none of which reached the level of statistical significance in our particular analysis. So, we focused our efforts on the 24 h post-IR and 7d post-IR time points, which will hereafter be referred to as “early” and “late” times respectively. Compared to our earlier study  we made significant improvements to our analytical approach, for example, we used a stringent filter to select differentially expressed IR-related molecular components (corrected p-value vs p-value). We also increased the numbers of TFs and their targets by using a promoter analysis with the aid of the TRANSFAC commercial database (instead of the publicly available TF-TG pairs used in our previous study). These improvements increased the number of the closed loop motifs from 87 to 4218 for the early post-IR time and from 140 to 957 for the late time point.
Rat retinal miRs showing higher connectivity in the large gene networks associated with early and late post-IR injury periods along with their reported activities in other systems and relevant citations
Early (24 h)
inhibition of miR-495 increased neovascularization and recovery of blood flow after cardiac ischemia in mice
miR-214 protected the mouse heart from ischemic injury by controlling Ca2+ overload and cell death
miR-298 was up-regulated in brain and blood after ischemic stroke
miR-206 was significantly deregulated during the conditions of unfolded protein response in H9c2 rat cardiomyoblasts
miR-221 was suggested as a biomarker for cerebrovascular disease. Stroke patients and atherosclerosis subjects showed significantly lower miR-221 serum levels than healthy controls
miR-873 was up-regulated after onset of focal cerebral ischemia in mice
miR-223 targeted glutamate receptors in mice brain. Overexpression of miR-223 decreased the levels of GluR2 and NR2B, inhibited NMDA-induced calcium influx in hippocampal neurons, and protected the brain from neuronal cell death following transient global ischemia and excitotoxic injury
miR-185 has been associated with inflammatory responses during brain ischemic stroke in mice and may provide underlying target for prevention and treatment of stroke
Inhibition of miR-329 increased neovascularization and blood flow recovery after ischemia in mice subjected to double femoral artery ligation
hypoxia-induced miR-138 is an essential mediator of endothelial cell dysfunction via targeting S100A1 Ca2+ sensor
We showed that transcription factors, Maf, Creb1 and Stat1, were the 3 principal hubs with high connectivity in the early phase IR-injury regulatory network, whereas Stat1, Lef1 and Bcl6 were the 3 principal hubs in the late phase IR-injury network. Each of these transcription factors is evidently a central coordinator because they regulate such large numbers of targets in the corresponding early and late phases of IR-injury. Reportedly, really important hubs tend to be shared by several tissues . Stat1 was the only common TF between the early and late post-IR times. Stat1 has been identified as a hub gene in several tumor-associated transcriptional networks  including a gene network within HeLa cells exposed to IFNγ . In addition, altered Stat1 levels have been reported in rats with focal cerebral ischemia  but also in glaucomatous rat retinal ganglion cells . The transcription factor Bcl6 has been identified as an important hub in gene regulatory networks associated with eight human tissues  and its critical role in preventing apoptosis in the retina during early eye development was previously reported . Increased phosphorylation of CREB in the brain was observed in two rat models of ischemic preconditioning  and Creb1 expression was detected in canine and human retinas affected with age-related macular degeneration (AMD) . Maf members form a distinct family of the basic leucine zipper (bZip) transcription factors and have been involved in various disease pathologies (reviewed in Ref ). A member of the Maf family, the leucine zipper protein Nrl, is neural retina-specific and has been shown to regulate the expression of rod-specific genes, including rhodopsin . Taken together, the six hub-TFs described above seem to regulate all biological processes in a particular phase following IR-injury, which indicated an important role for these regulators in the pathogenesis of this disorder in the retina.
Approximately 50% of the loop-motifs from the regulatory network associated with the early phase were involved in five cellular processes. All of these could ultimately lead to cell death, as indicated by the large number of loops shared with the cell death process. This implies that IR-injury initiates transcriptional and post-transcriptional regulatory interactions that contribute to neurodegeneration. The remaining loop-motifs were not associated with well-defined cellular processes. For comparison, 61% of the regulatory motifs from the network related to the late phase of IR-injury were linked to cellular processes, the largest of which were cell death, antigen presentation and immune responses. This indicates that the IR-injury related regulatory networks were consistent with previous studies in terms of affected cellular process [29,70,76-78]. However, the novel component in the present study is the integration of the regulatory elements, miRs and TFs, in closed loops, sub-networks and large regulatory networks that associated with particular biological processes during the early and late phases of ischemia-reperfusion injury in the retina.
The etiology of retinal ischemia-reperfusion injury is orchestrated by complex and still not well understood gene networks. This work represents the first large network analysis to integrate miRNA and mRNA expression profiles in context of retinal ischemia. Importantly, we highlighted the regulatory elements of miRs and TFs within these gene networks, and found specific miRs and TFs, associated with biological processes in the early and late phases of ischemia-reperfusion injury. It is likely that an appreciation of such regulatory networks will have prognostic potential. In addition, the computational framework described in this study can be used to construct miRNA-TF interactive systems networks for various diseases/disorders of the retina and other tissues.
This work was supported in part by grants from the National Eye Institute R01EY017594 and the National Institute of General Medical Sciences P20 GM103436.
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