Pharmacol Rev 56: 161, 2004. targets. Such bioinformatic analyses have not been systematically performed for PAD. We constructed global protein-protein interaction networks of angiogenesis (Angiome), immune response (Immunome), and arteriogenesis (Arteriome) using our previously developed algorithm GeneHits. The term PADPIN refers to the angiome, immunome, and arteriome in PAD. Here we analyze four microarray gene expression datasets from ischemic and nonischemic gastrocnemius muscles at posthindlimb ischemia (HLI) in two genetically different C57BL/6 and BALB/c mouse strains that display differential susceptibility to HLI to identify potential targets and signaling pathways in angiogenesis, immune, and arteriogenesis networks. We hypothesize that identification of the differentially expressed genes in ischemic and nonischemic muscles between the strains that recovers better (C57BL/6) vs. the strain that recovers more poorly (BALB/c) will help for the prediction of target genes in PAD. Our bioinformatics analysis identified several genes that are differentially expressed between the two mouse strains with known functions in PAD including TLR4, THBS1, and PRKAA2 and several genes with unknown functions in PAD including EphA4, TSPAN7, SLC22A4, and EIF2a. post-HLI (similar to BALB/c 3 days post-HLI) or scrambled antagomir-93-treated C57BL6 mice post-HLI (similar to C57BL6 3 days post-HLI). RNA microarrays were performed from the RNA extracted from these 3-day post-HLI gastrocnemius tissues. Data obtained from the microarray were used to identify the molecular events that control the perfusion AZD5582 recovery between mouse strains. Available microarray datasets in PAD patients include gene expression analysis of peripheral blood Cdh1 mononuclear cells (42) and human femoral arteries (14, 18). However, these cells or tissues are very different from the ischemic skeletal muscles that are the site of ischemia and the stimulus for vascular remodeling and growth. The only available gene transcriptional data comparing the ischemic vs. nonischemic gastrocnemius muscles from the two mouse strains at an informative early time point is described in Ref. 27. The main goal of this study is to determine the differential gene expression changes in the ischemic skeletal muscle between these two strains at an earlier time point to identify the potential molecules that could be responsible for better recovery in C57BL/6 mice and/or for worse recovery in BALB/c mice. Nonischemic muscle from both strains served as the baseline controls for this study. We have previously shown that at post-HLI the capillary density and perfusion recovery between these two mouse strains are comparable, which makes it a critical time point to investigate the upstream signaling changes between the two strains (43). By several changes in blood flow and perfusion recovery between these two mouse strains become significantly different, thereby making time points AZD5582 other than not relevant for the study. The failure of several strategies to improve blood flow in PAD suggests a critical need to identify the potential targets AZD5582 that can improve perfusion recovery in PAD. High-throughput data have been successfully used for other diseases, most prominently in cancer, to identify important novel targets. To integrate hundreds of angiogenesis-related molecules and infer angiogenesis-annotated genes and proteins, we have developed a machine learning algorithm to construct the angiome, a global protein-protein interaction network (PIN) relevant to angiogenesis (12). Such analyses for PAD-related angiogenesis, immune response, and arteriogenesis have not been performed. The primary goal of this study is to use the omic approach to identify important set of genes and signaling pathways in PAD, with the eventual goal of identifying new therapeutic targets. We used large-scale high-throughput gene expression datasets from ischemic and nonischemic muscles from the two mouse strains to construct PADPIN, the global PIN of angiogenesis, inflammation, and arteriogenesis in PAD. METHODS Animal model of HLI and perfusion imaging. Unilateral hindlimb ischemia was induced in 8C12 wk old male C57BL/6 (= 11) and BALB/c (= 12) mice by surgical ligation and excision of the femoral artery above the inguinal ligament of its bifurcation at saphenous and popliteal arteries as previously described (39). All the animal protocols in our experiments were reviewed and approved by the University of Virginia Institutional Animal Care and Use Committee. Laser Doppler perfusion imaging was performed immediately on ischemic and nonischemic feet of the AZD5582 mice immediately postoperation. Mice were anesthetized by intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) throughout the surgical and postoperative imaging process. Laser Doppler imaging to measure the blood flow was measured at time intervals post-HLI (39). Animal treatments, tissue isolation, and mRNA array. Animal treatments, tissue isolation, and mRNA array were performed as previously described (27). In brief, scrambled antagomir-93 sequences were synthesized according to the nucleotide modifications as previously described (37). Oligonucleotide sequences for scrambled antagomir-93 (6) are 5-AAGGCAAGCUGACCCUGAAGUU-3. Oligonucleotides were dissolved in.
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