BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 Background The cold-induced angiogenic response Angiogenesis can be initiated by a variety of stimuli. For example, angiogenesis is induced by airway smooth mus- cle strain in chronic asthmatics [1], by exercise [2-6] and in some species by cold exposure [7-10]. Many species are subjected to annual cycles of environmental cooling that represents a significant challenge for the cardiovascular system due to the effects of reduced oxygen diffusion and increased blood viscosity associated with low tempera- tures [7,11,12]. One way of overcoming limitations to aer- obic activity is to increase tissue capillary supply. Although the underlying process is unclear, cold-induced angiogenesis may respond to altered hormonal levels or changes in the mechanical environment of endothelial cells (ECs) [13]. Even within the mammalian literature, it is apparent that a number of types of capillary growth exist, e.g. pathological vs. physiological angiogenesis, albeit under the influence of a restricted set of genes [14]. In order to improve the signal to noise ratio from the wealth of published data and reduce the influence of host tissue in the readout, gene expression profiles for endothelial-specific up-regulated genes have recently been identified [15,16]. No such information exists for fish, although their use as an experimental model is increasing due to the additional interventions not possi- ble with mammals [17-22]. For the first time, we have used an in silico approach to identify whether orthologs between man and fish could be identified that were asso- ciated with angiogenesis, then used re-analysis of microar- ray data to see if these genes were differentially expressed on cold exposure and finally we determined what propor- tion of the up-regulated genes were angiogenic or EC-spe- cific. We anticipate this approach could be useful for similar studies in non-model species for which a genome sequence is not available but the human orthologs involved in the biological process are known. Results and Discussion Gene assignment by ortholog identification Orthologs (functional homologs), as a whole, perform an equivalent function in respective genomes [23-25]. Based on this assumption, carp orthologs of known angiogenic human genes provide genes pivotal in their cold ang- iogenic response. Cold carp data from a study by Gracey et al. 2004 [26] was to be used in this work to predict the identity of cold induced angiogenic genes. The genes assigned to carp sequences from the original article [26] were not applicable to this study as they used a BLASTX against multiple species databases and only looked for the best hit without using a control step. In addition, the data- bases searched were from 2004, several years out of date, and a more stringent method of sequence contig construc- tion was used in this work (see methods). A problem to be overcome was how best to assign human orthologs to 19,995 anonymous EST carp sequences col- lected from Genbank and CarpBASE (additional file 1). Many of these sequences were less than 500 bases and many of them contain partial or complete untranslated regions. An obvious first step was to cluster them, using CAP3 [27], to extend the sequence length and improve the quality of any subsequent analyses. This reduced the sequence count to a total of 16,650 sequence contigs and singletons (additional file 2). The next challenge was to identify their human orthologs. Choosing the ortholog assignment method Homologs, in the strictest sense, are characters that have been passed down following a speciation event from a common ancestor [25,28]. A gene in two species derived from a single gene in their common ancestor is defined as an ortholog (functional homolog) and usually has the same or similar function between species [25,28], i.e. the same gene in different organisms are orthologs [29]. Con- versely, paralogs are defined as genes derived from a single gene sequence duplication event and usually have differ- ent functions [25,28,30]. Orthologs and paralogs are not the only gene histories and several other events can take place, see [28] for a full list. In this work, the sole aim was to find the most likely functional human homologs of non-model transcripts and did not attempt to define or classify paralogs or any other forms of gene histories. Many bioinformatic programs and pre-computed data- bases of orthologs exist, a number of which are listed in a recent review [28]. Essentially, there are 3 methods of finding orthologs, tree based, graph based and a combina- tion of the two. Tree methods use sequence alignments and phylogenetic trees for predicting evolutionary rela- tionships, while graph based methods use pairwise sequence similarity search methods (such as BLAST) to predict orthologs. The review highlights the merits of each program or database and it provides the reader with a flow chart (their figure four) to assist in the best choice of tool for a particular application. After investigation of the rec- ommended programs it was decided that none were appli- cable to the high-throughput analysis needed to cope with the > 16 K incomplete anonymous carp nucleotide sequences. The main reasons were the inability of these programs to be queried with a high volume of data through their web servers or the databases were several years out of date. Further literature searches revealed a tool called BLAST on orthologous groups (BLASTO) [31] which searches a query sequence against a database of orthologous groups (a collection of homologous genes from at least two genomes [28]) as a single unit. Although this tool enabled searches against several combination method pre-com- Page 2 of 17 (page number not for citation purposes)