BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 Table 4: A selection of cold induced angiogenic genes predicted in this work Gene APOE FN1 GRN HMGB1 HSPG2 ITGAL MMP9 NCL PLAUR RHOA SOD2 Accession NP_000032 NP_997647 NP_002078 NP_002119 NP_005520 NP_002200 NP_004985 NP_005372 NP_002650 NP_001655 NP_001019636 Gene product apolipoprotein E fibronectin 1 granulin high-mobility group box 1 heparan sulfate proteoglycan 2 nucleolin plasminogen activator, urokinase receptor ras homolog gene family, member A superoxide dismutase 2, mitochondrial MAPK1 NP_002736 mitogen-activated protein kinase 1 integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide) matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) From the 135 putative angiogenic genes up regulated due to cold temperatures, 12 genes considered biologically interesting in terms of angiogenesis are listed. The four highlighted in bold are reviewed in the text. [87]. It has been shown that dominant-negative RHOA in ECs impairs tube formation and sprouting in vitro [88]. This is replicated in vivo where dominant-negative N19RhoA impairs vessel assembly and dominant-active V14RhoA stimulates ECs to form vessels [89]. RHOA has also been shown to induce cytoskeleton re-organisation, enhance migration and increase angiogenic capacity [90]. VEGF increases RHOA activity by enhancing its recruit- ment to the membrane and mediating the RhoA/Rho pathway during angiogenesis [91]. Over expression of dominant-active RhoA leads to increased tyrosine phos- phorylation of VEGFR2, which is the key inducer of ang- iogenesis [92]. Again, related to hypoxia and low oxygen diffusion at low temperatures, RHOA is up-regulated in hypoxia and has been shown to be required for the accu- mulation of HIF1A, which induces VEGF expression [93]. Granulin (GRN) Some of the cold-induced genes have not been widely studied as angiogenic factors but have nevertheless been implicated in the process. These include granulin and nucleolin. Granulin is not a direct acting angiogenic factor but has been shown to stimulate VEGF expression in breast carcinoma cells [94]. Granulin has also been shown by yeast two hybrid assays to bind the HIV Tat protein that is actively secreted by HIV infected cells and acts as an EC growth and angiogenic factor [95]. In addition, granulin has been shown to be expressed within proliferating ovar- ian carcinomas blood vessels and interact with perlecan. Therefore, it is thought to regulate tumour angiogenesis and influence cancer growth [96]. Finally, an anti-granu- lin antibody has been shown to inhibit tumour angiogen- esis in human hepatomas implanted into athymic mice [97]. Nucleolin (NCL) Nucleolin is one of the major proteins of the nucleolus but is also expressed on the cell surface where it binds a variety of ligands involved in several cell processes. Expression of nucleolin is particularly high on the surface of ECs in angiogenic blood vessels [98]. Inhibition of nucleolin in ECs prevents capillary tube formation and retards EC migration [99]. It is of interest that MMP9, also a predicted cold-responsive angiogenic gene in this study, is associated with nucleolin in angiogenesis. Mimicking hypoxia led to a 3-fold increase in MMP9 protein levels, due to enhanced translational efficiency caused by nucle- olin binding to the 3' UTR of MMP9 [100]. These data showed that MMP9 expression during angiogenesis can be post-transcriptionally regulated by nucleolin. Lastly, antibodies to nucleolin have been shown to suppress tumour growth and angiogenesis [101]. Page 11 of 17 (page number not for citation purposes)