Common polymorphisms in angiogenesis.

from linkage disequilibrium with the HLA complex, rather than from differences in VEGF regulation. Pharmacogenomic studies on angiogenesis inhibitors are currently somewhat rare. How- ever, one group has identified an association between VEGF promoter polymorphisms and (2)Chronic pancreatitis (2)Circulating HHV-8 DNA (2)Hot flashes (2)Progressive massive fibrosis (coal miners) (2)Hemifacial spasm (2)Autosomal-dominant polycystic kidney disease progression Boldface text indicates that the bulk of evidence confirms an association between one or more VEGF polymorphisms and the risk, severity, progression (including metastasis), age of onset, or outcome for the indicated trait. A (2) indicates that the bulk of evidence shows no association of the trait with the VEGF polymorphism(s) studied. Italic text indicates conflicting evidence. For other listed traits, only a single report is available and, thus, the association should be considered unconfirmed.

available from the authors upon request. Table 1. Continued (2)Inflammatory bowel disease (2)Familial Mediterranean fever (2)T ropical spastic paraparesis in HTL V1-infected individuals M.S. Rogers and R.J. D’Amato 6 Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from breast cancer response to bevicuzimab, with genotypes consistent with haplotype 4 associ- ated with dramatic extension of overall survival (Schneider et al. 2008). In addition, genotypes consistent with haplotype 1 reduced the inci- dence of treatment-associated hypertension (Schneider et al. 2008), an observation that has since been confirmed (Etienne-Grimaldi et al. 2010), and extended to sunitinib-treated metastatic renal cancer patients (Kim et al. 2009b). In colorectal cancer, VEGF promoter polymorphisms predict response and time to progression with anti-VEGF therapy (Pander et al. 2010). However, promoter polymor- phisms have similar predictive power with che- motherapy regimens lacking an anti-VEGF component (Chen et al. 2010a). Similar results are obtained in lung cancer (Heist et al. 2008). This suggests either that current cytotoxic chemotherapeutic regimens have a significant antiangiogenic component even in the absence of a targeted angiogenesis inhibitor, or that growth in an environment with altered VEGF availability affects the biological characteristics of a tumor in a way that alters its response to chemotherapy. VEGFR2 VEGFR2 is a tyrosine kinase receptor contain- ing several nonsynonymous SNPs of unknown functional significance, and several regulatory SNPs. For example, the 2604C allele decreases transcriptional activity and protects against age-related macular degeneration (Galan et al. 2010). In addition, there is evidence of gene – gene interactions with VEGF genotype (V an- Cleave et al. 2010). However, studies to identify VEGFR2 polymorphisms that might predict Lucentis response were negative (Kloeckener- Gruissem et al. 2011). In contrast, in colon cancer, the 2604CC promoter genotype is associated with increased microvessel density and decreased survival, whereas the translationally silent 1192C . T polymorphism is associated with decreased microvessel density and increased survival (Hansen et al. 2010). A tag SNP approach has generated some evidence for association between glioblastoma and VEGFR2 genotype (Andersson et al. 2010). However, breast and lung cancer studies are uniformly negative. In a small study of prostate cancer, H472Q affects the frequency of sorafanib-induced side effects, which are in turn predictive of patient survival (Jain et al. 2010). Similarly, in CML the H472Q polymorphism has been shown to affect the likelihood of complete cytogenetic response to imatinib therapy as well as the odds of treat- ment failure (Kim et al. 2010). The 472Q variant is overrepresented in endurance athletes, where it is also associated with an increase in VO 2 max and with muscle fiber type (Ahmetov et al. 2009). V ariation in this polymorphism is also associated with recurrent pregnancy loss (Su et al. 2011). Finally, as is true of VEGF , osteonecrosis of the femoral head has been associated with VEGFR2 genotype (Hong et al. 2010) likely as a result of the importance of angiogenesis to bone repair. VEGFR2 genotype is associated with several additional diseases; however, whether the me- chanism involved is angiogenesis related is uncertain. For example, the V297I variant is asso- ciated with atopy (Park et al. 2006), especially in combination with TNF genotype (Park et al. 2007) as well as with hemorrhagic stroke (Zhang et al. 2009). VEGFR2 SNPs are also associated with sarcoidosis (Pabst et al. 2010), coronary artery disease in both normal individuals (W ang et al. 2007) and in Kawasaki disease patients (Kariyazono et al. 2004), and saphenous vein graft patency (Ellis et al. 2007), underlin- ing the importance of the VEGFR2 gene in atherosclerosis. HIF-1a HIF-1a is a transcription factor that is involved with major cellular oxygen sensing pathways. In hypoxia, the protein is stabilized and up- regulates genes involved in erythropoiesis and angiogenesis such as erythropoietin, adreno- medullin, and VEGF . The oxygen sensitivity of this response can vary widely between indi- viduals (Brooks et al. 2009) and two polymor- phisms in the gene appear to affect function Common Polymorphisms in Angiogenesis Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 7 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from (P582S rs 11549465 þ1772C . T and A588T rs11549467þ1790G . A) by increasing protein levels and transcriptional activity (T animoto et al. 2003). The functional nature of these alleles is underlined by repeated reports that they are overrepresented in high-performance athletes and their association with maximal exercise- induced oxygen consumption. Interestingly, although there are only a few positive reports of association between HIF- 1a polymorphisms and cancer incidence, there are repeated reports in multiple tumor types of association with tumor size and disease aggres- siveness (Zhao et al. 2009). Interestingly, the A588T polymorphism is associated with gastric cancer in Tibetans (Li et al. 2009a). Impor- tantly, this group shows a high frequency of alleles that alter HIF-1 a regulation and are believed to contribute to high-altitude adapta- tion (Simonson et al. 2010). Thus, this associa- tion may only have relevance to this population. The P582S polymorphism is associated with an increase in coronary collateral arteries (Resar et al. 2005) and thus cardiovascular disease patients with this genotype tend to present with stable angina rather than acute myocardial infarction (Hlatky et al. 2007). In contrast, in hemodialysis patients, polymorphisms are asso- ciated with acute myocardial infarction and hypotension (Zheng et al. 2009). It is also asso- ciated with susceptibility to systemic sclerosis, a disease characterized by vascular inflammation and angiopathy (Wipff et al. 2009). Finally, dis- tinct polymorphisms of unknown significance are associated with osteonecrosis of the femoral head (Hong et al. 2007). Nitric Oxide Synthase (NOS3 /eNOS) Nitric oxide is a multipotent signaling molecule with significant effects on angiogenesis, vasodi- lation, vascular permeability, and inflamma- tion. In addition, as a free radical it has significant mutagenic potential. Endothelial nitric oxide synthase (eNOS /NOS3) is the major endothelial producer of nitric oxide. Three polymorphisms in NOS3 have been well studied: 2786T . C in the promoter, a varia- ble number tandem repeat in the fourth intron, and 894G . T in exon 7 which results in an amino acid change (E298D) in the protein. All of these polymorphisms can affect eNOS activ- ity (W ang et al. 2000; Marangoni et al. 2008). Although the polymorphisms were initially identified in the context of cardiovascular dis- ease, they have also been associated with several angiogenesis-dependent diseases. Because the E298D polymorphism has been associated with differences in collateral formation (Lam- blin et al. 2005), angiogenesis may be the mech- anism by which this polymorphism affects a subset of cardiovascular disease. Retinopathy of prematurity is associated with all three polymorphisms (Rusai et al. 2008; Y anamandra et al. 2010), whereas the cod- ing polymorphism affects endometriosis (Kim et al. 2009a), and the promoter polymorphism affects idiopathic osteonecrosis of the femoral head (Glueck et al. 2007). In cancers such as non – small cell lung can- cer (Fujita et al. 2010), acute lymphoblastic leu- kemia (ALL), and breast cancer there is evidence to suggest that NOS3 mutations can affect dis- ease susceptibility (Haas et al. 2009; Hao et al. 2010; Zintzaras et al. 2010). Such susceptibility might be a result of increased angiogenic activ- ity and a consequently decreased threshold for the angiogenic switch. In addition, increased free-radical production might increase the mutation rate in a tissue, thereby increasing the rate of cancer formation. This possibility is highlighted by reports of an interaction between NOS3 genotype and antioxidant consumption in the risk of breast (Li et al. 2009b) and prostate (Lee et al. 2009a) cancers. In contrast, progres- sion to advanced disease and metastasis are nec- essarily angiogenesis dependent. Thus, it is interesting to observe that in several cancers the genotype is not significantly different between controls and patients; however, if one focuses on early progression, metastasis, and / or disease-free survival following treatment there is a correlation with genotype. Finally, there is also evidence that NOS3 polymorphisms can influence response to and side effects from cancer therapy. In ALL the amino acid polymorphism can affect a patient’s IQ following cranial radiation (Krajinovic et al. M.S. Rogers and R.J. D’Amato 8 Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from 2005). Similarly, in the breast cancer setting, a reduced incidence of radiation-induced telan- giectasias was noted in women with lower-activ- ity NOS3 genotypes (Kuptsova et al. 2008). In NSCLC a decreased risk of pneumonitis was noted in carriers of the low-activity E298D allele (Hildebrandt et al. 2010); however, that effect is almost certainly not attributable to differences in angiogenesis, but rather to inflammatory dif- ferences. Similarly the decreased rectal cancer survival observed in carriers of the E298D allele following radiotherapy (Funke et al. 2009) are likely a result of decreased low reactive oxygen production rather than any angiogenic effects. Integrins Several integrins are expressed on endothelial cells, including avb3, which is upregulated in the angiogenic endothelium. During angio- genesis, this complex interacts directly with VEGFR2 and PDGFR b and cooperates with VEGFR2 to stimulate endothelial cell migra- tion. An early study showed that theb3 integrin L33P polymorphism increased the risk of mela- noma as well as ovarian and breast cancers (Bojesen et al. 2003). In the case of breast cancer, this risk appears to be particularly strong for younger women (W ang-Gohrke and Chang- Claude 2004) and extends to the risk of metasta- sis (Ayala et al. 2003; Langsenlehner et al. 2006). Polymorphisms in av integrin have been implicated in susceptibility to hepatitis B virus induced hepatocellular carcinoma (Lee et al. 2009b) and regional spread in gastric cancer (Scartozzi et al. 2010). Other integrins (e.g., avb5, a5b1) are also expressed on endothelial cell and involved in angiogenesis, but studies have not identified polymorphisms in these genes that regulate angiogenesis or angiogene- sis-dependent disease. IL-8/CXCR2 IL-8 is a VEGF-independent stimulator of angiogenesis which acts through the receptors CXCR1 and CXCR2, with the latter being the endothelial receptor (Li et al. 2003). The Il-8 -251T . A polymorphism leads to increased IL-8 production (Hull et al. 2000) and increases plasma IL-8 levels (Lee et al. 2005). There have now been over 50 studies con- ducted to assess the relationship between the IL-8 2251A allele and cancer. A meta-analysis of these studies found that the A allele increases the risk of nasopharyngeal carcinoma and that it increased the risk of a group of “other” less studied cancers (Gao et al. 2010). Importantly, when studies with population-based controls were compared with those with hospital-based controls it was found that hospital-based controls indicated an increased risk of cancer, whereas population-based controls indicated a decreased risk of cancer from the A allele (Gao et al. 2010). This result suggests that overall health status may be affected by an individual’s IL-8 allele and, thus, the immune functions of IL-8 may confound the ability to detect differ- ences in cancer resulting from angiogenic differ- ences. In this context, the effect of the A allele on tumor recurrence may be instructive. In stage III colon cancer recurrence is predicted by the high expressing A allele (Lurje et al. 2008), as is recur- rence in rectal cancer (Gordon et al. 2006). This allele also predicts incidence, recurrence, and overall survival in gastric adenocarcinoma (Lurje et al. 2010a) and increased risk of and aggressiveness in breast cancer (Snoussi et al. 2010). Interestingly, this polymorphism also represents a pharmacogenomic marker in anti- angiogenic therapy. It is associated with lower response to bevicuzimab and metronomic cyclophosphamide in ovarian cancer as is the CXCR2 þ785C . T polymorphism (Schulth- eis et al. 2008). At the distinct CXCR2 þ1208C . T polymorphism, the TT genotype is associated with both incidence and aggres- siveness in breast cancer (Snoussi et al. 2010). Proteases and Protease Inhibitors Several proteases, including matrix metallo- proteinases (MMPs), their tissue inhibitors (TIMPs), and members of the plasminogen sys- tem, are known to be involved in the invasion and remodeling required for angiogenic vessel outgrowth. In addition to remodeling matrix, some proteases and inhibitors have more direct Common Polymorphisms in Angiogenesis Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 9 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from activities. For example, the plasminogen frag- ment angiostatin has antiangiogenic activity (O’Reilly et al. 1994). Similarly TIMP-3 antago- nizes VEGF binding to VEGFR2 (Qi et al. 2003) and inhibits endothelial chemotaxis, collagen gel invasion, and vessel growth in the CAM assay (Anand-Apte et al. 1997). Certain loss- of-function mutations in TIMP-3 result in Sorsby’s fundus dystrophy, decreasing the anti- angiogenic properties of Bruch’s membrane and increasing susceptibility to choroidal neovascu- larization (W eber et al. 1994). Similarly, sus- ceptibility to age-related macular degeneration is affected by polymorphisms in the TIMP3 gene (Chen et al. 2010b). Longer length in an MMP9 promoter microsatellite is also associ- ated with susceptibility to wet ARMD (Fiotti et al. 2005). In men with proliferative diabetic retinopathy the MMP2 21306C allele is associ- ated with higher plasma MMP2 levels and with disease (Beranek et al. 2008) and the similar 2735C . T polymorphism is associated with psoriasis (V asku et al. 2002). In these diseases, it seems likely that gene polymorphism modu- lates disease susceptibility by modulating the angiogenic response, although other mecha- nisms are possible. Polymorphisms in several of these genes are also associated with cancer; however, in all of these cases, because substan- tial tissue remodeling is a hallmark of neoplastic disease it is difficult to determine whether dis- ease susceptibility is mediated by increased angiogenesis, increased tissue remodeling, or both. Promising Candidates Adrenomedullin is a potent vasodilator and a pro-angiogenic factor (Martinez 2006). An in / del in the 3 0 UTR of the adrenomedullin gene is associated with progression-free survival in bevicuzimab- and cyclophosphamide-treated ovarian cancer (Schultheis et al. 2008). Interest- ingly, this polymorphism is also associated with hypertension (Ishimitsu et al. 2001), a common side effect of anti-VEGF therapy. Full-length galectin-3 is involved in a num- ber of processes important to tumorgenesis including anchorage independent growth and tumor cell proliferation. The protein is also cleaved by MMPs and the cleaved form is involved in chemotaxis and angiogenesis. A P64H polymorphism adjacent to one of the MMP cleavage sites (between A62 and Y63) affects cleavage efficiency and angiogenesis stimulatory activity (Nangia-Makker et al. 2010). The H allele, which results in greater angiogenesis, is more common in Caucasian women than in Asian woman and has been hypothesized to explain a portion of the increased risk of breast cancer in Caucasian women (Balan et al. 2008). The H allele also increases the risk of breast cancer in each popu- lation independently (Balan et al. 2008). Proteinase-activated receptor 1 (PAR-1) has been shown to regulate the differential release of pro- and anti-angiogenic factors from platelets (Ma et al. 2005; Italiano et al. 2008). A promoter in /del at position 2506 in the gene is associated with outcome in gastric can- cer patients (Lurje et al. 2010a) and with tumor recurrence in esophageal carcinoma (Lurje et al. 2010b). GENOME-WIDE ASSOCIATION STUDIES IN HUMANS In principle, genome-wide association studies for polymorphisms affecting conditions such as cancer or cardiovascular disease might dis- cover angiogenesis response alleles. There are at least two potential examples of this. First, there have been repeated reports of genome- wide association between cancer and SNPs near the fibroblast growth factor receptor 2 gene (FGFR2) (Easton et al. 2007; Hunter et al. 2007; Thomas et al. 2009; Gaudet et al. 2010; T urnbull et al. 2010; Li et al. 2011), with the risk allele increasing the expression of the gene (Meyer et al. 2008). Overlap between array CGH-determined CNV suggests that several bFGF QTLs may be regulated by copy-number variation alleles (Cho et al. 2006). This protein is the receptor for several fibroblast growth fac- tors, including FGF2, a powerful angiogenesis stimulator (Shing et al. 1984; Klagsbrun and Shing 1985). A similar case is that of the phos- pholipase PLCE1, an intracellular regulator of M.S. Rogers and R.J. D’Amato 10 Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from growth factor signaling in both endothelial and other cell types, including cancer cells. An amino acid substitution in this gene is associ- ated with esophageal squamous cell carcinoma in Chinese subjects (W ang et al. 2010a). The relevance of both of these findings to angiogen- esis is questionable because both proteins are also mitogens and can confer survival signals. Nevertheless, it is interesting that polymorphisms in these genes, rather than others with similar characteristics, are observed to increase the risk of cancer. Thus, one might hypothesize that both the mitogenic and angiogenic activ- ities of these proteins are involved in conferring susceptibility to cancer. Indeed, it is possible that some of the genes and regions currently identified as cancer risk alleles have as-yet undiscovered roles in angiogenesis. A simpler case involves the HTRA1 gene in age-related macular degeneration (ARMD). HTRA1 is a serine protease expressed in the ret- inal pigment epithelium. It has been shown to selectively cleave eight RPE proteins. These include several ECM proteins that might regu- late angiogenesis, such as fibromodulin, clus- terin, ADAM9, and vitronectin (An et al. 2010). Promoter polymorphisms in HTRA1 have been associated specifically with wet ARMD (Dewan et al. 2006) suggesting that increased expression of this gene results in increased susceptibility to angiogenesis in the retina. However, the polymorphisms involved are part of a haplotype that also results in loss of function in the ARMS2 (LOC387715) gene, and this loss appears also to be required for ARMD susceptibility (Y ang et al. 2010). The absence of obvious angiogenesis- related gene associations in GW ASs of diseases that are clearly angiogenesis dependent might be explained in a number of ways. First, there remains a substantial amount of “dark matter” in genetics. That is, loci identified by current GW ASs and family studies explain only a small fraction of observed heritability (Manolio et al. 2009). It may be that angiogenesis re- sponse alleles substantially lie in the “dark matter. ” In this context it should be noted that the design of current GW ASs has resulted in enrichment for genes active very early in tumor initiation, which thus confer susceptibility to both pre-cancerous (and pre-angiogenic) lesions and frank cancer (V arghese and Easton 2010). As outlined above, candidate gene stud- ies have often found that angiogenesis gene polymorphisms exhibit stronger effects on tumor progression or metastasis than in tumor incidence. Thus, as GW ASs move toward more refined comparisons, it is likely that angiogenesis genes will appear in greater num- bers. Indeed, because cancer susceptibility

from the interplay of a large number of different processes, one means of identifying risk alleles is to use intermediate phenotypes that are affected by a smaller number of proc- esses, thus limiting the diluting effect of other processes (e.g., apoptosis susceptibility). Sec- ond, the involvement in a wide variety of proc- esses critical to life may limit the extent to which variation in angiogenesis-regulating genes is evolutionarily permitted. This, in turn, will limit the relative risk attributable to any given allele, and thus the detectability of that allele. Finally, current GW ASs rely heavily on a tag SNP approach. Although current knowledge necessitates such an approach, it is likely to be less effective than a function SNP approach. It is thus to be anticipated that, as GW AS techni- ques are refined, angiogenesis-regulating genes will appear with greater frequency in the results of such studies. LOCI IDENTIFIED IN ANIMAL STUDIES Several experiments in rodents, designed to map tumor-related traits, have identified loci that affect angiogenic response (T able 2). For example, in the estrogen-induced pituitary tumor model, a locus on rat chromosome 5, Edpm5, affects tumor mass and vascularity and is associated with the switch to the angio- genic phenotype (Pandey et al. 2004). Similarly, decreased proliferative capacity and altered la- tency associated with altered angiogenesis have been observed in MMTV-PyMT mice with spe- cific genotypes at any of three loci: Mmtg1, Mmtg2, and Mmtg3 (Le V oyer et al. 2001). In addition to altering growth or latency character- istics, differences in angiogenic responsiveness Common Polymorphisms in Angiogenesis Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 11 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from Table 2. Rodent angiogenic response QTLs QTL Location Effect of paternal allele Cross Reference Edpm5 Rat Chr 5M Resistance to estrogen- induced pituitary tumor angiogenesis Fisher 344 x Brown Norway Pandey et al. 2004 Skts9 Chr 16P Skin tumor resistance NIH /Ola x M. spretus Nagase et al. 1999 Mmtg1 Chr 4P Increased tumor burden I/ LnJ x FVB/N-TgN(MMTV-PyVT)634Mu1 Le V oyer et al. 2001 Mmtg2 Chr 4M Increased tumor burden I/ LnJ x FVB/N-TgN(MMTV-PyVT)634Mu1 Le V oyer et al. 2001 Mmtg3 Chr 7P Decreased tumor burden I/ LnJ x FVB/N-TgN(MMTV-PyVT)634Mu1 Le V oyer et al. 2001 Tgfbkm2 Chr 1D Normal yolk sac angiogenesis C57BL/6 Tgfb1 þ/2 x 129S2/Sv Tgfb1 þ/2 T ang et al. 2003 Tgfbm1 Chr 5P Normal yolk sac Angiogenesis C57BL/6 Tgfb1 þ/2 x NIH/OlaHsd Tgfb1 þ/2 T ang et al. 2005 Tgfbm3 Chr 12P Normal yolk sac angiogenesis C57BL/6 Tgfb1 þ/2 x NIH/OlaHsd Tgfb1 þ/2 T ang et al. 2005 AngVq1 Chr 10M Increased VEGF-induced angiogenesis C57BL/6J x DBA /2J Rogers et al. 2003, a AngVq2 Chr 2P Decreased VEGF-induced angiogenesis C57BL/6J x DBA /2J, C57BL/6J x A /J Rogers et al. 2003, a AngVq3 Chr 10P Decreased VEGF-induced angiogenesis C57BL/6J x DBA /2J, C57BL/6J x A /J Rogers et al. 2003, a AngVq4 Chr 7M Decreased VEGF-induced angiogenesis C57BL/6J x A /J a AngVq5 Chr Y Decreased VEGF-induced angiogenesis C57BL/6J x A /J, C57BL/6J x SJL a AngFq1 Chr 4P Increased bFGF-induced angiogenesis C57BL/6J x DBA /2J Rogers et al. 2004 AngFq2 Chr 13P Decreased bFGF-induced angiogenesis C57BL/6J x DBA /2J Rogers et al. 2004 AngFq3 Chr 15M Increased bFGF-induced angiogenesis C57BL/6J x DBA /2J Rogers et al. 2004 AngFq4 Chr 18D Increased bFGF-induced angiogenesis C57BL/6J x DBA /2J, C57BL/6J x 129P3 /J Rogers et al. 2004 AngFq5 Chr 7P Decreased bFGF-induced angiogenesis C57BL/6J x SJL /J b AngFq6 Chr 12P Increased bFGF-induced angiogenesis C57BL/6J x 129P3 /J, C57BL/6J x 129P1 /ReJ b AngFq7 Chr 14M Decreased bFGF-induced angiogenesis C57BL/6J x 129P3 /J b AngFq8 Chr Y Decreased bFGF-induced angiogenesis C57BL/6J x A /J, C57BL/6J x SJL b Quantitative trait loci (QTLs) for differences in angiogenic responsiveness along with their peak chromosomal location (P , proximal third; M, middle third; D, distal third), effect of the paternal (strain listed second) allele, and cross(es) in which the trait segregates. aMS Rogers, AE Birsner, and RJ D’Amato, unpubl. bMS Rogers, V Boyartchuk, AE Birsner, et al., unpubl. M.S. Rogers and R.J. D’Amato 12 Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from may alter tumor shape or eccentricity. This may

when in a given region of a growing tumor the fraction of angiogenic tumor cells drops below the threshold necessary to maintain angiogenesis (Udagawa et al. 2002). When this occurs, the tumor ceases to grow locally, result- ing in alteration of tumor shape. Thus, several lung tumor shape-determining loci ( Ltsd) may also be angiogenesis-response loci (T ripo- dis and Demant 2003). W e have used the mouse corneal micro- pocket assay (Rogers et al. 2007) to identify sev- eral quantitative trait loci (QTLs) that control response to bFGF and /or VEGF . Importantly, these studies contrast with knockout studies because the variation underlying the trait has survived the rigors of inbreeding and the breed- ing requirements of colony maintenance. As a result, it is likely to more closely model com- mon human polymorphisms that are responsi- ble for the bulk of human disease than would other approaches. W e have found results of the corneal micropocket assay to be stable with regard to animal age and cage environment, with two studies showing that genetic differen- ces explain more than 20 times the variance that is explained by environmental factors (Rogers et al. 2003, 2004). W e have used the BXD (C57BL/6J x DBA / 2J) recombinant inbred strain cross to identify bFGF response loci. As a result, we identified loci on chromosome 4, 13, 15, and 18 which we named AngFq1-AngFq4 for angiogenesis in response to bFGF (Rogers et al. 2004). Each of these loci has been confirmed with a congenic animal from the genome-tagged mouse set (Iakoubova et al. 2001). In addition, earlier mapping studies using F2 crosses between 129P3/J, 129P1ReJ, or SJL/ J and C57BL /6J identified four QTLs, one each on chromo- somes 7, 12, 14, and Y ( AngFq5-AngFq8). In the case of AngFq5, congenic animal generation combined with haplotype mapping identified the pink-eyed dilution mutation as a candidate that might explain the phenotype. This candi- date was confirmed using the distinct pJ allele in the same gene. Thus, unexpectedly, pink- eyed dilution was identified as a gene that affects angiogenic responsiveness. In the case of VEGF , mapping using the BXD strain set resulted in the identification of two QTLs, AngVq1 in the middle of chromo- some 10 and AngVq2 in the proximal portion of chromosome 2, each of which has been confirmed with a GTM mouse (Rogers et al. 2003; Rogers and D’Amato 2006). Importantly, a comparison of angiogenic response to bFGF and VEGF in the BXD strain set demonstrated substantial correlation. This may be a result of the observation that a substantial fraction of bFGF response can be inhibited by a VEGF inhibitor, indicating that a portion of the bFGF response is mediated by VEGF . Subse- quent work with the AXB and BXA strain sets as well as with chromosome substitution strains (Hill et al. 2006) has resulted in the identification of a second QTL on chromosome 10 (AngVq3) as well as QTLs on chromosomes 7( AngVq4) and Y ( AngVq5). Subsequent map- ping work has identified the albino mutation in tyrosinase as responsible for AngVq4 (MS Rog- ers, AE Birsner, and RJ D’Amato, unpubl.). Thus, in at least two cases, coat color loci can affect angiogenic response, indicating an over- lap in the two pathways. In addition to corneal angiogenesis, we have also extended this work to identify QTLs that control the neovascular area in a laser-induced mouse model of age-related macular degenera- tion (Nakai et al. 2009). In this case, because ret- inal pigmentation affects the amount of laser energy deposited, pigmentation-related alleles had to be controlled for. This mapping con- firmed AngVq1 and AngFq2. It also resulted in the identification of two new QTLs, AngCNVq1 and A ngCNVq2 on chromosomes 2 and 19, respectively. In addition to angiogenesis response QTLs, other QTLs affect the vasculature in inbred mice. For example, the extent and remodeling of collateral arteries are genetically controlled traits that can affect the outcome of ischemic injury (W ang et al. 2010b). Intestinal lymphatic vasculature can be affected by a QTL on chro- mosome 3 that includes the VCAM1 gene (Jurisic et al. 2010), and both basal and induced corneal lymphatics differ among mouse strains (Nakao et al. 2010). These traits appear, Common Polymorphisms in Angiogenesis Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 13 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from however, to be both mechanistically and genet- ically distinct from angiogenic responsiveness.

The study of the role of genetic variation in the angiogenic response is currently in its infancy. Nevertheless, it is now clear that poly- morphisms in angiogenesis-regulating genes can affect a large number of phenotypes, in- cluding a wide variety related to disease pro- cesses in man. In none of these cases does an absence of association necessarily imply that the gene/protein in question is not involved in disease. Rather, it may simply mean that func- tional polymorphisms in the gene have not been identified or do not exist at sufficient fre- quency in the study populations. Importantly, odds ratios for disease susceptibility in angio- genesis-regulating genes tend to be low, with meta-analysis typically demonstrating odds ratios in the 1.1 – 1.3 range. This is likely to be a result of purifying selection. Nevertheless, the large number of angiogenesis-regulating genes currently identified means that, even assuming no epistatic interaction among poly- morphisms, the overall risk due to angiogene- sis-regulating polymorphisms could be quite high. Epistatic interactions among members of interacting pathways may substantially increase risk. Indeed, the few studies that have looked at multiple angiogenesis-regulating polymor- phisms have found odds ratios associated with three risk alleles to be in the six to seven range for susceptibility (a range that would be expected to have required approximately 15 risk alleles without epistatic interactions) and 20 for tumor aggressiveness (Gerger et al. 2007; Sfar et al. 2009). Given the pervasive epistasis found in other studies of complex traits (Shao et al. 2008), it is likely that the sum of angiogenic response alleles plays a major role in angiogenesis-dependent dis- ease. Combined with the number of angiogenesis- regulated diseases and traits, it is not surprising that polymorphisms in just one angiogenesis reg- ulator are associated with human lifespan (Del Bo et al. 2008), and it is likely that the sum of angio- genesis-regulating variation plays a major role in determining the length and quality of life.

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Poly- morphisms of the endothelial nitric oxide synthase gene in breast cancer: A genetic association study and meta-analysis. J Hum Genet 55: 743 – 748. Common Polymorphisms in Angiogenesis Cite this article as Cold Spring Harb Perspect Med 2012;2:a006510 19 www .perspectivesinmedicine.org on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from September 28, 2011 2012; doi: 10.1101/cshperspect.a006510 originally published onlineCold Spring Harb Perspect Med    Michael S. Rogers and Robert J. D'Amato   Common Polymorphisms in Angiogenesis Subject Collection Angiogenesis Angiogenesis and Microvascular Permeability Ye Zeng and Bingmei M. Fu Edition Angiogenesis: Biology and Pathology, Second Diane R. Bielenberg and Patricia A. D'Amore Function Mechanics of Lymphatic Pumping and Lymphatic P. Padera Mohammad S. Razavi, Lance L. Munn and Timothy Development and Disease Vascular Organization: Lessons from Steve Spurgin and Ondine Cleaver Lymphangiogenesis in Pathological Conditions Prostanoids Regulate Angiogenesis and Watanabe, et al. Masataka Majima, Yasuhiro Matsuda, Shin-Ichi and Lymphangiogenic Signaling Epsin Endocytic Adaptor Proteins in Angiogenic Douglas B. Cowan, Hao Wu and Hong Chen and Disease Brain Barrier in Health−Regulation of the Blood Cara C. Rada, Kanako Yuki, Jie Ding, et al. In Vitro Modeling Brain Vasculature Immune Interactions Ruth Lyck, Hideaki Nishihara, Sidar Aydin, et al. Gut Microbiota and the Microvasculature Christoph Reinhardt Klytaimnistra Kiouptsi, Giulia Pontarollo and Morphogenesis, Maturation, and Stabilization Extracellular Matrix Regulation of Vascular George E. Davis and Scott S. Kemp Malformations (CCMs) Pathogenesis of Cerebral Cavernous Pericyte Interactions in the−Endothelial Cell Wang Min and Jenny Huanjiao Zhou Inflammation Targeting Angiogenesis via Resolution of Abigail G. Kelly and Dipak Panigrahy Lymphatic Clearance and Pump Function Jerome W. Breslin and Angiocrine Functions Notch Signaling in the Vasculature: Angiogenesis Sana S. Hasan and Andreas Fischer Platelets and (Lymph)angiogenesis Harvey G. Roweth and Elisabeth M. Battinelli Endothelium Signal Transduction and Gene Regulation in the Michel V. Levesque and Timothy Hla http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see Copyright © 2012 Cold Spring Harbor Laboratory Press; all rights reserved on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see Copyright © 2012 Cold Spring Harbor Laboratory Press; all rights reserved on August 24, 2026 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from