**4. Hepatocarcinoma angiogenesis induced by DRR**

 Risk factors induced DNA damages and dysregulated DRRs are regarded as molecular events [4]. In human, risk factors for hepatocarcinoma can manifest acute and chronic DNA damage. Acute and noticeable DNA damages often lead to severe chromosome aberration and even cell death, whereas chronic DNA damages are the earliest molecular change in hepatocytes and ultimately result in hepatocarcinoma [40]. In the past decades, angiogenesis induced by dysregulation of DRR pathways may act as a vital role in the process of hepatocarcinoma. Evidence from epidemiological and clinicopathological studies has shown that higher potential of angiogenesis is in the liver of patients with chronic DNA damage and low DRR capacity [40, 102–105]. For example, Pastukh et al*.* [102] investigated the association between recruitment of DNA repair enzymes involving in BER pathway and VEGF expression via a chromatin immunoprecipitation technique. They found that hypoxia-induced reactive oxygen species (ROS) stress caused promoter base modifications targeted to hypoxic response elements (HREs) and increased VEGF expression. During this modification, 8-oxoguanine (8-oxodG, an oxidative DNA damage product) in VEGF promoter was temporally correlated with binding of human 8-oxodG glycosylase 1 (hOGG1, a BER repair enzyme), HIF-1α, redox effector factor-1, endonuclease one, and breaks in DNA strands. If 8-oxodG was decreased in the promoter region of VEGF, VEGF expression would downregulate [102]. Recent molecular epidemiological studies have further proved that genetic variants in hOGG1 genes increase hepatocarcinoma risk and modify the prognosis of this malignancy [103–105]. Collectively, these data suggest that increasing ROS like 8-oxodG resulting from low DRR capacity may promote angiogenesis.

 Studies from high HBV and HCV infection and high AFB1 exposure area also display that the degrees of DNA damages are positively associated with MVD in tumor tissues from hepatocarcinoma [20, 55, 75, 79, 82]. For example, Lu et al*.* [20] investigated the effects of XRCC4 expression in tumor tissues on clinicopathological features and prognosis of hepatocarcinoma and found that decreasing XRCC4 expression was related to low DRR capacity, causing the formation of DNA adducts and TP53M. The dysregulation of XRCC4 may promote tumor proliferation and increase MVD. Several other studies further show that the low DRR capacity resulting from significant mutations in coding region of DNA repair genes (such as XRCC4, XRCC1, XPC, XPD, and XRCC7) increases MVD (**Table 4**) [21, 40, 52, 55, 59, 61, 62, 79, 80, 82]. Results from Lu et al*.* [20] and our studies [61, 62] showed that genetic alterations in the coding regions of XRCC4 gene (including Ala to Ser at codon 247 and Thr to Ala at codon 56) can decrease levels of XRCC4 protein expression and cause increasing amount of AFB1-DNA adducts and mutative frequency of TP53 gene in tissues with hepatocarcinoma. They also found that the amount of AFB1-induced DNA adducts, including 8,9-dihydro-8-(N7 -guanyl)-9 hydroxy-AFB1 (AFB1-N7 -Gua) and formamidopyridine AFB1 adduct (AFB1-FAPy), was positively associated with the number of microvessels (a biomarker for angiogenesis). Results from our studies [79, 106, 107] furthermore displayed that three low DNA repair markers related to AFB1, including tumor risk, TP53M frequency, and AFB1-FAPy adduct amount, were significantly correlated with the number of microvessels in liver tissues. These individuals with high AFB1-FAPy adduct level in liver tissues had an increasing risk of high MVD than those low adduct level (OR = 1.68, 95% CI = 1.45–2.87) [106]. Liu et al*.* [108] and Wang et al*.* [109] further proved that the upregulation of microRNA-429 and microRNA-24 expression in tissues with hepatocarcinoma not only increased the amount of AFB1-DNA adducts

 and the number of microvessels but also grew tumor metastasis risk via vessels and shorted patients' survival. Recent evidence has shown that microRNA-24/ microRNA-429 can modify the capacity of DDR via controlling Nbs1 (a regulator of DRR) [110, 111] and angiogenesis via regulating the crosstalk between the pro-contractile transforming growth factor-β/bone morphogenetic protein (TGF-β/ BMP) signal (inducing a quiescent 'contractile' phenotype) and the pro-synthetic platelet-derived growth factor (PDGF) signal (causing a proliferative 'synthetic' phenotype) [112, 113]. This suggests that microRNA-24/microRNA-429 may play an important regulative role between DRR capacity and angiogenesis. Taken together, this evidence proves that low DRR-induced MVD augmentation is regulated by the amount of DNA damage.

 Evidence from in vitro and in vivo studies further shows that dysregulation of DRRs and signaling to cell cycle checkpoints (CCCs) may modify hepatocarcinoma angiogenesis. CCCs involving in DRRs mainly encompass G1/S and G2/M checkpoint [114]. During G1/S checkpoint, both ATR and ATM act as central activators for DRR via inducing the phosphorylation of p53 protein which can activate p21 (a Cdk inhibitor). ATM/TP53/P21 pathway also plays an important function controlling G2/M procession [114]. The dysregulation of these factors and signal pathways can change the status of angiogenesis [115–119]. For example, Qin et al*.* [115] found that E2F1, an important cell cycle regulator, can modify angiogenesis via controlling VEGF expression by p53-dependent way. In this control model, deficient phenotype of E2F1 will result in VEGF overexpression, while its positive phenotype decreases VEGF expression [115]. Factors controlling cell shape and cytosol can regulate the cycle of vessel endothelial cells and angiogenesis [116, 117]. In mice model with the deficiency of BCL-2 (an important regulatory factor in DDRs), cells featured increasing DNA damage [118]; the inhibition of BCL-2 will result in the arrest of cells in S phrase and suppression of tumor angiogenesis [119]. In an integrated genomic study (including 5 hepatocarcinoma patients with hepatitis D visus [HDV] and 7 HDV-positive cirrhosis cases), Diaz et al*.* [120] investigated the association between HDV-related hepatocarcinoma and potential signal pathways involved in DNA damage and repair and cell cycle and found significant interactions of DDR/cell cycle-related genes, such as BRCA1, BARD1, CDK1, CDKN2C, CCNA2, CCNB1, CCNE2, GSK3B, H2AFX, MSH2, NPM1, PRKDC, and TOP2A. Results from the t-SNP (*t*-distributed stochastic neighbor embedding analyses) further exhibited that HUS1, BRCA1, BARD1, GADD45, DNA-damage-induced 14-3-3σ, and MSH2 gene involving in DRRs valuably scored with regulatory genes (such as ATM, TP53, NO, and epidermal growth factor), which involve in G2/M checkpoint and angiogenesis [120]. The dysregulation of HUS1 and corresponding genotoxin-activated checkpoint complex (also termed as Rad9- Rad1-Hus1complex) will cause abnormal DRR capacity and cell cycle in response to DNA damage and promote the alteration of hematogenous metastatic phenotype for hepatocarcinoma [121, 122]. The genetic alterations and abnormal expression of BRCA1 and GADD45 (two important regulatory factors in DRR and apoptosis pathways) in hepatocytes can also change TP53-dependent CCCs and VEGF expression [123, 124]. Altogether, these studies have proved that the dysregulation of DDRs can cause the abnormal regulation of CCCs and change the status of hepatocarcinoma angiogenesis.

Detailed molecular mechanisms of DRR dysregulation promoting hepatocarcinoma angiogenesis have still not been fully understood. Several possible pathways may play some important roles. First, DNA damage agents induce NO synthase and increase the expression of VEGF and HGF [125, 126]. Second, DNA damage agents like AFB1 cause the mutations of such genes as TP53, ras, and DNA repair genes. Activation of oncogenes and inactivation of tumor suppression genes and

 DNA repair genes lead to uncontrolled expression of genes involving in angiogenesis such as VEGF and Ang-1/2 [5, 6]. Third, genetic alterations in DRR pathways may alter the microenvironment of tumor and promote angiogenesis [127–129]. Fourth, the abnormal DRRs may accelerate the accumulation of DNA damages and trigger the dysregulation of angiogenesis-related genes and the progression of hepatocarcinoma. Finally, some metabolic products (such as AFBO) or nucleotide sequences (HBx) of DNA agents can bind to genomic DNA of hepatocytes and may increase the activation of VEGF HREs [22, 40, 41, 45]. Taken together, under the conditions of low DRR capacity and/or chronic risk factors, DNA damages will accumulate in hepatocytes and ultimately induce hepatocarcinogenesis and tumor angiogenesis.
