**2. Angiogenesis and regulation in hepatocarcinoma**

## **2.1 Angiogenesis process in hepatocarcinoma**

Several previous reviews have summarized the angiogenesis in hepatocarcinoma [5–7]. In brief, angiogenesis is a kind of crucial biological function and survival potential for normal organism development, growth, and adaptation to new environment. The dynamic balance between increasing and decreasing potential of angiogenesis is essential in the different physiological and pathological conditions, such as injury cure, damage repair, inflammatory procession, tumor progression, blindness, and ischemia. Hepatocarcinoma angiogenesis was extensively studied via cell models, experimental animal models, and human tumor samples [5–7]. Accumulating data have proved that local hypoxia in tumor tissues and the change in genome resulting from genetic or environmental risk factors will lead to the secretion and synthetics of angiogenetic regulative factors and triggering angiogenesis [8–10]. In hepatocarcinoma tissues, the process of angiogenesis consists of the following several stages: sprouting, extracellular matrix component (ECMs) reconstruction, endothelial cell (EC) migration and proliferation, lumen formation, and stabilization of newborn vessels (**Figure 1**) [11].

The establishment of conditions allowing ECs proliferation and migration, which often results from local hypoxia, first facilitates endothelial sprouting and budding. During this stage, hypoxia induces the secretion and synthetics of angiogenetic factors, such as nitric oxide (NO), vascular endothelial growth factor (VEGF), CD31, angiopoietin-1, and so on [11]. The NO-induced vasodilation and VEGF-caused high permeability result in the extravasation of plasma components (including fibrinogen and fibrin). Together with ECMs, these plasma components lay down and form provisional scaffolds for migrating ECs. The basement membranes and ECMs (mainly consisting of collagen I and IV and laminin) are next degraded, and subsequently, ECs migrate into local sites and proliferate. Increasing proliferation of ECs in the local hypoxia tissues leads to the formation of nascent vessels with lumen. After that, nascent vessels are recruited and structurally stabilized under the conditions of physical forces and a series of molecules such as platelet-derived growth factor β (PDFG-β), angiopoietin-1, angiopoietin-2, VEGF, and transforming growth factor β1 (TGF-β1) [7, 11, 12].

Vessels in hepatocarcinoma differ from other liver diseases or normal vessels [5, 11, 13]. First, tumor vessels typically appear as irregular diameter and abnormal branching patterns [5]. Second, pericytes of vessels are often incompletely covered or lost; furthermore, their basement membranes are also incomplete [11]. Third, tumor vessels sometimes form irregular channels and the walls of these channels are

#### **Figure 1.**

*Angiogenesis procession in hepatocarcinoma. The procession of angiogenesis consists of: (1) sprouting and budding; (2) ECM remodeling; (3) EC proliferation and migration; (4) lumen formation and three-D organization; and (5) stabilization of nascent vessels.* 

comprised of cancer cells. Moreover, the endothelial cells may be replaced by cancer cells partially or completely. Finally, angiogenesis in hepatocarcinoma not only appears abnormal architecture but also accompanies abnormal molecular expression and regulation [6, 14]. These characteristics result in abnormal structures and function for hepatocarcinoma; however, they can provide some important cues for early diagnosis and therapeutic strategies for cases with hepatocarcinoma.

#### **2.2 Angiogenesis regulation in hepatocarcinoma**

A series of angiogenic and antiangiogenic factors (**Tables 1** and **2**) regulate the angiogenesis process in hepatocarcinoma [5]. During the process of hepatocarcinoma angiogenesis, hypoxia and VEGF family play a vital role. Hypoxia in local



**Abbreviations***: VEGF, vascular endothelial growth factor; ECM, extracellular matrix component; EC, endothelial cell; PEDF, Pigment epithelium-derived factor; platelet and endothelial cell adhesion molecule 1; TIMPs, Tissue inhibitor of metalloproteases; IFN, interferon; MMPs, matrix metalloproteinases; Ang, angiopoietin; IL, interleukin; PIGF, placenta growth factor; HGF, hepatocyte growth factor; TGF, transforming growth factor; EGF, epidermal growth factor.* 

#### **Table 1.**

*Angiogenesis active regulative factors in hepatocarcinoma.* 



**Abbreviations:** *VEGF, vascular endothelial growth factor; ECM, extracellular matrix component; EC, endothelial cell; PEDF, Pigment epithelium-derived factor; platelet and endothelial cell adhesion molecule 1; TIMPs, Tissue inhibitor of metalloproteases; IFN, interferon; MMPs, matrix metalloproteinases; Ang, angiopoietin.* 

#### **Table 2.**

*Angiogenesis inhibitive regulative factors in hepatocarcinoma.* 

tumor tissues, an important pathophysiological phenomenon caused by rapid growth of tumor, leads to the expression of hypoxia-inducible factor (HIF)-1α, which is a key inducible factor for angiogenesis in hypoxia tissues [7, 14]. On the one hand, HIF-1α can induce the expression of hypoxia-response-related genes like NO, VEGF, transforming growth factor (TGF) α and β, adrenomedullin (ADM), LDL-receptor-related protein 1 (LRP1), and leptin; on the other hand, local hypoxia status in tumor tissues also downregulates the expression of antiangiogenic factors such as thrombospondin-1 (TS1) and -2 (TS2) [15–17]. Additionally, growing literature has shown that lots of factors, including genetic or acquired alterations in the oncogenes (i.e., Ras, c-Jun, and Myc) and tumor suppressor genes (i.e., TP53), Hepatitis B Virus X (HBx) protein, chromobox 4, and DNA damage induced by chronic inflammation and AFB1 exposure, can increase the expression proangiogenic factors [18–23]. For example, HBx protein has a potential for increasing HIF-1α expression via promoting transcriptional and translational activity and therefore accelerating angiogenesis during carcinogenesis process of hepatocarcinoma [24]. Recent studies have reported that chromobox 4 (a known transcriptional regulator and also a SUMO E3 enzyme) can promote angiogenesis via stabilizing HIF-1 in hepatocarcinoma [18, 19]. VEGF (including its glycoprotein family members VEGF-A, -B, -C, and -D) is another important angiogenic factor that always upregulates in most cases with hepatocarcinoma [5]. The upregulation of VEGF in hepatocarcinoma is proved not only to increase tumor neovascularization but also to accelerate tumor growth via in vitro cell experiments and animal

models. The role of VEGF is mediated mainly by two receptors: VEGF-R1 (also called Flt-1) and VEGF-R2 (also termed as KDR/Flk-1). Both VEGF-R1 and VEGF-R2 have tyrosine kinase activity and are normally expressed in hepatic parenchyma cells including endothelial cells of portal and sinusoidal tracts [5, 6]. In hepatocarcinoma, both mRNA and protein amount of them are increasing noticeably in the tumor tissues compared to peri-tumor tissues [25]. Some other factors, such as angiopoietin 1 and 2, involve in the regulation of angiogenesis in hepatocarcinoma (**Tables 1** and **2**) [5, 6, 13]. Together, increasing angiogenic potential but decreasing antiangiogenic potential facilitates hepatocarcinoma angiogenesis.
