Vascular Endothelial Growth Factor (VEGF) in Liver Disease

Abstract

Vascular endothelial growth factor (VEGF) is the most potent stimulating factor for angiogenesis. Its expression is related to inflammation and hypoxia. In normal conditions, VEGF is important in the wound healing process. The binding of VEGF with its receptors triggers angiogenesis and lymphangiogenesis and increases vascular permeability. Liver diseases comprise acute and chronic ones. Liver diseases cause inflammation and hypoxia, which increase VEGF level. If they occur chronically, persistent high VEGF levels will promote the risk of chronic liver diseases, including hepatic viral infections, alcoholic and nonalcoholic fatty liver diseases, liver cirrhosis, and finally hepatocellular carcinoma (HCC). High VEGF level is also associated with progressive disease course and poorer outcomes. Tissue remodeling by replacement of normal liver tissue with fibrous tissue occurs. Due to the importance of VEGF in angiogenesis and liver diseases, therapeutic agents targeting VEGF have been developed. Drugs that neutralize VEGF and modulate VEGF receptors have been approved for treating various disorders, including liver disease. Additionally, VEGF is a promising modality for diagnosing liver cirrhosis and HCC. VEGF may also be utilized to predict the outcome of the liver and to monitor the therapeutic response of patients.

1. Introduction

A hypothesis regarding blood vessel growth stimulating factors had been proposed nearly 70 years ago. This was based on the development of organs and diseases. The substance induces vessel growth in positive manner, such as normal retinal vasculature and negative ones, such as tumor cells [1]. In 1989, vascular endothelial growth factor (VEGF) was finally identified, isolated, and cloned [1, 2]. Gene coding human VEGF is located in chromosome 6p21.3. Its consists of 8 exons and is separated by seven introns [3, 4]. This structure makes a high genetic variation to become possible. Approximately 140 variations have been identified and affect the substance itself [4]. There are several subtypes of VEGF, including VEGF-A, VRGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF), with VEGF-A being the most frequently studied one. VEGF-A has isoforms, with the most common ones being VEGF-A₁₂₁, VEGF-A₁₆₅, VEGF-A₁₈₉, and VEGF-A₁₆₅. Each isoform has different heparin-binding ability. When VEGF binds its receptor, angiogenesis activity and vascular permeability are increased [1, 5, 6, 7, 8]. VEFG also acts as an anti-apoptotic factor for endothelial cells, thus enhances angiogenesis [5, 7, 8, 9].

Liver cirrhosis represents the fate of almost all liver diseases. The prevalence of liver cirrhosis is estimated at 0.15% of the total population in USA. However, the exact prevalence is difficult to predict since many cases are asymptomatic. Liver cirrhosis is considered as a precursor for hepatic cellular carcinoma (HCC). HCC is one of the most common solid organ tumors globally [10] and the most common primary malignancy of the liver. It comprises approximately 80% of liver malignant lesions. Over 500,000 new cases are diagnosed annually worldwide. The incidence rate is increasing from time to time. In USA, the incidence had doubled from 1.4 per 100,000 in 1975–1977 to 4.8 per 100.000 in 2005–2007 [11]. Approximately 2 million deaths are recorded annually due to liver diseases. Half of them are caused by complications of liver cirrhosis and the rest is due to viral hepatitis and HCC. Liver cirrhosis and HCC account for 3.5% of global deaths. In developed countries, liver cirrhosis is most commonly caused by alcohol and non-alcoholic fatty liver (NAFLD) while hepatitis B is the most common etiology of liver cirrhosis in China, other Asian, and African countries [10, 11, 12]. Liver cirrhosis and HCC are the third most common cause of death in European countries. The overall 5-year survival is less than 12%. Both conditions also increase the rate of liver transplantation [5, 10, 11]. In USA, chronic liver disease-related hospitalization is constantly increased from 3056 in 2012 to 3757 in 2016 per 100,000 cases with total inpatient hospitalization costs increased from $14.9 billion to $18.8 billion. Among all chronic liver diseases, alcoholic and non-alcoholic fatty liver diseases are dominant with an increasing trend. The presence of liver cirrhosis and HCC further worsens the socioeconomic burden of chronic liver diseases [13].

Liver cirrhosis and HCC progression are associated with angiogenesis. Angiogenesis increases hepatic resistance and the risk of liver failure, leading to manifestations such as gastroesophageal varices, upper gastrointestinal bleeding, ascites, spontaneous bacterial peritonitis, and hepatic encephalopathy. Angiogenesis also plays a critical role in HCC growth and metastases. VEGF is the main pro-angiogenic factor in the liver. Its expression is increased in pathological conditions of the liver. The underlying triggers such as hypoxia, inflammation, and mechanical stress have been proven to increase VEGF levels in liver diseases [2]. In this article, we will discuss VEGF mechanism of action, its role in liver diseases, and its importance in the management of liver diseases.

2. Mechanism of action of VEGF

Hypoxia and inflammation are the most frequent triggers for VEGF production. Inflammation exerts tissue damage and activates endothelial cells. Both conditions triggers VEGF production in concordance with the tissue repair mechanism. Hypoxia itself may trigger VEGF production by the role of hypoxia-inducible factors (HIF). Hypoxia also triggers further inflammation and creates a viscous cycle between inflammation and angiogenesis [14, 15]. VEGF binds to its receptor with the aid of neuropilins as co-receptor and activates tyrosine kinase. There are three subtypes of VEGF receptor and binding of VEGF-A elicits the most potent signaling for angiogenesis (Figure 1). The receptors are found in a wide variety of cell types including endothelial cell, hematopoietic stem cell, monocyte, macrophage, and lymphatic endothelial cell. Tyrosine kinase then activates the signaling pathway through mediators such as phosphatidylinositol kinase, mitogen-activated kinase, and protein kinase C. These mediators promote angiogenesis, lymphangiogenesis, and vascular permeability, accordingly [2, 6, 8, 15, 16]. Nitric oxide is the first substance produced after binding between VEGF and its receptor. The later process increases intracellular calcium, activates calmodulin, and increases NO synthesis. Elevated NO is in line with increased vascular permeability and endothelial cell survival [2, 14]. The extravasation of vascular content including extracellular matrix components marks the initial angiogenesis process. Endothelial cell proliferation, tube formation, and branching of new vessels will occur. When the repair mechanism is completed, angiogenesis will be stopped by the action of inhibitors such as plasminogen activator inhibitors [14]. Overall, angiogenesis is regulated by a balance between stimulating and inhibiting factors [8].

Figure 1.

Binding of VEGF subtypes with VEGF receptor subtypes elicits various processes including angiogenesis. PlGF: Placental growth factor, VEGF: Vascular endothelial growth factor, NP: Neuropilin [6].

3. VEGF and liver disease

Angiogenesis is a process of new blood vessel formation. As blood vessels carry important nutrients to organs and dispose of unnecessary metabolites, angiogenesis plays important homeostatic role [1, 14]. In normal conditions, angiogenesis is important in liver regeneration from several conditions including partial hepatectomy and liver transplantation [5, 17]. This is called physiological angiogenesis and involves liver sinusoidal endothelial cells. The process starts at 48–72 hours after the damage and peaking at 4–5 days. Angiogenesis may occur from pre-existing blood vessels or directly from endothelial cell proliferation [5, 9].

Unregulated angiogenesis causes a negative impact and results in diseases including tumors. Unregulated angiogenesis may result from an imbalance between pro- and anti-angiogenesis. In this situation, VEGF is the culprit. Several abnormalities regarding VEGF coding genes are one of the underlying pathogenesis of the diseases [1, 5, 17]. Baitello et al. conducted a study to determine the role of genetic variations in liver disease, particularly HCC. They observed that VEGF polymorphism C936T and A1154G are associated with elevated VEGF level and incidence of HCC [18]. VEGF promotes angiogenesis and increases vascular permeability. Tissue hypoxia is the major signaling for VEGF expression [1, 5, 17]. In liver, angiogenesis involves hepatic stellate cell (HSC), a specific which plays a central role in tissue remodeling. Prolonged inflammation and tissue damage trigger VEGF expression together with angiogenesis. In angiogenesis, HSC is activated and normal tissue is replaced with fibrous tissue. This impairs tissue oxygenation, cerates hypoxia state, and triggers further inflammation. This cycle should be halted by eliminating any points from the pathway [14].

Elevated VEGF level is proposed in alcoholic liver disease. Luo et al. investigated liver tissue of rats with alcoholic liver disease. They found that mRNA level of VEGF is elevated significantly in liver tissue of rats with the alcoholic liver disease compared to liver tissue of normal rats. A similar finding was reported for mRNA level of HIF. The degree of disease was positively correlated with VEGF and HIF mRNA levels. The trigger of VEGF overexpression, in this case, is different from other liver diseases. In alcoholic liver disease, VEGF overexpression is triggered by leptin that is released from adipocytes [14, 19]. Kasztelan-Szczerbinska et al. confirmed the previous study. The level of plasma VEGF in patients with alcoholic liver disease in their study is significantly higher compared to healthy control [15]. Serum VEGF level may also distinguish between alcoholic liver disease and chronic hepatic viral infections. A higher level was observed in alcoholic liver disease. However, further studies are mandatory before extrapolating this result in general population [20]. Similar to nonalcoholic fatty liver disease (NAFLD), the expression of VEGF is up-regulated by a different pathway. Leptin as an adipocytokine plays a central role in promoting VEGF and other pro-inflammatory cytokines expression. VEGF expression is elevated through the recruitment and stabilization of HIF by leptin. This leads to angiogenesis and fibrogenesis, and progression from NAFLD to non-alcoholic steatohepatitis (NASH) [14, 17]. The severity of steatosis in NASH is associated positively with VEGF level [17].

Pathological angiogenesis has been observed in chronic liver diseases for a long period of time. This phenomenon is observed in chronic hepatitis B and C, autoimmune hepatitis, and primary biliary cirrhosis. The damage suffered by the liver triggers inflammation and initiates the wound healing process with increased expression of several growth factors including VEGF. Elevated VEGF level promotes angiogenesis then angiogenesis leads to fibrosis and liver tissue remodeling distinctive of liver cirrhosis. The latter process involves hepatic stellate cells which produce an extracellular matrix. If the damage occurs chronically, high VEGF expression also becomes chronic, followed by chronic angiogenesis and fibrogenesis. Hypoxia resulted from extensive fibrogenesis further increases VEGF expression as stated above, which is mediated by HIF. Lately, it is found that not only VEGF level is increased but also VEGF receptor [5, 14, 17]. Hepatitis B virus itself surprisingly can induce VEGF release without the presence of inflammation and hypoxia state. The positive correlation is reported between serum VEGF level and severity of chronic liver diseases [14].

A study by Franchitto et al. supports the previous facts. Patients with chronic viral hepatitis and primary biliary cirrhosis have abundant hepatic progenitor cells in their liver. Furthermore, VEGF and its receptor’s expression is increased in those progenitor cells. The number of progenitor cells expressing VEGF is correlated with angiogenesis, fibrogenesis, and carcinogenesis in subjects in their study [21]. VEGF is level not only elevated in primary liver disease but also in diseases with liver complications. Nihei, et al. conducted a study in children with Kawasaki disease. They found that inflammatory growth factors are elevated in all patients. More than half of the patients in their study had liver dysfunction as a complication from Kawasaki disease and VEGF was significantly elevated in patients with liver dysfunction compared to those without liver dysfunction [22].

Massive formation of portosystemic collateral vessels particularly in the esophagus and gut is the underlying pathogenesis of variceal bleeding. Collateral vessels shunt blood from portal to systemic circulation and cause substances that are normally detoxified by the liver to enter the systemic circulation. This leads to encephalopathy and sepsis in patients with liver disease. VEGF also contributes to portal hypertension. Angiogenesis increases blood flow in splanchnic organs draining into the portal vein and further increases portal venous flow. Nitic oxide furtherly enhances vasodilatation and blood flow. VEGF is known to promote nitric oxide level [5, 14, 17, 23]. Tissue remodeling also increases liver tissue resistance and ends with portal hypertension [14]. An animal study conducted by Huang et al. shows that rats with portal hypertension have increased VEGF expression as high as 40% compared to healthy rats as control. Portal pressure was also positively correlated with VEGF level [23]. Spider angiomas also result from elevated VEGF level. A study proved that subjects with liver cirrhosis and spider angiomas have higher plasma VEGF level compared to liver cirrhotic patients without spider angiomas [24].

Liver cirrhosis is the end-point of chronic liver disease and predisposing lesion to HCC. Chronic damage to liver maintains a high VEGF level over time and is associated with continuous angiogenesis and fibrogenesis. In the end, liver tissue is replaced by abnormal fibrous tissue [12]. Li et al. reported that plasma VEGF level is elevated significantly in liver cirrhotic patients compared to control group [24]. Abdelmoaty et al. also conducted a study regarding serum VEGF level in patients with liver cirrhosis. Serum VEGF level was significantly increased in patients with liver cirrhosis compared to healthy individuals. This result is in line with the result from previous study. Serum VEGF level was also positively related to degree of liver dysfunction based on Child-pugh score [25].

In cancers, increased expression of VEGF is positively associated with its growth and risk of metastases but negatively associated with the outcome of disease. VEGF triggers angiogenesis and angiogenesis itself nurtures the cancer cells [1, 6, 7, 8]. HCC is a highly vascularized cancer thus its progression and outcome are closely related to angiogenesis [5, 21, 26, 27]. Additionally, VEGF acts in an autocrine fashion in HCC. A study by Sharma et al. showed that both VEGF and its receptor expressions are elevated in HCC cell lines. This marks the ability of cancer tissue to grow independently from normal angiogenesis pathway [28]. The high angiogenesis activity in HCC is suspected due to increased oxygen demand by cancer cells during their growth trigger hypoxia state. Hypoxia further increases pro-angiogenesis factors including VEGF. VEGF has a good discrimination ability between HCC and chronic liver diseases. Therefore, it can be utilized as one of the diagnostic modality to detect HCC at its early stage [8]. Li et al. conducted a study in patients with HCC, benign liver lesions, and normal controls. The result showed that plasma VEGF level in HCC patients is significantly elevated compared to patients with benign liver lesions and normal subjects. In HCC group itself, subjects with large tumor size, distant metastasis, portal vein thrombosis, and arterial-portal vein shunting had higher plasma VEGF level compared to their counterparts [29]. The above result is confirmed by Zhang et al. In their study, plasma VEGF level was higher in HCC patients with multiple lesions, lesion larger than 5 cm, bilobar tumor distribution, and metastasized cancer [30]. In contrast, Uematsu et al. found different results in their study. Serum VEGF level was increased in patients with HCC and significantly higher compared to healthy volunteers but the difference was not significant if being compared with liver cirrhosis [27].

4. VEGF and management of liver disease

As HCC possesses high morbidity and mortality rates, diagnosis at its early stage is important to improve the patient’s outcome. Hamdy et al. reported that VEGF is a promising diagnostic modality for HCC from their study. A VEGF cut off point of ≥280 pg./mL has sensitivity of 60.27% and specificity of 100% in discriminating HCC and chronic liver diseases from healthy subjects while a cutoff point of ≥482 pg./mL has sensitivity of 52.59% and specificity of 100% in discriminating HCC from chronic liver diseases [26]. Mukozu et al. in their study also proposed VEGF as novel marker for HCC diagnosis in patients with chronic hepatitis C virus infection. They reported that serum VEGF is better compared to alpha-fetoprotein in discriminating between HCC and liver cirrhosis. The sensitivity and specificity of VEGF were reported to be 98% and 46%, respectively. The values were obtained with a VEGF cutoff of 108 pg./mL [31]. Jinno et al. supported the previous findings. They proved that plasma VEGF level in subjects with HCC is significantly higher compared to healthy control, subjects with chronic hepatitis, and subjects with liver cirrhosis. Furthermore, plasma VEGF level in stage IV-B HCC patients was significantly higher among all stage groups. This implies that besides diagnosing HCC, VEGF is also useful in diagnosing metastasized HCC [32]. Another study from Japan reported concordance results. Serum VEGF level is higher in advanced HCC such as stage IV-B disease, giant and multinodular lesion, and distant metastasized disease [20].

Considering the role of VEGF in liver diseases, management focusing on VEGF manipulation has become popular [1, 5, 33]. Judah Folkman had hypothesized a strategy for managing cancers and other diseases with anti-angiogenesis [1]. The strategy comprises VEGF, its receptors, and it signaling pathways interventions. Nowadays, there are drugs targeting VEGF such as bevacizumab, ziv-aflibercept, rapamycin, and ramucirumab [1, 2, 5, 6, 7]. Bevacizumab and ramucirumab are neutralizing antibodies to VEGF. Approved in 2004, bevacizumab has become the most widely used anti-VEGF in the field of oncology. Ziv-aflibercept is soluble VEGF receptor that prevents the binding of VEGF with its natural receptor [1, 2, 6, 7].

Other agents such as tyrosine kinase receptor inhibitors (sunitinib, sorafenib, and imatinib) have been approved as therapeutic agents [5, 7, 33]. Among all, sorafenib which was developed in 1990 has become the most commonly used agent for HCC treatment [33]. The list of anti-angiogenic agents may be observed in Table 1 [2]. In vivo studies proved that the agent may decrease pathologic angiogenesis as high as 52% in patients with liver diseases. Combination with other anti-angiogenesis agent is also urged and shows better outcome in patients. Platelet-derived growth factor (PDGF) signaling inhibitor is one of the treatment modality in the combination regime [5].

Agents	Mechanism of action	Approved by FDA
Bevacizumab, ramucirumab	Monoclonal antibody against VEGF	Yes
Ziv-aflibercept	Decoy VEGF receptor	Yes
Sorafenib, sunitinib, apatinib, axitinib, cabozantinib, lenvatinib, nintedanib, pazopanib, regorafinib, imantinib	Tyrosine kinase inhibitor	Yes
Cediranib, lucitanib, semaxanib, tivozanib	Tyrosine kinase inhibitor	No

Table 1.

List of anti-angiogenesis agents and their mechanism of action [2, 6].

FDA: Food and Drug Administration, VEGF: vascular endothelial growth factor.

Single anti-angiogenesis therapy is effective in several cancers including HCC in the advanced stage [7]. Some side effects should be put in consideration when administering anti-angiogenesis therapy. Hypertension, renal dysfunction, proteinuria, thrombosis, bleeding, and arrhythmia are the most common side effects reported. Hypertension is the most common side effect, occurring in 25% of patients treated with anti-angiogenesis. This is strongly related with decreased NO level due to anti-angiogenesis agents. Similar mechanisms underlie further side effects [2, 6]. Resistance against anti-angiogenesis therapy is another threatening problem even though this phenomenon has not been proven consistently. However, long-term follow-up showed the tendency of growing resistance to this treatment [6].

Serum VEGF level is also useful in monitoring a patient’s response toward therapies. Matsui et al. measured serum VEGF level in patients with HCC receiving chemotherapy. The chemotherapeutic agents used were leucovorin, cisplatin, and 5-fluorouracil. The results showed that serum VEGF level is higher in patients with partial response or stable disease compared to progressive disease [20]. A similar result is reported by Li et al. Even though the treatment modality in their study was different (transcatheter arterial chemoembolization/TACE), the result showed that patients with high pre-therapeutic plasma VEGF level are associated with poor response to treatment [29]. Plasma VEGF level is suggested to be a modality for monitoring prognosis after liver transplantation in HCC cases. A plasma VEGF level of >44 pg/mL is associated with worse overall and disease-free survival. Additionally, it is also associated with higher disease recurrence and poorer disease outcomes [30]. However, an anomaly was submitted by Shigesawa et al. They observed HCC patients receiving anti-angiogenesis agent for 8 weeks and found that serum VEGF level is significantly lower in patients who experienced deterioration compared to those without deterioration [34]. Ramadan et al. found similar result with Shigesawa et al. Patients with hepatitis C virus-associated HCC had higher VEGF level after receiving treatments compared to those untreated ones. The recurrence rate became higher in line with elevated VEGF level [16]. These findings raise suspicion regarding the possibility of treatment resistance.

5. Conclusions

Liver diseases are conditions that may occur both acutely or chronically. Liver cirrhosis and HCC are the end-points of chronic liver diseases which carry heavy socioeconomic burden. Angiogenesis plays a significant role in liver diseases, including alcoholic fatty liver disease, NAFLD, chronic hepatic viral infections, and their progressions. The most potent mediator for angiogenesis is VEGF. A high level of VEGF is associated with an increased incidence of liver disease and a worse clinical course. Inflammation and hypoxia from chronic liver diseases are the triggering factors for VEGF release. The binding of VEGF with its receptors triggers angiogenesis, lymphangiogenesis, and vascular permeability increment. If occur for a long period, liver tissue remodeling is observed as a precursor lesion of HCC. Due to the importance of angiogenesis, anti-angiogenesis therapy targeting VEGF is becoming popular. Several agents that neutralize VEGF and modulate its receptors have been approved to treat various diseases. Besides, VEGF is also a promising modality for the diagnosis of liver diseases and for predicting disease outcomes. The therapeutic response of patients may also be monitored using VEGF level.

Conflict of interest

The authors declare no conflict of interest.