Vascular interference: A blockade to tumor epithelial growth
Authors: Susan E. Crawford
Journal: Hepatology (2004)

In this issue of HEPATOLOGY, Yoshiji et al. postulate that vascular endothelial growth factor (VEGF) drives the growth of hepatic tumors by stimulating angiogenesis.1 They demonstrate that antibodies directed against VEGF receptor type 1(VEGFR-1) and type 2 (VEGFR-2), in combination, inhibit the development of adenomas and hepatocellular carcinoma (HCC) in mice by inducing apoptosis of endothelial cells. Even more striking was the ability of this therapeutic regimen to block HCC lung metastases. VEGFR-2, the receptor linked to endothelial mitogenicity, had higher therapeutic efficacy than VEGFR-1 alone. These important findings point to VEGF receptor blockade as an effective strategy to disrupt the paracrine activities of endothelial cells and tumor epithelial cells in HCC growth and metastases. VEGF is one of the most widely studied proangiogenic mediators in human cancer.2-4 In many tumors, including HCCs, increased circulating and intratumoral levels of VEGF are associated with less favorable clinical outcomes.5-7 The ability of VEGF to support the vascular network of tumors has prompted the development of pharmacological interventions aimed at blocking VEGF protein, its receptors, VEGFR-1 (flt-1) or VEGFR-2 (FLK-1/KDR), or by interfering with its tyrosine kinase activity.5, 8 Monoclonal antibodies directed against VEGF have undergone Phase III clinical trials with mixed tumor responses, while several receptor tyrosine kinase inhibitors appear to be hampered by a spectrum of unexpected side effects.8 Given the importance of VEGF in tumor growth, results of Yoshiji et al.1 strongly suggest that antibodies targeting the VEGF receptors will reign as effective therapeutic agents with potent antitumor activity to combat HCC. VEGF and its receptors play important roles in vasculogenesis and physiological angiogenesis of developing organ systems through endothelial cell-dependent and independent mechanisms.5, 9, 10 Once development halts in most organs, the adult vasculature is maintained in a quiescent state by the presence of angiogenic inhibitors within the tissue matrix.2, 3, 11 The switch to a proangiogenic state in a tumor entails alterations in oncogenes or tumor suppressors in conjunction with the gain of vascular stimulators, the loss of endogenous inhibitors of angiogenesis, or, as observed in many cases, concurrent changes in each pool of angiogenic factors.2, 3, 11 Most HCC studies have focused on the imbalance triggered by the gain of stimulators, although data are emerging to suggest that loss of endogenous inhibitors can contribute to dysregulated angiogenesis. Normal hepatocytes express endostatin precursor molecule collagen XVIII, and its level diminishes with HCC tumor progression12 and treatment with adenoviral vectors encoding inhibitor endostatin. angiostatin-like molecule, or pigment epithelium-derived factor, suppressed experimental HCC growth in vivo.13-15 In HCC tumor tissue, antiangiogenic thrombospondin localized to rough endoplasmic reticulum of HCC cells, endothelial cells and fibroblasts16; however, the absence of data regarding expression of inducers makes it difficult to know if the pool of inducers were still able to overwhelm thrombospondin's antiangiogenic activity. When circulating endostatin levels were measured in some patients with HCC, rising levels correlated with higher VEGF and increased tumor microvascular density, suggesting that the inducers successfully overcame the inhibitory activity.17 Clearly, the most accurate determination of net angiogenic activity in any organ system requires evaluation of both inducers and inhibitors of neovascularization using functional angiogenesis assays. VEGF, vascular endothelial growth factor; HCC, hepatocellular carcinoma. Unlike most adult organ systems, the liver retains is unique ability to regenerate, given the appropriate stimulus. Regeneration of the liver is an example where cross-talk between endothelial cells and hepatocytes must be carefully orchestrated through regulation of angiogenic and proliferative signaling pathways. If one pathway falters due to the gain of a stimulator or the loss of an inhibitory mechanism, uncontrolled growth of the liver could ensue. Partial hepatectomy is a potent stimulator of regeneration and hepatocyte proliferation by altering cell cycle proteins, transcription factors, and angiogenic inducers such as VEGF.18-20 Posthepatectomy, endothelial cells appear to be early participants in the regeneration process, since VEGF levels peak nearly 14 hours prior to maximal levels of DNA synthesis.21 Hepatocyte proliferation is coincident with release of growth factors such as hepatocyte growth factor (HGF) and induction of immediate early transcription factors like Stat3.20 Other functions have been attributed to these molecules related to angiogenesis. HGF, itself is a potent angiogenic factor,22 while its antagonist, HGF/NK4 is capable of blocking VEGF-induced angiogenesis using in vitro and in vivo assays.23 Stat3 protein binds to the VEGF promoter in vivo and constitutively activated Stat3-induced VEGF expression and angiogenesis in culture.24 The use of the regenerating liver has led to the discovery of many important molecules involved in hepatic carcinogenesis, many of which could have gone unrecognized if studies were limited to the quiescent state. A tumor-permissive environment is a prerequisite for sustained tumor growth. Once tumor cells overcome the endogenous inhibitory barriers in an organ, tumor progression is highly dependent on the interaction between tumor epithelial cells, endothelial cells, and their surrounding extracellular matrix. An imbalance in any one compartment can promote tumor growth and metastases.2, 3, 25 When matrix deposition is in excess in human or rodent livers, the frequency of HCC increases dramatically. Matrix disturbances coupled with tumor cell–derived VEGF have been shown to participate in early development of experimental HCC. Human HCC tumors also rely on VEGF, as Flt-1 messenger RNA (mRNA) has been shown to positively correlate with tumor VEGF mRNA.26 Epithelial-targeted factors such as HGF, a mitogen for hepatocytes, can also modulate the vascular bed. When HGF was overexpressed in hepatocytes under a metallothionein promotor, the frequency of liver tumors accelerated.27 These findings accompanied a parallel increase in VEGF expression and angiogenesis, thus supporting other studies demonstrating an intimate link between epithelial cell growth and the adjacent vasculature.10, 26 Hepatic stellate cells are hypoxia sensitive and best known for their role in modulation of the matrix through secretion of matrix metalloproteinases and pro-fibrogenic proteins, such as TGF-beta.28 Turnover of the extracellular matrix involves the balance of metalloproteinases and their inhibitors.28 Matrix degradation is one of the first critical steps in stimulating physiological or pathological angiogenesis. In the liver, vascularization of the matrix may be an early marker of dysregulated angiogenesis. In support of this concept is the finding that VEGF expression has been found to be highly expressed in both tumor and non-tumor-bearing regions of the same liver. Moreover, there is histological evidence of marked neovascularization within the broad bands of fibrous tissue encircling both benign regenerative nodules (Fig. 1) and in HCC (Fig. 2A). In some cases, the matrix angiogenesis can exceed intratumoral vessel density (Fig. 2A). These data suggest that acquisition of proangiogenic activity in HCC may require recruitment of the matrix and its infiltrating vessels. Another epigenetic mechanism responsible for modulating angiogenesis and tumor development is hypoxia. Hypoxic conditions associated with tumor growth can have profound effects on nonparenchymal cells. Similar to tumor cells, activated hepatic stellate cells exposed to hypoxia show a rapid induction of VEGF mRNA and protein and preferential stimulation of Flt-1 mRNA.29 In some instances, VEGF induction is mediated through HIF-1 alpha and COX-2. These observations, taken together, suggest that the hepatic stellate cells cannot be regarded as innocent bystanders in tumor-related angiogenesis. Their juxtaposition to proliferating tumor cells (Fig. 2B) make them likely candidates to modulate, directly or indirectly, the net angiogenic activity in the liver by secreting inducers and stimulators of endothelial cells.30 Microscopic section of human liver demonstrating regenerative nodules with focal cellular atypia. Broad bands of fibrous tissue (*) encase the nodules associated with a mononuclear cell infiltrate. Within the fibrous tissue, there are numerous endothelial cell-lined vessels (arrows) that are highlighted by a brown stain (immunohistochemical stain using an antibody directed against Factor VIII–related antigen; 10×). RN, regenerative nodules. F,. (A) Histological evaluation of the hepatocellular carcinoma tumor-matrix interface reveals brisk neovascularization (arrows) within the matrix. In many sections of the tumor, the matrixassociated microvascular density (a hallmark of angiogenesis) was greater than the intratumoral vascular density (immunohistochemical stain using an antibody directed against Factor VIII–related antigen, 20×).(B) Microscopic section of a human hepatocellular carcinoma demonstrating a dense band of activated stellate cells encircling the tumor nodule (immunohistochemical stain using an antibody directed against smooth muscle-specific actin, 20×). S, stellate cells; T, tumor nodule. The importance of endothelial cells and VEGF in modulating hepatocyte growth was underscored in a compelling study by N. Ferrara's laboratory.31 Liver weight clearly increased in mice receiving VEGF-A; however, isolated hepatocytes failed to grow unless liver sinusoidal endothelial cells were included in the assay, supporting the codependency of these two cell types. Moreover, in vivo studies highlighted the cellular specificity of VEGF-R1 and VEGF-R2. VEGF-R1 induced a significant increase in hepatocyte proliferation, whereas VEGF-R2 stimulated sinusoidal endothelial cell proliferation,31 thereby confirming earlier work where only mutants lacking KDR affinity effectively blocked endothelial cell mitogenesis.5 The current study by Yoshiji et al. highlights a similar disparity in the ability of the two receptors to block liver tumor growth in vivo. Blockade with VEGF-R2 was consistently more effective in diminishing tumor number and size, and was associated with a more significant decrease in CD31 mRNA, an endothelial cell marker. The results advocate the use of combination therapy in the treatment of experimental HCC and emphasize the power of vascular interference to disable the tumor epithelial cell. The author thanks Dr. Veronica Stellmach for her insightful discussions.