What we do …

Angiogenesis

Angiogenesis refers to new microvessel formation via sprouting or splitting from preexisting vessels. Upon exposure to proangiogenic signals like vascular endothelial growth factor (VEGF), endothelial cells activate, become mobile and protrude filopodia, forming tip cells, which initiate new sprouts. Stalk cells follow tip cells, proliferate to support sprout elongation, and establish a vessel lumen. Microvessel loops are then formed when tip cells anastomose with neighbouring sprouts. After the microvessel forms a lumen, endothelial cells secrete growth factors, such as platelet-derived growth factor (PDGF), to attract pericytes and smooth muscle cells that stabilize the nascent vessel. Endothelial cells and pericytes become sensitized to the chemotactic and proliferative effects of the various growth factors by upregulating their receptors, predominantly VEGF receptor-2 and PDGF receptor-beta.

Angiogenesis in liver disease

Abnormal neovessel formation represents a critical and clinically important hallmark feature of chronic liver diseases, such as liver cirrhosis and liver cancer, for which few therapeutic options are available. Recognition of the importance of angiogenesis in the pathophysiology of chronic liver disease is relatively recent, and was given particular impetus by the observation that the proangiogenic growth factor VEGF and its signaling pathway is switched on and promotes an extensive VEGF-driven neovascularization in the mesenteric vascular bed during liver cirrhosis, thereby contributing to the development of increased splanchnic blood flow (Read More), working in a cooperative fashion with splanchnic vasodilatation, and aggravating portal hypertension (Read More). Pathological angiogenesis is also behind the establishment and maintenance of the abnormal angioarchitecture distinctive of the cirrhotic liver, being functionally linked with fibrogenesis and inflammation (Read More). Accordingly, diminishing pathological angiogenesis has multiple beneficial effects in chronic liver disease (Read More).

Angiogenesis, collaterals and varices

Importantly, VEGF-dependent angiogenesis also plays a crucial part in the formation of portosystemic collateral vessels and gastroesophageal varices (Read More). These varices are fragile and particularly prone to leak blood and even rupture, causing upper gastrointestinal tract bleeding. This hemorrhage is often torrential and difficult to staunch, and, despite many advances made in this field, it continues to be the cause of significant morbidity and mortality in patients. Furthermore, because portosystemic venous shunts bypass the liver, noxious substances that are normally metabolized by the liver can escape from collaterals to the central venous system, leading to other potentially lethal consequences, such as portosystemic encephalopathy, spontaneous bacterial peritonitis or systemic infections.

Angiogenesis is not always a bad thing

Therapeutic targeting of angiogenesis has been proposed as a promising strategy for liver disease. However, the clinical benefit of antiangiogenic drugs is restricted because of significant adverse effects, including collapse of normal vasculature, vascular leakage and bleeding, as exemplified in patients with hepatocellular carcinoma and cirrhosis. Most of these limitations arise from the fact that current anti-VEGF approaches are not selective for pathological VEGF production, but instead inhibit also physiological VEGF required for vascular homeostasis of healthy vessels and formation of vascular supply in many physiological settings. Deciphering mechanisms that regulate VEGF expression is therefore critical to achieve specific inhibition of pathological generation of VEGF without affecting its physiological production. This is a major goal of our research.

CPEB proteins regulate pathologic VEGF expression and angiogenesis

In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we have investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of liver disease. We have identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and non-redundant functions of CPEB1 and CPEB4. Activation of CPEB1 promotes alternative nuclear processing within non-coding 3′-untranslated regions of VEGF and CPEB4 mRNAs, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promotes cytoplasmic polyadenylation of VEGF mRNA, increasing its translation and generating high levels of VEGF protein, which induces pathologic angiogenesis in chronic liver disease (Read More).

CPEB proteins as targets for therapy

From a translational point of view, our studies highlight that CPEBs could be promising angiogenesis-disrupting targets in disease. Thus, targeting CPEBs could lead to safer treatment outcomes by specifically reducing excessive pathological VEGF production instead of indiscriminately perturbing both pathological and physiological VEGF synthesis, minimizing potential adverse side-effects. Reduction of pathological angiogenesis in early disease stages could also prevent further disease progression and reduce the risk for developing overt liver cirrhosis. Accordingly, development and evaluation of CPEB inhibitors is currently underway. As better and more specific inhibitors of pathologic angiogenesis are developed, combination strategies continue to evolve, and increased understanding of the complex biology of angiogenesis takes place, antiangiogenic therapy will certainly be evaluated in future clinical trials (Read More).

Vascular stem cells, new culprits uncovered 

Recent studies from our lab highlight the functional significance of pathologic neovascularization derived from vascular stem/progenitor cells as an important mechanism of formation of new blood vessels in adults, in the setting of chronic liver disease, and identifies these stem cells as potential new therapeutic targets. Thus, we have demonstrated the existence in the vascular wall of adult mesenteric blood vessels of a distinctive population of vascular stem/progenitor cells. These cells display some of the most widely accepted criteria for stem cell recognition, including quiescence and slow-cycling properties, high proliferative potentiality, capability of growing as cellular spheres in suspension culture, expression of specific biomarkers of stem cells, and the ability to self-renew and generate daughter cells in response to liver disease induction. This vascular stem cell progeny is able to differentiate into endothelial and smooth muscle cell lineages, and readily contribute physically and functionally to neovascularization in vivo during chronic liver disease. Hence, chronic liver disease-associated abnormal neovascularization might conceivably be a heterogeneous process, arising through a combination of both neoangiogenesis and neovasculogenesis. Accordingly, therapeutic targeting of both vascular stem cell-derived neovascularization (vasculogenesis) and new vessel growth mechanisms that utilize non-stem cell constituents (angiogenesis) may effectively block abnormal neovessel formation and improve antiangiogenic therapeutics (Read More).

Liver cancer

Liver cancer is among the most lethal and prevalent cancers worldwide, and the second most common cause for cancer-related death. In contrast to other cancers, its incidence is rising dramatically, and is predicted to increase even more in the coming years mainly due to the global epidemic of obesity/overweight, which has become a major socioeconomic challenge and strong risk factor for liver cancer. The worst is that obesity does not only affect adults but also children. There is also an urgent need for development of novel therapeutic strategies to treat liver cancer. And for that it is essential to decipher mechanisms underpinning the detrimental impact of obesity on the progression of chronic liver disease, from simple steatosis to fibrosis and liver cancer, which are mostly still unknown. This is a major goal of our research.

Transition to liver cancer

To address our goal, we use transdisciplinary approaches and work collaboratively with other internationally reknown groups towards a common purpose: advance fundamental understanding on liver cancer (with a focus not only on the tumor but also on the dynamic interplay tumor-microenvironment, and not only on cancer itself but also on all disease stages preceding and leading to tumorigenesis), and develop new diagnostic and therapeutic tools, which are critical for science, health and economic and social welfare. In this regard, we have recently discovered that the RNA binding protein CPEB4 protects against overnutrition-associated fatty liver by regulating a new adaptive branch of the unfolded protein response that helps to re-establish cell equilibrium. Accordingly, in absence of CPEB4, the liver becomes hypersensitive to steatosis due to excess high-fat diet consumption. Published in Nature Cell Biology.

New therapeutic opportunities for liver disease

What is new about this study? This study describes an innovative therapeutic strategy for treatment of one of the most clinically relevant complications of chronic liver disease, the formation of portosystemic collateral vessels and gastroesophageal varices. This treatment is based on the short interfering RNA (siRNA) technology, which has emerged as one of the most promising platforms for therapeutic product development. In particular, in collaboration with Silence Therapeutics, we have developed a novel and effective siRNA delivery system (siRNA^KDR-lipoplexes), based on clinical stage components, to efficiently and specifically target the kinase insert domain receptor KDR in vascular endothelial cells in vivo after systemic intravenous administration. The KDR receptor plays a key role in portosystemic collateralization and contributes to disease progression and aggravation, as we have previously shown, making it an attractive therapeutic target.

What is the key finding? Our results demonstrate that therapy with siRNA^KDR-lipoplexes markedly ameliorates the development of portosystemic collateral vessels and impairs the pathological angiogenic potential of endothelial cells in a murine model of portal hypertension (a syndrome that occurs in chronic liver disease). Our findings also demonstrate that the mechanisms underlying the decrease in collateralization after KDR knockdown include reduced endothelial cell proliferation and decreased angiogenesis and vascular remodeling.

Which could hopefully be the future impact of this work? This treatment could be a promising and plausible therapeutic modality for attenuating the formation of portosystemic collateral vessels in a clinical setting. Of note, this therapeutic intervention may potentially prevent the formation of large varices from small varices. This is important because new collaterals and varices develop each year during the evolution of chronic liver disease, and, currently, no treatment can prevent this development. Of interest, a related type of formulation developed by Silence Therapeutics has been shown to be clinically acceptable for systemic administration of siRNAs and is currently successfully tested in several clinical trials in patients with advanced solid tumors, further supporting the translational relevance and therapeutic potential of this approach for chronic liver disease in a clinical context. Given the emerging roles of angiogenesis in a number of human pathologies, including inflammation, obesity and tumor growth, siRNA^KDR-lipoplexes may also provide a novel strategy to treat a wide spectrum of diseases.

Pigment epithelium-derived factor, protective factor with therapeutic potential

Pigment epithelium derived factor (PEDF) is one of the strongest natural inhibitors of pathological angiogenesis known to date, and this property has turned it into a promising therapeutic tool for slowing the progression of many neovascular pathologies. Recent studies from our research group demonstrate that both the negative regulator of angiogenesis PEDF and the angiogenesis inducer VEGF are unidirectionally upregulated in mesentery and liver in experimental models of cirrhosis, being closely linked in time and space with mesenteric neovascularization and liver fibrogenesis, suggesting that PEDF induction might reflect a compensatory mechanism aimed at reducing the adverse effects of VEGF. Exogenous PEDF overexpression by adenovirus-mediated gene transfer moves the balance between VEGF and PEDF in favor of inhibition and angiogenesis, translating into portal pressure decrease and partial correction of excessive angiogenesis in experimental cirrhosis. Therefore, exogenous PEDF supplementation could be a promising and plausible therapeutic modality for preventing further disease progression and consequently reducing the risk for developing overt liver cirrhosis in patients. Of note, targeting early pathogenic changes might afford an opportunity to intervene on chronic liver disease at a stage before irreversible fibrosis. Another prominent advantage of using angioinhibitors that are endogenously present in the body, such as PEDF, is that these molecules would not be expected to activate drug resistant genes and, thus, may offer a promising breakthrough for effective antiangiogenesis therapy (Read More).

Vasohibin, new feedback inhibitor of angiogenesis

Vasohibin-1 is a recently identified endogenous inhibitor of angiogenesis that is selectively induced by the proangiogenic growth factor VEGF as a consequence of a specific negative-feedback regulator mechanism of pathological angiogenesis in cirrhosis. As we have seen, ectopical, and non-VEGF regulated, vasohibin-1 overexpression by adenoviral-mediated gene transfer leads to disruption of the VEGF-vasohibin-1 negative-feedback loop, dampening VEGF production to intermediate steady-state levels sufficient to maintain vascular homeostasis or physiological angiogenesis associated with wound healing, but not to drive pathological angiogenesis. This effect translated into efficient suppression of mesenteric and intrahepatic pathological neovascularization and reduction of portal pressure and portosystemic collateral vessel formation in cirrhotic rats, without any apparent adverse side effect. The protective action of overexpressing VASH1 protein in vivo by gene therapy with an adenoviral vector encoding VASH1 was not restricted to inhibition of angiogenesis, but included a potent ability to attenuate intrahepatic fibrogenesis through suppression of hepatic stellate cell activation. These results strongly suggest that supplementation with VASH1 might be a novel and promising therapeutic strategy for halting chronic liver disease progression (Read More).