Current Pharmaceutical Design - Volume 14, Issue 36, 2008

In this decade, novel strategy against cancer, antiangiogeneic therapy, was attempted. A major microenvironmental event in tumor growth and expansion is the ‘angiogenic switch’, an alteration in the balance of pro-angiogenic and anti-angiogenic molecules that leads to tumor neovascularization. Angiogenesis, a process by which new vascular networks are formed from pre-existing capillaries or circulating endothelial ceslls, is required for tumors to grow, invade and metastasize. Tumor vessels are genetically quite stable, and less likely to accumulate mutations that allow them to develop drug resistance in a rapid manner. Therefore, targeting vasculature that supports tumor growth, rather than cancer cells themselves, is considered the most promising approach to cancer therapy. In this issue, we describe the possible antiangiogenesis basis of this therapeutic strategy. Circulating endothelial cell (CEC) and progenitor (CEP) number and viability are modulated in various pathological conditions including cancer. Martin-Padura and Bertolini [1] described CEC and CEP play an important role in cancer progression and metastasis. Indeed, emerging clinical data support that CEC and CEP kinetics and viability might predict the efficacy on anticancer drug combinations that include antiangiogenic agents. On the basis of these observations, CEC and CEP measurements have attractive potential diagnostic and therapeutic applications for malignant diseases. Among ‘angiogeneic cytokine’, macrophage migration inhibitor factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects that has been implicated in tumor growth and progression. Bifulco et al. [2] reviewed MIF's function in multiple processes fundamental to tumorigenesis such as tumor proliferation, evasion of apoptosis, angiogenesis and invasion. These pleiotropic functional aspects are paralleled by MIF's unique signaling properties, which involve activation of the ERK-1/2 and AKT pathways. These properties reflect features central to growth regulation, apoptosis and cell cycle control than is typical for an immune cytokine. The significance of these pro-tumorigenic properties has found support in several in vitro and in vivo models of cancer and in the positive association between MIF production and tumor aggressiveness and metastatic potential in a variety of human tumors. Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, and is involved in the pathogenesis of angiogenic eye disease such as proliferative diabetic retinopathy. Abe et al. [3] reviewed a functional role for PEDF in tumor growth and angiogenesis. Recent studies reported the antitumor potential of PEDF in various cancer based on its antiangiogenic properties and PEDF direct inhibitory effect via tumor cell apoptosis. The discovery and evaluation of antiangiogenic substances initially relied on methods such as various models that use the cornea to assess blood vessel growth. Although they are important for understanding the mechanisms of blood vessel induction, these models do not represent tumor angiogenesis and are poorly suited to drug discovery. Amoh et al. [4] have utilized multicolored fluorescent proteins to develop imaging models of tumor angiogenesis, which are clinically-relevant imageable models to visualize and quantify angiogenesis and efficacy of inhibitors. Angiogenesis is a complex process which is critical for the growth, invasion, and metastasis of tumors. Fujita et al. [5] reviewed recent progress in this clinical filed. In the past ten years numerous new agents have been developed as angiogenesis inhibitors. Angiogenesis inhibitors can be classified by their targeted area of the angiogenic process; (1) VEGF and its receptors VEGFR (e.g. Bevacizumab); (2) tyrosine kinases within endothelial cells (Sunitinib); (3) proliferation of endothelial cells (Endostatin); (4) MMPs (Marimastat); (5) intercellular interactions via integrins (Cilengitide) and (6) combinations of the above mechanisms (Thalidomide). Some show anti-tumor effects with objective responses and stable disease, and some disappeared from clinical use due to unexpected side effects or insufficient efficacies. Further investigations of combined therapies including angiogenesis inhibitors will shed light on the treatment of advanced and metastasized malignancies.

Circulating endothelial cell (CEC) and progenitor (CEP) number and viability are modulated in various pathological conditions including cancer. There is increasing evidence showing that CEC and CEP play a role in cancer progression and metastasis in different animal models. At the clinical level, emerging data support that CEC and CEP kinetics and viability might predict the efficacy on anticancer drug combinations that include antiangiogenic agents. On the basis of these observations, CEC and CEP measurements have attractive potential diagnostic and therapeutic applications for malignant diseases.

Macrophage migration inhibitor factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects that has been implicated in tumor growth and progression. MIF's function is unique among cytokines and its effects extend to multiple processes fundamental to tumorigenesis such as tumor proliferation, evasion of apoptosis, angiogenesis and invasion. These pleiotropic functional aspects are paralleled by MIF's unique signaling properties, which involve activation of the ERK-1/2 and AKT pathways and the regulation of JAB1, p53, SCF ubiquitin ligases and HIF-1. These properties reflect features central to growth regulation, apoptosis and cell cycle control than is typical for an immune cytokine. The significance of these pro-tumorigenic properties has found support in several in vitro and in vivo models of cancer and in the positive association between MIF production and tumor aggressiveness and metastatic potential in a variety of human tumors.

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, and is involved in the pathogenesis of angiogenic eye disease such as proliferative diabetic retinopathy. However, a functional role for PEDF in tumor growth and angiogenesis remains to be determined. Melanoma is one of the most highly invasive and metastatic tumors. Malignant Melanoma is an increasingly common malignancy and also one the most invasive and metastatic tumors, and its mortality rates have been rapidly increasing above those of any other cancer in recent years. Surgical resection and systemic chemotherapy are the main therapeutic strategies for the treatment of malignant melanoma. However, these approaches are insufficiently effective and may be associated with significant adverse effects. Angiogenesis, a process by which new vascular networks are formed from pre-existing capillaries, is required for tumors to grow, invade and metastasize. Tumor vessels are genetically stable, and less likely to accumulate mutations that allow them to develop drug resistance in a rapid manner. Therefore, targeting vasculatures that support tumor growth, rather than cancer cells, is currently considered the most promising approach to malignant melanoma therapy. Now, novel anti-angiogenic agents with tolerable side effects are actually desired for the treatment of patients with malignant melanoma. In this paper, we review the current understanding of anti-angiogenic therapy for malignant melanoma, especially focusing on PEDF, which was recently identified as the most potent endogenous inhibitor of angiogenesis in the mammalian eye.

We have utilized multicolored fluorescent proteins to develop three imaging models of tumor angiogenesis. In one model, the nonluminous induced capillaries are clearly visible by contrast against the very bright tumor green fluorescent protein (GFP) fluorescence examined either intravitally or by whole-body imaging in real time. Intravital images of an orthotopic model of human pancreatic tumors expressing GFP visualized angiogenic capillaries at both primary and metastatic sites. Whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period in an orthotopic model of human breast cancer expressing GFP. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold and enables vessels to be externally visualized in GFP-expressing tumors growing on internal organs. In another model, dual-color fluorescence imaging was effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice that express GFP in all cells. This dual-color model visualizes with great clarity the details of the tumor-stroma interaction, especially tumorinduced angiogenesis. The GFP-expressing tumor vasculature, both nascent and mature, are readily distinguished interacting with the RFP-expressing tumor cells. The third model involves a transgenic mouse in which the regulatory elements of the stem cell marker nestin drive GFP (ND-GFP). The ND-GFP mouse expresses GFP in nascent blood vessels. RFPexpressing tumors transplanted to nestin-GFP mice enable specific visualization of nascent vessels. The ND-GFP mouse was utilized to develop a rapid in vivo/ex vivo fluorescent angiogenesis assay by implanting Gelfoam which was vascularized by fluorescent nascent blood vessels. This process could be markedly stimulated or inhibited by specific compounds. We also observed, using ND-GFP mice, that the hair follicle is angiogenic and that the hair-follicle vascular network is a prime target for chemotherapy drugs which cause hair loss (chemotherapy-induced alopecia). These fluorescent models, generally termed AngioMouse^®, can quantitatively determine efficacy of antiangiogenesis compounds.

Angiogenesis is a complex process which is critical for the growth, invasion, and metastasis of tumors. In the past ten years numerous new agents have been developed as angiogenesis inhibitors. Angiogenesis inhibitors can be classified by their targeted area of the angiogenic process; (1) VEGF and its receptors VEGFR (e.g. Bevacizumab); (2) tyrosine kinases within endothelial cells (Sunitinib); (3) proliferation of endothelial cells (Endostatin); (4) MMPs (Marimastat); (5) intercellular interactions via integrins (Cilengitide) and (6) combinations of the above mechanisms (Thalidomide). Some showed anti-tumor effects with objective responses and stable disease, and some disappeared from clinical use due to unexpected side effects or insufficient efficacies. Further investigations of combined therapies including angiogenesis inhibitors will shed light on the treatment of advanced and metastasized malignancies.

The introduction of stem cells and/or progenitor cells into damaged myocardium has promising therapeutic potential in ischemic heart diseases and dilated cardiomyopathy. However, understanding the biologic mechanisms and the outcomes of transplanted cells during cardiac regenerative therapy remains mostly limited to histological assessment. Positron emission tomography (PET) is a sensitive molecular imaging modality that can non-invasively assess stem cell retention, survival, and function after transplantation. Two radiolabel approaches have been explored to implement PET: 1) direct cell labeling with a radionuclide; and 2) reporter gene-based cell labeling. Direct cell labeling has previously been used for early tracking of transplanted stem cells into the myocardium in several therapeutic clinical trials. Stem cells can also be labeled after transfection with a reporter gene, which can subsequently be visualized by using a PET reporter probe that binds to the reporter gene, therefore allowing serial in vivo evaluation of cell viability and proliferation in longterm follow-up studies. Recently, some studies successfully used this method to visualize implanted stem cells by PET imaging in animals. With the projected rapid growth of cell therapy for heart disease, PET is expected to play a major role in monitoring relevant changes that occur at every stage in cardiac regenerative therapy. These two cell tracking approaches used for PET imaging are reviewed here and compared against other imaging modalities.

Subtype selectivity of phosphodiesterase 4 (PDE4) has been proposed to be the most salient feature for the development of drugs for asthma and inflammation. The present review provides an account of various strategies to overcome the side effects of the PDE4 inhibitors. Subtype selectivity and recent developments of molecular modeling approaches towards PDE4 were addressed using QSAR and docking, followed by a detailed structural analysis of more than three dozen available X-ray structures of PDE4B and PDE4D. Usually, the lack of a 3-dimensional structure of a target protein is a bottleneck for rational drug design approaches. However, in this case the availability of 39 X-ray structures along with co-crystals has not improved the therapeutic ratio of drugs through rational drug design approaches. The investigation of structures led to find significant variations in the M-loop region, which is the integral part of the active site of PDE4B and PDE4D. These differences can be accounted for by varying conformation of the Pro430 residue and a Thr436/Asn362 mutation in the M-loop that causes variations in adjacent residue properties and also the pattern of hydrogen- bonding interactions. The impact of the M-loop region on inhibitor binding has been further scrutinized by MOLCAD surfaces and hydrophobicity. These have shown that PDE4B is more hydrophobic in nature than PDE4D in the M-loop region. A review of the above aspects given the emphasis on a new PDE4 inhibitor which can access both metal and solvent pockets may possibly lead to ligands with enhanced potency. The lining of the Q2 pocket that involves the M-loop region may be considered for the design of potent subtype-selective inhibitors.

Neurodegenerative diseases comprise a heterogeneous spectrum of neural disorders that cause severe and progressive cognitive and motor deficits. A histological hallmark of these disorders is the occurrence of disease-specific cell death in specific regional subpopulations of neurons, such as the loss of dopaminergic neurons in the substantia nigra in Parkinson's disease. Neurodegenerative disease can also possibly occur from the loss or dysfunction of selected glial cell subsets, such as the dysfunction of supportive glial cells around somatic motor neurons in amyotrophic lateral sclerosis. The central nervous system (CNS), unlike many other tissues, has a very limited capacity for self-repair. Mature nerve cells lack the ability to regenerate, although endogenous neural stem cells exist in the adult brain that do have very limited ability to generate new functional neurons in response to injury. Rapid advances in stem cell biology have opened an alternative, fascinating perspective of neurogenesis by activation of endogenous neural stem cells and/or transplantation of in vitro-expanded stem cells and/or their neuronal- or glial-differentiated progeny. Embryonic stem (ES) cells, because of their ability to provide seemingly unlimited supply of specific cell types, their amenability to genetic engineering manipulations, and their broad developmental potential, are expected to become a cell source and biological delivery system for use in a variety of neurodegenerative diseases, and are likely to play a role in the development of novel cell-based therapies for these indications. However, before the full potential of ES cells can be realized for regenerative medicine, we need to understand mechanisms regulating their proliferation, differentiation into therapeutically relevant cells, and most importantly in the case of neuronal and glial lineages, to characterize their functional properties. In the present review we will be focusing on the factors and methodologies responsible for differentiation of ES cell into different neural precursors and neural cell lineages with particular emphasis on the potential research and clinical applications of ES cells in the field of neurodegenerative disease.