Angiogenesis research: guidelines for translation to clinical application

Angiogenesis research is being translated to the clinic. Certain guidelines may facilitate this effort. Recruitment of endothelial cells by a tumor is an early event in angiogenesis, a process regulated at genetic and epigenetic levels. The microvascular endothelial cell has become an important second target in cancer therapy. Angiogenesis inhibitors are either "direct" or "indirect" and their optimal dosing depends on a different logic than conventional chemotherapy. Conversely, antiangiogenic scheduling of chemotherapy can by-pass drug resistance. Like all solid tumors, hematologic malignancies are angiogenesis-dependent. Further, angiogenesis is modulated by proteins and cells from the hematopoietic and hemostatic systems. Clinical testing of angiogenesis inhibitors has accentuated the need for surrogate markers of tumor angiogenesis activity. Microvessel density, so valuable as a prognostic indicator of metastatic risk, cannot determine efficacy of an angiogenesis inhibitor. In the future, angiogenesis inhibitors may be added to chemotherapy or to radiotherapy, or to other modalities. Also, combinations of angiogenesis inhibitors may be administered together.     

Angiogenesis is regulated by angiopoietins during experimental autoimmune encephalomyelitis and is indirectly related to vascular permeability

The regulation of angiogenesis was studied over the course of the animal model of multiple sclerosis, acute experimental autoimmune encephalomyelitis (EAE) in mice using immunohistochemistry. During EAE, angiogenesis peaked 21 days after disease induction, with significant increases in gray matter and adjacent to the leptomeninges. Angiogenesis correlated with clinical and pathologic scores. Spinal cord expression of angiopoietin 1 (Ang-1) by neurons and glia was reduced at Day 14, but expression by inflammatory cells restored earlier levels at Day 21. Angiopoietin 2 expression increased markedly at Day 21 and was mostly associated with inflammatory cells. Levels of the angiopoietin receptor Tie-2 were reduced at Day 14, but recovered by day D21. Double labeling demonstrated Ang-1 expression on infiltrating CD3-positive T cells; Ang-2 was expressed by monocytes/macrophages. During EAE, the expression of vascular endothelial growth factor peaked at Day 14 and began to decrease by Day 21. Double labeling showed expression of Tie-2 and vascular endothelial growth factor receptor 2 but not Ang-2 in blood vessels at Day 21. Vascular permeability increased early in EAE, but was reduced by Day 21. Although individual values did not correlate with angiogenesis, the volume of permeable tissue showed a weak positive correlation with angiogenesis. These temporal changes in angiogenic factors suggest an integral role during EAE-related angiogenesis.     

Angiogenesis in ischemic disease

Angiogenic growth factors and their endothelial receptors function as major regulators of blood vessel formation. The VEGF/VEGFR and the Angiopoietin/Tie2 receptor systems represent key signal transduction pathways involved in the regulation of embryonic vascular development. Inactivation of any of the genes encoding these molecules results in defective vascular development and lethality between embryonic day 8.5 and 12.5. In addition, VEGF and its receptors are also critically involved in the regulation of pathological blood vessel growth in the adult during various angiogenesis-dependent diseases that are associated with tissue hypoxia, such as solid tumor growth and ischemic diseases. It is now well established that therapeutic angiogenesis can be achieved in animal models of hind limb and myocardial ischemia by exogenously adding VEGF and/or other angiogenic growth factors. Available clinical data from human trials also suggests that patients with severe cardiovascular diseases could potentially benefit from such therapies. However, much more work needs to be done to compare the potency of different angiogenic factors or the combination thereof, as well as the best way of delivery, either as recombinant proteins, as naked DNA or via adenoviral vectors. Nevertheless, the therapeutic efficacy of simply injecting naked plasmid DNA or proteins into ischemic tissue to deliver secreted angiogenic factors is an encouraging finding. Time will show whether the adverse side effects of therapeutic angiogenesis, mainly vascular permeability and edema formation, can be minimized and angiogenic factors can be used as an effective therapy in patients for the treatment of ischemic diseases such as arterial occlusive disease, myocardial infarction, and, eventually, also stroke.     

Angiogenesis in neurological disorders: a review

Angiogenesis, recruitment of new blood vessels, is an essential component of the metastatic pathway. These vessels provide the principal route by which tumor cells exit the primary tumor site and enter the circulation. For many tumors, the vascular density can provide a prognostic indicator of metastatic potential, with the highly vascular primary tumors having a higher incidence of metastasis than poorly vascular tumors. The discovery and characterization of tumor-derived angiogenesis modulators greatly contributed to our understanding of how tumors regulate angiogenesis. However, although angiogenesis appears to be a rate-limiting event in tumor growth and metastatic dissemination, a direct connection between the induction of angiogenesis and the progression to tumor malignancy is less well understood. In this review, we discuss the observations concerning the modulation of angiogenesis and their implications in various neurological disorders, as well as their potential impact on cancer therapy.     

Cerebral ischemia and angiogenesis

Angiogenesis occurs in a wide range of conditions. As ischemic tissue usually depends on collateral blood flow from newly produced vessels, acceleration of angiogenesis should be of therapeutic value to ischemic disorders. Indeed, therapeutic angiogenesis reduced tissue injury in myocardial or limb ischemia. In ischemic stroke, on the other hand, angiogenic factors often increase vascular permeability and thus may deteriorate tissue damage. In order to apply safely the therapeutic angiogenesis for ischemic stroke treatment, elucidating precise mechanism of brain angiogenesis is mandatory. In the present article, we review previous reports which investigated molecular mechanisms of angiogenesis. Endothelial cell mitogens, enzymes that degrade surrounding extracellular matrix, and molecules implicated in endothelial cells migration are induced rapidly in the ischemic brain. Their possible neuroprotective or injury exacerbating effects are discussed. Because therapeutic potential of angiogenic factors application had gained much attention, we here extensively reviewed relevant previous reports. In the future however, there is a need to consider angiogenesis in relation with regenerative medicine, as angiogenic factors sometimes possess neuron producing property.     

Inhibition of angiogenesis as a strategy for tumor growth control

Angiogenesis is a complex sequence of events leading to the formation of new capillaries. Although essential to maturation and wound healing, most angiogenesis in the adult is associated with pathological events, such as the development of solid tumors. One approach to the inhibition of angiogenesis is the antagonism of basic fibroblast growth factor, a major angiogenic protein. Evidence is reviewed to suggest that inhibiting angiogenesis results in the suppression of tumor growth.     

Tumour angiogenesis: Its mechanism and therapeutic implications in malignant gliomas

Angiogenesis is a key event in the progression of malignant gliomas. The presence of microvascular proliferation leads to the histological diagnosis of glioblastoma multiforme. Tumour angiogenesis involves multiple cellular processes including endothelial cell proliferation, migration, reorganisation of extracellular matrix and tube formation. These processes are regulated by numerous pro-angiogenic and anti-angiogenic growth factors. Angiogenesis inhibitors have been developed to interrupt the angiogenic process at the growth factor, receptor tyrosine kinase and intracellular kinase levels. Other anti-angiogenic therapies alter the immune response and endogeneous angiogenesis inhibitor levels. Most anti-angiogenic therapies for malignant gliomas are in Phase I/II trials and only modest efficacies are reported for monotherapies. The greatest potential for angiogenesis inhibitors may lie in their ability to combine safely with chemotherapy and radiotherapy.     

Inhibition of angiogenesis as a strategy for tumor growth control

Angiogenesis is a complex sequence of events leading to the formation of new capillaries. Although essential to maturation and wound healing, most angiogenesis in the adult is associated with pathological events, such as the development of solid tumors. One approach to the inhibition of angiogenesis is the antagonism of basic fibroblast growth factor, a major angiogenic protein. Evidence is reviewed to suggest that inhibiting angiogenesis results in the suppression of tumor growth.     

Antiangiogenic therapy in brain tumors

Angiogenesis, the recruitment of new blood vessels, is an essential component of tumor progression. Malignant brain tumors are highly vascularized and their growth is angiogenesis-dependent. As such, inhibition of the sprouting of new capillaries from pre-existing blood vessels is one of the most promising antiglioma therapeutic approaches. Numerous classes of molecules have been implicated in regulating angiogenesis and, thus, novel agents that target and counteract angiogenesis are now being developed. The therapeutic trials of a number of angiogenesis inhibitors as antiglioma drugs are currently under intense investigation. Preliminary studies of angiogenic blockade in glioblastoma have been promising and several clinical trials are now underway to develop optimum treatment strategies for antiangiogenic agents. This review will cover state-of-the-art antiangiogenic targets for brain tumor treatment and discuss future challenges. An increased understanding of the angiogenic process, the diversity of its inducers and mediators, appropriate drug schedules and the use of these agents with other modalities may lead to radically new treatment regimens to achieve maximal efficacy.     

Angiogenesis in malignant gliomas

One event that accompanies glioma progression is the upregulation of angiogenesis. Low-grade gliomas are moderately vascularized tumors whereas high-grade gliomas show prominent microvascular proliferations and areas of high vascular density. To analyze the molecular mechanisms underlying glioma angiogenesis, we studied the expression of vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGFR-1 and VEGFR-2 during normal brain development and glioma-induced angiogenesis. Our results suggest a paracrine control of angiogenesis and endothelial cell proliferation that is tightly regulated and transient in the embryonic brain, switched off in the normal adult brain, and turned on in tumor cells (VEGF) and the host vasculature (VEGFR-1 and ?2) during tumor progression. It is unknown how VEGF and VEGF receptors are upregulated during glioma angiogenesis, but there is recent evidence that VEGF as well as endogenous inhibitors of angiogenesis could be under control of the tumor suppressor genes p53 and VHL. © 1995 Wiley-Liss, Inc.     

Induction of angiogenesis by platelet-activating factor in the rat striatum

Angiogenesis, the formation of new blood vessels, has been studied following injection of platelet-activating factor into the rat striatum. Immunohistochemical analysis for laminin showed that platelet-activating factor induced significant neovascularization in the striatum. A dose-dependent loss of striatal neurons accompanied this angiogenic activity. The platelet-activating factor-induced neovascularization and neurodegeneration were attenuated by the platelet-activating factor receptor antagonist, BN52021, or the inducible nitric oxide synthase inhibitor, aminoguanidine. Additionally, these agents significantly reduced levels of the angiogenic agent vascular endothelial growth factor, which was markedly enhanced in platelet-activating factor-injected striatum. Double immunofluorescence staining demonstrated that expression of vascular endothelial growth factor and inducible nitric oxide synthase were predominantly localized to microglia, suggesting that inflammatory responses mediated by activated microglia could contribute to platelet-activating factor-induced angiogenesis and neuronal loss.     

Angiogenesis and neuronal remodeling after ischemic stroke

Increased microvessel density in the peri-infarct region has been reported and has been correlated with longer survival times in ischemic stroke patients and has improved outcomes in ischemic animal models.This raises the possibility that enhancement of angiogenesis is one of the strategies to facilitate functional recovery after ischemic stroke.Blood vessels and neuronal cells communicate with each other using various mediators and contribute to the pathophysiology of cerebral ischemia as a unit.In this mini-review,we discuss how angiogenesis might couple with axonal outgrowth/neurogenesis and work for functional recovery after cerebral ischemia.Angiogenesis occurs within 4 to 7 days after cerebral ischemia in the border of the ischemic core and periphery.Post-ischemic angiogenesis may contribute to neuronal remodeling in at least two ways and is thought to contribute to functional recovery.First,new blood vessels that are formed after ischemia are thought to have a role in the guidance of sprouting axons by vascular endothelial growth factor and laminin/β1-integrin signaling.Second,blood vessels are thought to enhance neurogenesis in three stages:1)Blood vessels enhance proliferation of neural stem/progenitor cells by expression of several extracellular signals,2)microvessels support the migration of neural stem/progenitor cells toward the peri-infarct region by supplying oxygen,nutrients,and soluble factors as well as serving as a scaffold for migration,and 3)oxygenation induced by angiogenesis in the ischemic core is thought to facilitate the differentiation of migrated neural stem/progenitor cells into mature neurons.Thus,the regions of angiogenesis and surrounding tissue may be coupled,representing novel treatment targets.     

血管生成相关基因在脑动-静脉畸形不同部位差异表达的初步研究

目的:应用功能分类基因芯片研究脑动-静脉畸形(BAVM)不同结构部位的血管生成相关基因表达。方法:6例BAVM,分别在“供血动脉”、“引流静脉”、“畸形团血管”和“周边脑组织”4个不同部位取材,进行血管新生芯片杂交。以“周边脑组织”为自身对照,计算“畸形团血管”、“供血动脉”和“引流静脉”与之的比值。随意选取8个基因进行逆转录-聚合酶链反应(RT-PCR)验证。结果:经过分析和比较,共筛选出44个明显异常的基因,包括了生长因子和受体相关基因18个(含4个下调基因),黏附分子相关基因6个,基质蛋白相关基因6个,转录因子基因5个,细胞因子和趋化因子基因9个(含1个下调基因)。RT-PCR结果与之基本一致。结论:为BAVM的机制研究开辟了许多新的切入点。     

Border zone neovascularization in cerebral ischemic infarct

Immunocytochemical and quantitative studies on vascular reaction (angiogenesis) in cortical border zone of infarct were undertaken. Intensity and temporal profile of angiogenesis was assessed in 60 patients aged between 48 and 69 (younger group), and between 70 and 92 (older group), with cerebral infarct in the area of middle cerebral artery vascularization, who died during the first six weeks following the stroke. We have found that angiogenesis was a multistage process in which four stages were distinguished: phase of primary activation of endothelial cells, two consecutive phases of active angiogenesis and final phase of only sporadic proliferation of vessels. The distinction of phases in a multiphase angiogenic cascade helped us to evaluate the correlation with survival time and the age of patients. The most pronounced intensification of angiogenesis and increased density of CD 31 positive capillaries in penumbra were observed in the second phase, especially in younger patients. The duration of the penumbral neovascularization decreased in the older age patient. Our results indicate that sprouting angiogenesis is a quantitatively significant source of vessels in the cerebral infarct border zone. However, non-therapeutically stimulated angiogenesis developed only 3-4 days after the stroke, that is beyond the period of reversible changes in ischemic penumbra recognized as a "therapeutical window" in the human brain. The angiogenic therapy opens a new way towards the revascularization of ischemic brain infarct.     

Induction of angiogenesis in the beta-amyloid peptide-injected rat hippocampus

Angiogenesis, the formation of new capillary blood vessels, has been studied following the stereotaxic injection of beta-amyloid peptide (Abeta1-42) into rat hippocampus. Immunohistochemical analysis for laminin showed that neovascularization was only slightly increased, relative to control, in the hippocampus 1 day post-Abeta1-42 injection. However, 7 days following peptide injection neovascularization was markedly up-regulated (by 2.2-fold) compared to control. Immunoreactivity for the angiogenic stimulatory agent vascular endothelial growth factor (VEGF) was also significantly increased in the hippocampus 7 days after Abeta1-42 injection. Double immunofluorescence staining demonstrated that the increased level of VEGF immunoreactivity was localized to both astrocytes and microglia, suggesting inflammatory responses contributed to angiogenesis. The findings of beta-amyloid stimulated angiogenesis and the involvement of peptide-induced inflammatory processes may have relevance to the pathology of Alzheimer's disease.     

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.     

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.     

Angiogenic signaling in Alzheimer's disease

Genome-wide expression profiling has identified significant alterations in the abundance of specific mRNA populations in Alzheimer's disease brain when compared to age-matched controls. Increases in the expression of certain brain genes are in contrast to the majority of expressed RNAs (55-67%), which are down-regulated. The data presented here shows, that at the level of mRNA abundance, there is marked up-regulation in a family of stress-related genes that have significant potential to promote angiogenesis. This supports the hypothesis of an advancement in angiogenic signaling in Alzheimer's disease brain. Angiogenesis, perhaps as the result of dysfunctional cerebral vasculature, may be both a consequence and a contributory factor to the etiopathology of the Alzheimer's disease process.     

Temporal profile of angiogenesis and expression of related genes in the brain after ischemia.

Angiogenesis is an intricately regulated phenomenon. Its mechanisms in the ischemic brain have not been clearly elucidated. The authors investigated expression of angiogenesis-related genes using a complementary DNA (cDNA) array method as well as Western blotting and immunohistochemistry, and compared these studies with a temporal profile of angiogenesis in mouse brains after ischemia. The number of vessels significantly increased 3 days after injury, and proliferating endothelial cells increased as early as 1 day. This means that angiogenesis occurs immediately after the injury. Ninety-six genes implicated in angiogenesis were investigated with a cDNA array study. It was found that 42, 29, and 13 genes were increased at 1 hour, 1 day, and 21 days, respectively. Most of the well-known angiogenic factors increased as early as 1 hour. Vessel-stabilizing factors such as thrombospondins also increased. At 1 day, however, thrombospondins decreased to lower levels than in the control, indicating a shift from vascular protection to angiogenesis. At 21 days, many genes were decreased, but some involved in tissue repair were newly increased. Western blotting and immunohistochemistry showed findings compatible with the cDNA array study. Many molecules act in an orchestrated fashion in the brain after ischemia and should be taken into account for therapeutic angiogenesis for stroke.