VEGF expression is developmentally regulated during human brain angiogenesis

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.     

Lipocalin‐2 enhances angiogenesis in rat brain endothelial cells via reactive oxygen species and iron‐dependent mechanisms

Inflammation is a key part of central nervous system pathophysiology. However, inflammatory factors are now thought to have both beneficial and deleterious effects. Here, we examine the hypothesis that lipocalin‐2 (LCN2), an inflammatory molecule that can be up‐regulated in the distressed central nervous system, may enhance angiogenesis in brain endothelial cells. Adding LCN2 (0.5–2.0 μg/mL) to RBE (Rat brain endothelial cells). 4 rat brain endothelial cells significantly increased matrigel tube formation and scratch migration, and also elevated levels of iron and reactive oxygen species. Co‐treatment with a radical scavenger (U83836E), a Nox inhibitor (apocynin) and an iron chelating agent (deferiprone) significantly dampened the ability of LCN2 to enhance tube formation and scratch migration in brain endothelial cells. These findings provide in vitro proof of the concept that LCN2 can promote angiogenesis via iron‐ and reactive oxygen species‐related pathways, and support the idea that LCN2 may contribute to the neurovascular recovery aspects of inflammation.

     

Expression of brain-specific angiogenesis inhibitor 3 (BAI3) in normal brain and implications for BAI3 in ischemia-induced brain angiogenesis and malignant glioma

Murine brain-specific angiogenesis inhibitor 1 and 2 (mBAI1, mBAI2) are involved in angiogenesis after cerebral ischemia. In this study, mBAI3 was cloned and characterized. Northern and Western blot analyses demonstrated a unique developmental expression pattern in the brain. The level of mBAI3 in brain peaked 1 day after birth, unlike mBAI1 and mBAI2, which peaked 10 days after birth. In situ hybridization analyses of the brain showed the same localization of BAI3 as BAI1 and BAI2, which includes most neurons of cerebral cortex and hippocampus. In the in vivo focal cerebral ischemia model, BAI3 expression decreased from 0.5 h after hypoxia until 8 h, but returned to control level after 24 h. The expression of vascular endothelial growth factor following ischemia showed an inverse pattern. The decreased expressions of BAIs in high-grade gliomas were observed, but BAI3 expression was generally lower in malignant gliomas than in normal brain. Our results indicate that the expression and distribution of BAI3 in normal brain, but not its developmental expression, are very similar to those of BAI1 and BAI2, and that BAI3 may participate in the early phases of ischemia-induced brain angiogenesis and in brain tumor progression.     

Coordinated interaction of neurogenesis and angiogenesis in the adult songbird brain

Neurogenesis proceeds throughout life in the higher vocal center (HVC) of the adult songbird neostriatum. Testosterone induces neuronal addition and endothelial division in HVC. We asked if testosterone-induced angiogenesis might contribute importantly to HVC neuronal recruitment. Testosterone upregulated both VEGF and its endothelial receptor, VEGF-R2/Quek1/KDR, in HVC. This yielded a burst in local HVC angiogenesis. FACS-isolated HVC endothelial cells produced BDNF in a testosterone-dependent manner. In vivo, HVC BDNF rose by the third week after testosterone, lagging by over a week the rise in VEGF and VEGF-R2. In situ hybridization revealed that much of this induced BDNF mRNA was endothelial. In vivo, both angiogenesis and neuronal addition to HVC were substantially diminished by inhibition of VEGF-R2 tyrosine kinase. These findings suggest a causal interaction between testosterone-induced angiogenesis and neurogenesis in the adult forebrain.     

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

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Induction of angiogenesis by bovine brain derived class 1 heparin-binding growth factor

The angiogenic capacity of the class 1 heparin-binding growth factor from bovine brain, an anionic endothelial cell mitogen of Mr 16 000, has been evaluated. Its ability to induce the growth of new blood vessels has been assessed by means of two established assay systems. On the embryonic chick chorioallantoic membrane dose-response studies demonstrate that 160 ng (10 pmol) of mitogen is required to induce angiogenesis in greater than 50% of the eggs within 72 h. In the presence of 1 unit of exogenous heparin only 40 ng of mitogen (2.5 pmol) is needed to induce a similar response. Moreover, this occurs within 48 h, indicating that heparin also augments the angiogenic response by enhancing the rate of induction of angiogenesis. Eighty nanograms (5 pmol) of mitogen also induces the ingrowth of new blood vessels into the rabbit cornea, both in the presence and in the absence of heparin. These results establish that the class 1 heparin-binding growth factor from bovine brain is an angiogenesis factor. Importantly, the neovascularization induced by this angiogenesis factor is enhanced by heparin. The mechanistic implications for neovascularization under certain normal and pathological conditions are discussed.     

A new method of lectin histochemistry for the study of brain angiogenesis

Summary In an attempt to analyse the kinetics of angiogenesis in the brain, we developed a new lectin-histochemical staining technique for identifying the vasculature. Three horseradish-peroxidase-conjugated lectins, i.e., Griffonia simplicifolia agglutinin 1 (GS1), Ricinus communis agglutinin 1 (RCA1) and soybean agglutinin (SBA), selectively stained vascular walls in brain-tissue sections. When these lectins were injected into the circulation of ether-anesthetized animals via the pulsating left ventricle, they bound specifically to the inner surface of endothelial cells and revealed the three-dimensional architecture of the vascular network within thick tissue preparations. When this technique, referred to a lectin angiography, was combined with 5-bromo-2-deoxyuridine (BudR) immunohistochemistry, proliferating capillary cells could be easily identified in three-dimensional structures of the developing vasculature. Because of its simplicity and wide applicability, lectin angiography should be useful for analysing the kinetics of angiogenesis in developmental, regenerative, and pathological conditions in various tissues and organs.     

VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.     

Regulation of brain endothelial cells migration and angiogenesis by P-glycoprotein/caveolin-1 interaction

We have investigated the involvement of P-glycoprotein (P-gp)/caveolin-1 interaction in the regulation of brain endothelial cells (EC) migration and tubulogenesis. P-gp overexpression in MDCK-MDR cells was correlated with enhanced cell migration whereas treatment with P-gp inhibitors CsA or PSC833 reduced it. Transfection of RBE4 rat brain endothelial cells with mutated versions of MDR1, in the caveolin-1 interaction motif, decreased the interaction between P-gp and caveolin-1, enhanced P-gp transport activity and cell migration. Moreover, down-regulation of caveolin-1 in RBE4 cells by siRNA against caveolin-1 stimulated cell migration. Interestingly, the inhibition of P-gp/caveolin-1 interaction increased also EC tubulogenesis. Furthermore, decrease of P-gp expression by siRNA inhibited EC tubulogenesis. These data indicate that the level of P-gp/caveolin-1 interaction can modulate brain endothelial angiogenesis and P-gp dependent cell migration.     

Therapeutic angiogenesis using tumor cell‐conditioned medium

Stem cell‐conditioned medium (CM), which contains angiogenic factors that are secreted by stem cells, represents a potential therapy for ischemic diseases. Along with stem cells, tumor cells also secrete various angiogenic factors. Here, tumor cells as a cell source of CM for therapeutic angiogenesis was evaluated and the therapeutic efficacy of tumor cell CM in mouse hindlimb ischemia models was demonstrated. CM obtained from a human fibrosarcoma HT1080 cell line culture was compared with CM obtained from a human bone marrow‐derived mesenchymal stem cell (MSC) culture. HT1080 CM contained higher concentrations of angiogenic factors compared with MSC CM, which was attributable to the higher cell density that resulted from a much faster growth rate of HT1080 cells compared with MSCs. For use in in vitro and in vivo angiogenesis studies, HT1080 CM was diluted such that HT1080 CM and MSC CM would have the same cell number basis. The two types of CMs induced the same extent of human umbilical vein endothelial cell (HUVEC) proliferation in vitro. The injection of HT1080 CM into mouse ischemic limbs significantly improved capillary density and blood perfusion compared with the injection of fresh medium. Although the therapeutic outcome of HT1080 CM was similar to that of MSC CM, the preparation of CM by tumor cell line culture would be much more efficient due to the faster growth and unlimited life‐time of the tumor cell line. These data suggest the potential application of tumor cell CM as a therapeutic modality for angiogenesis and ischemic diseases. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:456–464, 2016     

Neurovascular abnormalities in brain disorders: highlights with angiogenesis and magnetic resonance imaging studies

The coupling between neuronal activity and vascular responses is controlled by the neurovascular unit (NVU), which comprises multiple cell types. Many different types of dysfunction in these cells may impair the proper control of vascular responses by the NVU. Magnetic resonance imaging, which is the most powerful tool available to investigate neurovascular structures or functions, will be discussed in the present article in relation to its applications and discoveries. Because aberrant angiogenesis and vascular remodeling have been increasingly reported as being implicated in brain pathogenesis, this review article will refer to this hallmark event when suitable.     

Amyloid triggers extensive cerebral angiogenesis causing blood brain barrier permeability and hypervascularity in Alzheimer's disease

Evidence of reduced blood-brain barrier (BBB) integrity preceding other Alzheimer's disease (AD) pathology provides a strong link between cerebrovascular angiopathy and AD. However, the "Vascular hypothesis", holds that BBB leakiness in AD is likely due to hypoxia and neuroinflammation leading to vascular deterioration and apoptosis. We propose an alternative hypothesis: amyloidogenesis promotes extensive neoangiogenesis leading to increased vascular permeability and subsequent hypervascularization in AD. Cerebrovascular integrity was characterized in Tg2576 AD model mice that overexpress the human amyloid precursor protein (APP) containing the double missense mutations, APPsw, found in a Swedish family, that causes early-onset AD. The expression of tight junction (TJ) proteins, occludin and ZO-1, were examined in conjunction with markers of apoptosis and angiogenesis. In aged Tg2576 AD mice, a significant increase in the incidence of disrupted TJs, compared to age matched wild-type littermates and young mice of both genotypes, was directly linked to an increased microvascular density but not apoptosis, which strongly supports amyloidogenic triggered hypervascularity as the basis for BBB disruption. Hypervascularity in human patients was corroborated in a comparison of postmortem brain tissues from AD and controls. Our results demonstrate that amylodogenesis mediates BBB disruption and leakiness through promoting neoangiogenesis and hypervascularity, resulting in the redistribution of TJs that maintain the barrier and thus, provides a new paradigm for integrating vascular remodeling with the pathophysiology observed in AD. Thus the extensive angiogenesis identified in AD brain, exhibits parallels to the neovascularity evident in the pathophysiology of other diseases such as age-related macular degeneration.     

A new method of lectin histochemistry for the study of brain angiogenesis. Lectin angiography

In an attempt to analyse the kinetics of angiogenesis in the brain, we developed a new lectin-histochemical staining technique for identifying the vasculature. Three horseradish-peroxidase-conjugated lectins, i.e., Griffonia simplicifolia agglutinin 1 (GS1), Ricinus communis agglutinin 1 (RCA1) and soybean agglutinin (SBA), selectively stained vascular walls in brain-tissue sections. When these lectins were injected into the circulation of ether-anesthetized animals via the pulsating left ventricle, they bound specifically to the inner surface of endothelial cells and revealed the three-dimensional architecture of the vascular network within thick tissue preparations. When this technique, referred to a lectin angiography, was combined with 5-bromo-2-deoxyuridine (BudR) immunohistochemistry, proliferating capillary cells could be easily identified in three-dimensional structures of the developing vasculature. Because of its simplicity and wide applicability, lectin angiography should be useful for analysing the kinetics of angiogenesis in developmental, regenerative, and pathological conditions in various tissues and organs.     

Release of regulators of angiogenesis following Hypocrellin-A and -B photodynamic therapy of human brain tumor cells

Photodynamic therapy (PDT) is an innovative strategy for the treatment of solid neoplasms of the brain. Aside from inducing cell death in tumor cells, PDT induces endothelial cell death and promotes formation of blood clots; however, exact mechanisms that trigger these phenomena remain largely unknown. We now used Western blotting to analyze secretion of regulators of angiogenesis to the supernatants of one glioma, one macrophage, and one endothelial cell line following Hypocrellin-A and -B photodynamic therapy. We observed induction of proangiogenic VEGF (vascular endothelial growth factor) and of antiangiogenic sFlt-1, angiostatin, p43, allograft inflammatory factor-1, and connective tissue growth factor. Release of thrombospondin-1 was diminished in a glioma cell line supernatant. Endostatin release was induced in glioma cells and reduced in macrophages and endothelial cells. These data show that a wide range of antiangiogenic factors are secreted by brain tumor cells following Hypocrellin photochemotherapy. However, VEGF release is also induced thus suggesting both favorable and deleterious effects on tumor outgrowth.     

Glioma angiogenesis

Brain tumors exhibit marked and aberrant blood vessel formation indicating angiogenic endothelial cells as a potential target for brain tumor treatment. The brain tumor blood vessels are used for nutrient delivery, and possibly for cancer cell migration. The process of angiogenesis is complex and involves multiple players. The current angiogenesis inhibitors used in clinical trials mostly target single angiogenic proteins and so far show limited effects on tumor growth. Besides the conventional angiogenesis inhibitors, RNA-based inhibitors such as small-interfering RNAs (siRNAs) are being analyzed for their capacity to silence the message of proteins involved in neovascularization. More recently, a new family of non-coding RNAs, named angiomirs [microRNAs (miRNAs) involved in angiogenesis] has emerged. These small RNAs have the advantage over siRNAs in that they have the potential of silencing multiple messages at the same time and therefore they might become therapeutically relevant in a “one-hit multiple-target” context against brain tumor angiogenesis. In this review we will discuss the emerging technologies in anti-angiogenesis emphasizing on RNA-based therapeutics.