Pathways of macromolecular extravasation across microvascular endothelium in response to VPF/VEGF and other vasoactive mediators

OBJECTIVE: The goal of these studies was to define the anatomic pathways by which circulating macromolecules extravasate from the hyperpermeable microvessels that supply tumors and from normal venules that have been rendered hyperpermeable by vasoactive mediators. METHODS: Extravasation pathways of circulating macromolecular tracers were followed by several morphological techniques: light and fluorescence microscopy, transmission electron microscopy of routine as well as ultrathin and serial sections, computer-assisted three-dimensional reconstructions, and morphometry. RESULTS AND DISCUSSION: Macromolecules extravasated across tumor microvessels or across normal venules rendered hyperpermeable by VPF/VEGF, histamine, or serotonin by three primary pathways: 1) Vesiculo-vacuolar organelles (VVOs), clusters of cytoplasmic vesicles and vacuoles that span endothelial cytoplasm from lumen to ablumen; 2) trans-endothelial cell (EC), pores, and 3) fenestrae. We also present data concerning the structure and function of VVOs as well as evidence that VVOs form as the result of linking together and fusion of caveolae-sized unit vesicles. Under suitable conditions VVOs also afforded a pathway for macromolecular transport in the reverse direction, i.e., from vascular ablumen to lumen. Finally, in addition to opening VVOs to the passage of macromolecules, mediators such as VPF/VEGF may also induce structural rearrangements of VVOs, transforming them into trans-EC pores or fenestrae.     

The dormant in vivo phenotype of early stage primary human melanoma: termination by overexpression of vascular endothelial growth factor

Early stage primary human cutaneous melanoma is known to remain relatively avascular and dormant for up to a decade, after which it may give rise to more rapidly growing, vascular and metastatically-competent primary tumor. Clinical dormancy of early stage human melanomas can be recapitulated experimentally by injection of cell lines established from such tumors into nude mice. For example, WM1341B cells, which were isolated from a thin vertical growth phase (VGP) human melanoma, are non-tumorigenic in nude mice even though some of the cells remain viable for at least three weeks at the site of orthotopic injection. These cells produce little or no vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a potent stimulator of angiogenesis. In order to determine whether their in vivo dormant behaviour may therefore be related to an inability to induce tumor angiogenesis, subpopulations of WM1341B cells were engineered to constitutively overexpress the VEGF/VPF121 isoform. This apparently single modification was sufficient to induce overt and progressively growing tumors by several independent VEGF/VPF121 producing clones, which could be largely blocked by systemic treatment of mice with a monoclonal anti-VEGF neutralizing antibody (A 4.6.1). No evidence for an autocrine mechanism of growth stimulation by VEGF was found. Taken together, these results support the notion that defective angiogenesis may, at least in part, account for dormant phenotype of some early stage primary melanomas. Since the induction of an overt tumorigenic phenotype in several VEGF/VPF transfected WM1341B clones appears to depend exclusively on their expression of VEGF/VPF, such sublines should be useful for screening the activity of known or potential VEGF/VPF ligand or VEGF/VPF receptor antagonists in an in vivo context. This revised version was published online in June 2006 with corrections to the Cover Date.     

Endostatin inhibits angiogenesis by stabilization of newly formed endothelial tubes

Endostatin decreased vascular endothelial growth factor (VEGF)-induced formation of endothelial tubes and microvessels sprouting from aortic rings and blocked their network. After cessation of treatment, the survival time of endostatin plus VEGF-treated tubes was approximately doubled in comparison to VEGF alone. Endostatin antibody blocked VEGF-induced endothelial tube formation and disrupted existing tubes. Endostatin immunostaining was localized between endothelium and basement membrane and in inter-endothelial junctions of new, but not of quiescent, blood vessels. In tumors grown in SCID mice, endostatin immunostaining was stronger accompanying blood vessel maturation and was significantly prominent in vessels of tumor marginal zone where angiogenesis is highly active. These data indicate a new antiangiogenic action of endostatin stabilizing and maturating endothelial tubes of newly formed blood vessels. Thus, strategies accelerating vascular stabilization and maturation could be promising in tumor therapy. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell-mediated delivery of fibroblast growth factor-2 and vascular endothelial growth factor onto the chick chorioallantoic membrane: endothelial fenestration and angiogenesis.

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) exert their angiogenic activity by interacting with endothelial cells in a distinct manner. In this study, we investigated the morphological features of endothelial cells of the chick embryo chorioallantoic membrane (CAM) microvasculature after stimulation with FGF2 or VEGF. In order to provide a continuous delivery of the growth factor, we utilized a recently developed gelatin sponge/CAM assay in which a limited number of FGF2- or VEGF-transfected cells were adsorbed onto gelatin sponges and applied on the top of the CAM on day 8 of development. Their angiogenic activity was compared to that exerted by a single bolus of the corresponding growth factor. All the angiogenic stimuli induced a comparable vasoproliferative response, as demonstrated by the appearance of similar numbers of immature blood vessels within the sponge on day 12. No angiogenic response was observed in CAMs implanted with the corresponding parental cell lines or vehicle. Electron microscopy demonstrated that VEGF-overexpressing cells modified the phenotype of the endothelium of the blood vessels at the boundary between the implant and the surrounding CAM mesenchyme. The endothelial lining of 30% of these vessels showed segmental attenuations, was frequently interrupted and became fenestrated, mimicking what is observed in tumor vasculature. In contrast, the vessels consisted of continuous endothelium sealed by tight junctions in all the other experimental conditions. These results indicate that FGF2 and VEGF interact with endothelial cells of the CAM in a distinct manner. Both growth factors induce a potent angiogenic response, but only VEGF delivered in a continuous manner by its transfectants can modify the phenotype of the otherwise quiescent endothelium of CAM blood microvessels. The gelatin sponge/CAM assay may constitute a new model to study the mechanisms leading to endothelial fenestration in tumor growth.     

Permeability of muscle capillaries to interstitially microinjected ferritin

Horse spleen ferritin (molecular weight, ～750,000; diameter, 110–120 Å) was microinjected into the interstitium of rat skeletal muscle. Specimens obtained 1–20 min postinjection were examined in the electron microscope with respect to the transcapillary flux of the tracer. At all time points, a concentration gradient of ferritin across the periendothelial basement membrane was recorded. The ferritin particles were apparently halted against the outer surface of the basement membrane. Accordingly, the capillary endothelium was exposed to ferritin at a relatively low concentration. A limited labelling of endothelial plasmalemmal vesicles was observed, indicating a restricted transendothelial transport of ferritin molecules that had permeated the basement membrane. There were no indications of transjunctional permeation of ferritin. It is concluded that, in addition to the endothelium, the periendothelial basement membrane of continuous blood capillaries constitutes a substantial barrier to the permeation from the interstitium to the plasma of macromolecules of the size of ferritin.     

Excessive tumor-elaborated VEGF and its neutralization define a lethal paraneoplastic syndrome

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and key regulator of both physiologic and pathologic (e.g., tumor) angiogenesis. In the course of studies designed to assess the ability of constitutive VEGF to block tumor regression in an inducible RAS melanoma model, mice implanted with VEGF-expressing tumors sustained high morbidity and mortality that were out of proportion to the tumor burden. Documented elevated serum levels of VEGF were associated with a lethal hepatic syndrome characterized by massive sinusoidal dilation and endothelial cell proliferation and apoptosis. Systemic levels of VEGF correlated with the severity of liver pathology and overall clinical compromise. A striking reversal of VEGF-induced liver pathology and prolonged survival were achieved by surgical excision of VEGF-secreting tumor or by systemic administration of a potent VEGF antagonist (VEGF-TRAP(R1R2)), thus defining a paraneoplastic syndrome caused by excessive VEGF activity. Moreover, this VEGF-induced syndrome resembles peliosis hepatis, a rare human condition that is encountered in the setting of advanced malignancies, high-dose androgen therapy, and Bartonella henselae infection. Thus, our findings in the mouse have suggested an etiologic role for VEGF in this disease and may lead to diagnostic and therapeutic options for this debilitating condition in humans.     

The Glomerular Sieve and the Mechanisms of Proteinuria

Abstract: The glomerular capillary wall acts as a sieve, allowing the passage of water and small solutes but holding up circulating macro-molecules the size of albumin or larger. Recent studies indicate that such macromolecules are held up at the level of the endothelial fenestrae by a functional barrier which depends critically upon the maintenance of normal haemo-dynamic conditions. The effectiveness of this barrier probably relates to molecular sieving phenomena across the basement membrane, perhaps in conjunction with the formation of a concentration-polarisation layer beneath the endothelium, and possibly in association with charge effects. Accordingly, proteinuria may result not only from structurat damage to the capillary wall in glomerular disease but also from general or local changes in glomerular blood flow or ultrafiltration flux, causing disturbances in the functional barrier at the endothelial fenestrae. Because it appears that the epithelial layer offers the major restriction to water flux across the wall, protein leakage at sites of focal loss bf the epithelial layer (as found in a variety of human and experimental proteinuric states) may result from a focal "blow-out" of ultrafiltration flux, thereby dis rupting the functional barrier at the endothelial fenestrae and dragging macromolecules across to the urinary space.     

Impact of oncogenes in tumor angiogenesis: Mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells

Targeted disruption of the single mutant K-ras allele in two human colorectal carcinoma cell lines (DLD-1 and HCT-116) leads to loss of tumorigenic competence in nude mice with retention of ability to grow indefinitely in monolayer culture. Because expression of the mutant K-ras oncogene in these cell lines is associated with marked up-regulation of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), we sought to determine whether this potent angiogenesis inducer plays a role in K-ras-dependent tumorigenic competence. Transfection of a VEGF₁₂₁ antisense expression vector into DLD-1 and HCT-116 cells resulted in suppression of VEGF/VPF production by a factor of 3- to 4-fold. The VEGF/VPF-deficient sublines, unlike the parental population or vector controls, were profoundly suppressed in their ability to form tumors in nude mice for as long as 6 months after cell injection. In contrast, in vitro growth of these sublines was unaffected, thus demonstrating the critical importance of VEGF/VPF as an angiogenic factor for HCT-116 and DLD-1 cells. Transfection of a full-length VEGF₁₂₁ cDNA into two nontumorigenic mutant K-ras knockout sublines resulted in a weak but detectable restoration of tumorigenic ability in vivo in a subset of the transfectants, with no consistent change in growth properties in vitro. The findings indicate that mutant ras-oncogene-dependent VEGF/VPF expression is necessary, but not sufficient, for progressive tumor growth in vivo and highlight the relative contribution of oncogenes, such as mutant K-ras, to the process of tumor angiogenesis.     

Angiogenesis in histologically benign squamousmucosa is a sensitive marker for nearbycervical intraepithelial neoplasia

Cervical intraepithelial neoplasia (CIN) lesions elicit a neovascularization response at the stromal–epithelial junction. In this study, the angiogenic properties of histologically benign cervical epithelium adjacent to CIN were compared to samples of normal cervix from patients with no evidence of CIN. Vessels were detected by immunohistochemical staining for von Willebrand factor (vWf). Expression of a potent angiogenic factor, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), was measured by in situ hybridization. Statistical comparisons were made using the Student t-test. In histologically benign cervix adjacent to CIN, the microvessel count was significantly increased (164.5 versus 47.3 vessels/cm; p < 0.004) and vessels were more closely apposed to the epithelium (15.3 versus 22.4 μm to the basal cell nuclei; p < 0.0001) than in normal cervix. Expression of VEGF/VPF was rare in normal cervix but was detectable in more than 90% of sample s of benign epithelium adjacent to CIN. Therefore, vWf staining and VEGF/VPF expression are more sensitive at identifying nearby CIN than conventional histology. This revised version was published online in August 2006 with corrections to the Cover Date.     

Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature.

Antibody-based therapy of solid tumors has met with limited success, chiefly because solid tumors are relatively impermeable to macromolecules. This problem could be circumvented by attacking the readily accessible endothelial cells of the tumor vascular bed. We have developed a model to test this "vascular targeting" approach in which cytokine gene transfection of the tumor cells causes them to induce an experimental marker selectively on tumor vascular endothelium. An anti-tumor endothelial cell immunotoxin caused complete occlusion of the tumor vasculature and dramatic regressions of large solid tumors. By contrast, a conventional anti-tumor cell immunotoxin of equivalent in vitro potency produced only minor, transient antitumor effects but, when combined, the two immunotoxins induced permanent complete remissions in over half of the animals. These experiments indicate that immunotoxins directed against recently described markers on vascular endothelial cells in human tumors could provide a general treatment for solid tumors in humans.     

Expression of VEGF(121) in gastric carcinoma MGC803 cell line

INTRODUCTION Vascular endothelial growth factor (VEGF) which is also known as vascular permeability factor (VPF) is a heparin-binding, dimeric polypeptide growth factor and a potent mitogen for endothelial cells.VEGF can stimulate the endothelial cell growth and enhance the motility through its two known receptors flt-1 and KDR[1]. Acting through these receptors, VEGF may stimulate angiogenesis and promote tumor progression. VEGF12l, as one of the four VEGF protein isoforms containing the least number of amino acids, has all the biological function of VEGF and is the ideal isoforms for further studying VEGF at molecular levels[2]. In this study, we cloned     

In vitro and in vivo angiogenesis in PC12 pheochromocytoma cells is mediated by vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels from existing vascular endothelium, is essential for tumor growth. Vascular endothelial growth factor (VEGF) is an endotheliumspecific mitogen and regulator of angiogenesis. Angiogenesis has been associated to the malignant phenotype of pheochromocytomas and is readily observed in experimental pheochromocytomas. Although VEGF gene expression has already been demonstrated in the rat PC12 cell line, the detailed mechanisms of action are not known. We have, therefore, studied angiogenesis in the rat PC12 pheochromocytoma cell line in vitro and in vivo. VEGF gene expression and accumulation of VEGF protein in cytoplasm and conditioned medium of PC12 cells was found. Conditioned medium from PC12 cells significantly increased proliferation of VEGF-dependent endothelial cells from human umbilical veins, and this effect reversed upon addition of a neutralizing anti-VEGF antibody. Dexamethasone and nerve growth factor (NGF) increased VEGF mRNA expression and accumulation of VEGF protein of PC12 subclones with established metastatic activity in vivo. PC12 cells xenotransplanted to nude mice had marked VEGF expression and induced host angiogenesis, confirmed by the presence of CD34-positive endothelial cells in the experimental PC12 tumors. When NGF-primed PC12 cells were immobilized in Matrigel supplemented with rising concentrations of the growth factor and xenotransplanted, increasing NGF resulted in tumors with smaller areas of necrosis and increased vital tumor volume. These results suggest that VEGF is a mediator of angiogenesis in the PC12 pheochromocytoma cell line, and that dexamethasone and NGF affect VEGF expression. Our data further suggest that NGF may contribute to angiogenesis in experimental pheochromocytoma.     

Endothelial surface charge of intestinal mucosal capillaries and its modulation by dextran.

Capillary endothelial surface charge was investigated by perfusing the intestinal circulation of anesthetized rats in situ with 0.1 or 10 mg/ml native ferritin (NF, pI = 3.8-4.2) or with 0.1 mg/ml cationized ferritin (CF, pI greater than 10.0) for 5 min, with or without 5% Dextran 40. Ferritin binding was quantified by electron microscopy. All electron micrographs of capillaries perfused with NF showed some NF binding. Mean NF particle densities (particles/microns) were significantly greater at vesicle necks (PDv) than elsewhere on the endothelial surface (PD). Capillaries perfused with CF showed binding in only 60% of the transverse sections examined. The binding was very marked in a large proportion of these vessels. Mean CF particle densities were significantly greater at fenestrae (PDf) than elsewhere. These results demonstrate that mucosal capillaries have a variable negative electrostatic charge on the endothelial surface and support the hypothesis that some vesicle diaphragms act as preferential attractors for anionic macromolecules. Such structures could promote transendothelial vesicular transport of albumin. Dextran significantly decreased PD for NF from 25.4 +/- 2.4 (SEM) to 13.1 +/- 0.9 and PD, PDv, and PDf for CF from 45.9 +/- 4.6 to 31.0 +/- 2.7, from 62.1 +/- 6.2 to 43.6 +/- 5.4, and from 152.9 +/- 15.5 to 114.5 +/- 8.6, respectively. The above responses result from dextran's weak interaction with the endothelial surface. Dextran reduces access of ferritin molecules to the cell surface by steric hindrance and/or electrostatic shielding of the glycocalyx.     

Molecular differentiation and specialization of vascular beds

Transport in the large and complex bodies of vertebrate organisms is mediated by extensive and highly branched tubular networks that are formed by endothelial cells. Blood vessels are responsible for systemic circulation, while the lymphatic vasculature drains extravasated plasma, proteins, particles, and cells from the interstitium. Endothelial cells of blood vessels and lymphatic vessels can be distinguished by the expression of certain molecular markers, which accompany or even contribute to functional and morphological differences. Even within the blood vessel network, some molecules and pathways selectively mark the endothelium of arteries, veins and capillaries and are thought to contribute to the differentiation of these vessels. Moreover, microvessels can acquire organ-specific specialization in response to local tissue-derived signals. This review summarizes molecular markers and pathways that are specifically expressed in the endothelium of certain vascular beds and vessel types. Special attention will be given to known functional roles in the morphogenesis of these vessels.     

Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.

Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.     

Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization.

Neovascular diseases of the retina are a major cause of blindness worldwide. Hypoxia is thought to be a common precursor to neovascularization in many retinal diseases, but the factors involved in the hypoxic neovascular response have not been fully identified. To investigate the role of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in retinal neovascularization, the expression of VEGF/VPF mRNA and protein were studied in a mouse model of proliferative retinopathy. RNA (Northern) blot analysis revealed that retinal VEGF/VPF mRNA expression increased 3-fold between 6 and 12 hr of relative retinal hypoxia and remained elevated during the development of neovascularization. In situ hybridization localized VEGF/VPF mRNA to cells bodies in the inner nuclear layer of the retina. Immunohistochemical confocal microscopy demonstrated that VEGF/VPF protein levels increase with a time course similar to that of the mRNA. The cells in the inner nuclear layer of the retina that produce VEGF/VPF were identified morphologically as Müller cells. These data suggest that VEGF/VPF expression in the retina plays a central role in the development of retinal ischemia-induced ocular neovascularization.     

VEGF gene alternative splicing: pro- and anti-angiogenic isoforms in cancer

Tumor growth and progression depend on angiogenesis, a process of new blood vessels formation from a preexisting vascular endothelium. Tumors promote angiogenesis by secreting or activating angiogenic factors that stimulate endothelial proliferation and migration and capillary morphogenesis. The newly formed blood vessels provide nutrients and oxygen to the tumor, increasing its growth. Thus, angiogenesis plays a key role in cancer progression and development of metastases. An important growth factor that promotes angiogenesis and participates in a variety of physiological and pathological processes is the vascular endothelial growth factor (VEGF-A or VEGF). Overexpression of VEGF results in increased angiogenesis in normal and pathological conditions. The existence of an alternative site of splicing at the 3′ untranslated region of the mRNA results in the expression of isoforms with a C-terminal region which are downregulated in tumors and may have differential inhibitory effects. This suggests that control of splicing can be an important regulatory mechanism of angiogenesis in cancer.     

Binding of charged ferritin to alveolar wall components and charge selectivity of macromolecular transport in permeability pulmonary edema in rats

Rat lungs were inflated and incubated in either anionic or cationic ferritin, and alveolar and capillary basement membranes were examined by electron microscopy. Cationic ferritin bound to heparan sulfate proteoglycans on the external surface of the alveolar basement membrane, whereas cationic ferritin bound to the lamina densa of the capillary basement membranes. Anionic and cationic ferritin was also perfused through the pulmonary circulation of lungs isolated from control rats and rats previously injected with alpha-naphthylthiourea, which produces permeability pulmonary edema. Neither anionic nor cationic ferritin leaked from the pulmonary capillaries in perfused controls; cationic, but not anionic, ferritin adhered to endothelial cell surfaces. In lungs with alpha-naphthylthiourea pulmonary edema, perfused for 2-15 minutes, anionic ferritin leaked from pulmonary capillaries into the alveolar interstitium and alveolar space, while cationic ferritin remained within the capillary lumen. Five times as much anionic ferritin appeared in the capillary basement membranes on the thick side of the alveolar wall, as in the alveolar basement membranes on the thin side of the alveolar wall. In alpha-naphthylthiourea lungs perfused for 45-60 minutes, cationic ferritin also leaked through the injured endothelium and bound twice as much to the alveolar as the capillary basement membranes. The negatively charged pulmonary capillary endothelium, the positively charged capillary basement membranes, and the negatively charged alveolar basement membranes may influence the transport of macromolecules from the pulmonary circulation in permeability pulmonary edema.