Dian Feng

Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis

This Chapter has reviewed the literature concerning VPF/VEGF as a potent vascular permeabilizing cytokine. In accord with this important role, microvessels have been found to be hyperpermeable to plasma proteins and other circulating macromolecules at sites where VPF/VEGF and its receptors are overexpressed, i.e., in tumors, healing wounds, retinopathies, many important inflammatory conditions and in certain physiological processes, such as ovulation and corpus luteum formation. Moreover, microvascular hyperpermeability to plasma proteins was shown to have an important consequence: the laying down of a fibrin-rich extracellular matrix. This provisional matrix, in turn, favors and supports the ingrowth of fibroblasts and endothelial cells which, together, transform the provisional matrix into the mature stroma characteristic of tumors and healed wounds. Finally, we have considered the pathways by which these and other circulating macromolecules cross the endothelium of normal and VPF/VEGF-permeabilized microvessels. These pathways include VVOs and trans-endothelial openings that have been variously interpreted as inter-endothelial cell gaps or trans-endothelial cell pores. At least some trans-endothelial cell pores may arise from VVOs. In conclusion, these data provide new insights into the mechanisms of angiogenesis and stroma formation, insights which are potentially applicable to a wide variety of disease states and which may lead to identification of new targets for therapeutic intervention.

T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema

Within the vascular system, the mucin‐type transmembrane glycoprotein T1α/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic‐specific homeobox gene Prox1. In this study, we examined the role of T1α/podoplanin in vascular development and the effects of gene disruption in mice. T1α/podoplanin is first expressed at around E11.0 in Prox1‐positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1α/podoplanin−/− mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1α/podoplanin is also expressed in the basal epidermis of newborn wild‐type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1α/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA‐mediated inhibition of T1α/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1α/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

Vascular Permeability Factor/Vascular Endothelial Growth Factor Induces Lymphangiogenesis as well as Angiogenesis

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A164, we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A–expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A164 also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new “giant” lymphatics generated by VEGF-A164 were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.

Vascular permeability factor/vascular endothelial growth factor:A multifunctional angiogenic cytokine

VPF/VEGF is a multifunctional cytokine that contributes to angiogenesis by both direct and indirect mechanisms. On the one hand, VPF/VEGF stimulates the endothelial cells lining nearby microvessels to proliferate, to migrate and to alter their pattern of gene expression. On the other hand, VPF/VEGF renders these same microvascular endothelial cells hyperpermeable so that they spill plasma proteins into the extravascular space, leading to profound alterations in the extracellular matrix that favor angiogenesis. These same principles apply in tumors, in several examples of non-neoplastic pathology, and in physiological processes that involve angiogenesis and new stroma generation. In all of these examples, microvascular hyperpermeability and the introduction of a provisional, plasma-derived matrix precede and accompany the onset of endothelial cell division and new blood vessel formation. It would seem, therefore, that tumors have made use of fundamental pathways that developed in multicellular organisms for purposes of tissue defense, renewal and repair. VPF/VEGF, therefore, has taught us something new about angiogenesis; namely, that vascular hyperpermeability and consequent plasma protein extravasation are important--perhaps essential--elements in its generation. However, this finding raises a paradox. While VPF/VEGF induces vascular hyperpermeability, other potent angiogenic factors apparently do not, at least in sub-toxic concentrations that are more than sufficient to induce angiogenesis (Connolly et al., 1989a). Nonetheless, wherever angiogenesis has been studied, the newly generated vessels have been found to be hyperpermeable. How, therefore, do angiogenic factors other than VPF/VEGF lead to the formation of new and leaky blood vessels? We do not as yet have a complete answer to this question. One possibility is that at least some angiogenic factors mediate their effect by inducing or stimulating VPF/VEGF expression. In fact, there are already clear example of this. A number of putative angiogenic factors including small molecules (e.g. prostaglandins, adenosine) as well as many cytokines (e.g. TGF-alpha, bFGF, TGF-beta, TNF-alpha, KGF, PDGF) have all been shown to upregulate VPF/VEGF expression. Further studies that elucidate the crosstalk among various angiogenic factors are likely to contribute significantly to a better understanding of the mechanisms by which new blood vessels are formed in health and in disease.

Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is an angiogenic cytokine with potential for the treatment of tissue ischemia. To investigate the properties of the new blood vessels induced by VPF/VEGF, we injected an adenoviral vector engineered to express murine VPF/VEGF164 into several normal tissues of adult nude mice or rats. A dose-dependent angiogenic response was induced in all tissues studied but was more intense and persisted longer (months) in skin and fat than in heart or skeletal muscle (≤3 weeks). The initial response (within 18 hours) was identical in all tissues studied and was characterized by microvascular hyperpermeability, edema, deposition of an extravascular fibrin gel, and the formation of enlarged, thin-walled pericyte-poor vessels (“mother” vessels). Mother vessels developed from preexisting microvessels after pericyte detachment and basement membrane degradation. Mother vessels were transient structures that evolved variably in different tissues into smaller daughter vessels, disorganized vessel tangles (glomeruloid bodies), and medium-sized muscular arteries and veins. Vascular structures closely resembling mother vessels and each mother vessel derivative have been observed in benign and malignant tumors, in other examples of pathological and physiological angiogenesis, and in vascular malformations. Together these data suggest that VPF/VEGF has a role in the pathogenesis of these entities. They also indicate that the angiogenic response induced by VPF/VEGF is heterogeneous and tissue specific. Finally, the muscular vessels that developed from mother vessels in skin and perimuscle fat have the structure of collaterals and could be useful clinically in the relief of tissue ischemia.

The Vesiculo–Vacuolar Organelle (VVO): A New Endothelial Cell Permeability Organelle

A newly defined endothelial cell permeability structure, termed the Vesiculo–Vacuolar organelle (VVO), has been identified in the microvasculature that accompanies tumors, in venules associated with allergic inflammation, and in the endothelia of normal venules. This organelle provides the major route of extravasation of macromolecules at sites of increased vascular permeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), serotonin, and histamine in animal models. Continuity of these large sessile structures between the vascular lumen and the extracellular space has been demonstrated in kinetic studies with ultrastructural electron-dense tracers, by direct observation of tilted electron micrographs, and by ultrathin serial sections with three-dimensional computer reconstructions. Ultrastructural enzyme-affinity cytochemical and immunocytochemical studies have identified histamine and VPF/VEGF bound to VVOs in vivo in animal models in which these mediators of permeability are released from mast cells and tumor cells, respectively. The high-affinity receptor for VPF/VEGF, VEGFR-2, was localized to VVOs and their substructural components by pre-embedding ultrastructural immunonanogold and immunoperoxidase techniques. Similar methods were used to localize caveolin and vesicle-associated membrane protein (VAMP) to VVOs and caveolae, indicating a possible commonality of formation and function of VVOs to caveolae.

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.

Reinterpretation of endothelial cell gaps induced by vasoactive mediators in guinea-pig, mouse and rat: many are transcellular pores.

1 In response to vascular permeabilizing agents, particulates circulating in the blood extravasate from venules through endothelial cell openings. These openings have been thought to be intercellular gaps though recently this view has been challenged. 2 To define the precise location of endothelial cell gaps, serial section electron microscopy and three‐dimensional reconstructions were performed in skin and cremaster muscle of guinea‐Pigs, mice and rats injected locally with agents that enhance microvascular permeability: vascular permeability factor, histamine or serotonin. Ferritin and colloidal carbon were injected intravenously as soluble and particulate macromolecular tracers, respectively. 3 Both tracers extravasated from venules in response to all three permeability enhancing agents. The soluble plasma protein ferritin extravasated primarily by way of vesiculo‐vacuolar organelles (VVOs), interconnected clusters of vesicles and vacuoles that traverse venular endothelium. In contrast, exogenous particulates (colloidal carbon) and endogenous particulates (erythrocytes, platelets) extravasated from plasma through transendothelial openings. 4 Serial electron microscopic sections and three‐dimensional reconstructions demonstrated that eighty‐nine of ninety‐two openings were transendothelial pores, not intercellular gaps. Pore frequency increased 3‐ to 33‐fold when carbon was used as tracer. 5 The results demonstrate that soluble and particulate tracers extravasate from venules by apparently different transcellular pathways in response to vasoactive mediators. However, some pores may derive from rearrangements of VVOs.

Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.

Permeability properties of tumor surrogate blood vessels induced by VEGF-A

Malignant tumors generate new blood vessels by secreting growth factors, particularly members of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) family. Overall, the new blood vessels that form are hyperpermeable to plasma proteins, a property that is thought to be important for generating new stroma. However, tumor blood vessels are structurally heterogeneous and include microvessels of at least the following distinct types: mother vessels (MV), glomeruloid microvascular proliferations (GMP), arterio-venous-like vascular malformations and capillaries. Our goal was to determine whether macromolecular tracers leaked from all or from only a subset of these vessel types and to elucidate the extravasation pathways. As blood vessels are only a minor component of tumors, and therefore, difficult to study in situ, we used an adenoviral vector to express VEGF-A164, the most important member of the VPF/VEGF family, in mouse tissues. So expressed, VEGF-A164 induces large numbers of surrogate vessels of each type found in tumors in a highly reproducible manner. Overall permeability to plasma proteins was assessed qualitatively with Evans blue dye and quantitatively with a dual tracer method employing radioactive albumin. Leaky vessels were identified by confocal microscopy (FITC-dextran) and by electron microscopy (ferritin). MV, and to a lesser extent GMP, were found to be hyperpermeable but capillaries and vascular malformations were not. Ferritin extravasated primarily by two trans-cellular routes, vesiculo-vacuolar organelles (VVOs) and fenestrae. This occurred despite a considerable reduction in VVO frequency as VVO membranes translocated to the plasma membrane during MV formation. However, reduction in the number and complexity of VVOs was offset by extensive endothelial cell thinning and a greatly shortened extravasation pathway. Extrapolating these findings to tumors predicts that only a subset of tumor vessels, MV and GMP, is hyperpermeable, and that measures of overall vessel permeability greatly underestimate the permeability of individual MV and GMP.