Kathryn Pyne

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.

Ultrastructural Localization of Platelet Endothelial Cell Adhesion Molecule (PECAM-1, CD31) in Vascular Endothelium

The distribution of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium has been disputed. Originally reported to be highly concentrated at interendothelial cell contacts, recent studies have claimed that CD31 is distributed evenly over the entire endothelial cell surface. We re-investigated this question with two different murine anti-CD31 antibodies (MEC 13.3 and M-20), using a pre-embedding immunonanogold electron microscopic procedure that allowed precise label quantitation. MEC 13.3 reacted strongly with the luminal and abluminal plasma membranes of the endothelial cells lining microvessels in normal tissues and in angiogenic vessels induced by a tumor and vascular endothelial growth factor (VEGF-A164). Lateral plasma membranes were significantly less labeled. Conversely, M-20 strongly labeled the cytoplasmic face of the lateral plasma membranes of endothelial cells, although sparing specialized junctions, and only weakly labeled the luminal and abluminal plasma membranes. Both antibodies stained a significant minority of vesicles and vacuoles comprising the vesiculovacuolar organelle (VVO). Neither antibody was reactive in CD31-null mice. We conclude that CD31 is distributed over the entire endothelial cell surface, exclusive of specialized junctions, and in VVOs, but is not equally accessible to different antibodies in all locations.

Platelets Exit Venules by a Transcellular Pathway at Sites of F–Met Peptide–Induced Acute Inflammation in Guinea Pigs

Platelets maintain the integrity of vascular endothelium, but also appear outside of blood vessels in pathological states such as acute inflammation. However, it is widely believed that platelets extravasate from blood vessels only as the result of endothelial injury and that, on contacting extravascular collagen, they undergo a morphologically defined activation sequence and release their granule contents. We here report that platelets may cross intact venular endothelium without exhibiting this release reaction or injury. Platelets became adherent to the luminal surface of venular endothelium within ∼15 min of intradermal injection of 10–5M N–formyl–methionyl–leucyl–phenylalanine in guinea pig flank skin. Individual intact platelets were noted in large endothelial cell cytoplasmic vacuoles from which they subsequently migrated abluminally. They then crossed the vascular basal lamina and entered the dermis without exhibiting evidence of a release reaction. Serial electron–microscopic sections confirmed that the cytoplasmic vacuoles within which platelets crossed endothelial cells were independent of interendothelial cell junctions which remained normally closed. Platelets extended pseudopods and gave other evidence of cell motility. These findings require a paradigm shift in our thinking about platelet movement and functions.

Histamine-releasing activity (HRA): III. HRA induces human basophil histamine release by provoking noncytotoxic granule exocytosis

Histamine-releasing activity (HRA) is an approximately 10,000-15,000 dalton, protease-sensitive factor that induces the rapid liberation of histamine from human basophils. Production of HRA by human peripheral blood mononuclear cells in vitro is augmented by concanavalin A or antigen, suggesting a mechanism whereby lymphocytes may regulate basophil mediator release in vivo. In order to determine whether HRA provokes conventional exocytosis of basophil granules or, alternatively, results in mediator release by some other mechanism such as vesicular transport or cytotoxicity, we investigated the ultrastructural features of human blood basophils purified over Percoll and exposed to HRA in vitro. HRA preparations induced a noncytotoxic pattern of basophil degranulation very similar to that previously observed in basophils triggered to release histamine in response to specific antigen, C5a, or mannitol. Thus, cytoplasmic granules were extruded singly through multiple separate points of fusion between perigranular membranes and the plasma membrane. Degranulating basophils exhibited plasma membrane activation but lacked a polarized configuration. By contrast, those basophils exposed to HRA that did not exhibit evidence of degranulation displayed a single elongated cellular process. The development of this polarized cellular configuration, similar in some respects to that of uropod-bearing motile guinea pig basophils, may have reflected chemokinetic or chemotactic effects of preparations containing HRA activity.

Regular articleHistamine-releasing activity (HRA): III. HRA induces human basophil histamine release by provoking noncytotoxic granule exocytosis

Histamine-releasing activity (HRA) is an approximately 10,000-15,000 dalton, protease-sensitive factor that induces the rapid liberation of histamine from human basophils. Production of HRA by human peripheral blood mononuclear cells in vitro is augmented by concanavalin A or antigen, suggesting a mechanism whereby lymphocytes may regulate basophil mediator release in vivo. In order to determine whether HRA provokes conventional exocytosis of basophil granules or, alternatively, results in mediator release by some other mechanism such as vesicular transport or cytotoxicity, we investigated the ultrastructural features of human blood basophils purified over Percoll and exposed to HRA in vitro. HRA preparations induced a noncytotoxic pattern of basophil degranulation very similar to that previously observed in basophils triggered to release histamine in response to specific antigen, C5a, or mannitol. Thus, cytoplasmic granules were extruded singly through multiple separate points of fusion between perigranular membranes and the plasma membrane. Degranulating basophils exhibited plasma membrane activation but lacked a polarized configuration. By contrast, those basophils exposed to HRA that did not exhibit evidence of degranulation displayed a single elongated cellular process. The development of this polarized cellular configuration, similar in some respects to that of uropod-bearing motile guinea pig basophils, may have reflected chemokinetic or chemotactic effects of preparations containing HRA activity.