Evolutionary origins of the blood vascular system and endothelium.

Every biological trait requires both a proximate and evolutionary explanation. The field of vascular biology is focused primarily on proximate mechanisms in health and disease. Comparatively little attention has been given to the evolutionary basis of the cardiovascular system. Here, we employ a comparative approach to review the phylogenetic history of the blood vascular system and endothelium. In addition to drawing on the published literature, we provide primary ultrastructural data related to the lobster, earthworm, amphioxus, and hagfish. Existing evidence suggests that the blood vascular system first appeared in an ancestor of the triploblasts over 600 million years ago, as a means to overcome the time-distance constraints of diffusion. The endothelium evolved in an ancestral vertebrate some 540-510 million years ago to optimize flow dynamics and barrier function, and/or to localize immune and coagulation functions. Finally, we emphasize that endothelial heterogeneity evolved as a core feature of the endothelium from the outset, reflecting its role in meeting the diverse needs of body tissues.

Why are tumour blood vessels abnormal and why is it important to know?

Tumour blood vessels differ from their normal counterparts for reasons that have received little attention. We report here that they are of at least six distinct types, we describe how each forms, and, looking forward, encourage the targeting of tumour vessel subsets that have lost their vascular endothelial growth factor-A (VEGF-A) dependency and so are likely unresponsive to anti-VEGF-A therapies.

RNA is closely associated with human mast cell lipid bodies.

Both novel and multiple ultrastructural studies based on different principles show relationships of cytoplasmic lipid bodies and ribonucleic acid (RNA) of potential importance to RNA metabolism in human mast cells. The methods include general ultrastructural morphological observations, imaging of RNA with an EDTA regressive stain, imaging of the incorporation of radio labeled uridine by ultrastructural autoradiography, postembedding immunogold labeling of uridine, ribosomes and small nuclear ribonuclear proteins and ultrastructural in situ hybridization detection of poly(A)-positive messenger RNA. Altogether these studies implicate human mast cell lipid bodies in RNA metabolism and are analogous to earlier similar studies which showed that human mast cell granules also curtain RNA.

Ribosomes and secretory granules in human mast cells: close associations demonstrated by staining with a chelating agent.

The distribution of ribosomes was investigated in mature human mast cells with a chelation-based staining protocol known to bleach DNA-rich structures, leaving RNA-rich structures unbleached. With this method, electron-dense ribosomes were adjacent to, attached to, and within secretory granules, which were also bleached with the chelation method that we used. The finding of these ribosome-secretory granule relationships suggests that secretory granules in mature human mast cells may participate in RNA metabolism.

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.

Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery.

Glomeruloid bodies are a defining histological feature of glioblastoma multiforme and some other tumors and vascular malformations. Little is known about their pathogenesis. We injected a nonreplicating adenoviral vector engineered to express vascular permeability factor/vascular endothelial growth factor-164 (VPF/VEGF(164)) into the ears of athymic mice. This vector infected local cells that strongly expressed VPF/VEGF(164) mRNA for 10 to 14 days, after which expression gradually declined. Locally expressed VPF/VEGF(164) induced an early increase in microvascular permeability, leading within 24 hours to edema and deposition of extravascular fibrin; in addition, many pre-existing microvessels enlarged to form thin-walled, pericyte-poor, "mother" vessels. Glomeruloid body precursors were first detected at 3 days as focal accumulations of rapidly proliferating cells in the endothelial lining of mother vessels, immediately adjacent to cells expressing VPF/VEGF(164). Initially, glomeruloid bodies were comprised of endothelial cells but subsequently pericytes and macrophages also participated. As they enlarged by endothelial cell and pericyte proliferation, glomeruloid bodies severely compromised mother vessel lumens and blood flow. Subsequently, as VPF/VEGF(164) expression declined, glomeruloid bodies devolved throughout a period of weeks by apoptosis and reorganization into normal-appearing microvessels. These results provide the first animal model for inducing glomeruloid bodies and indicate that VPF/VEGF(164) is sufficient for their induction and necessary for their maintenance.

Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells.

Isolated human lung mast cells were used to identify subcellular sites of basic fibroblast growth factor using a postembedding immunogold method. The factor was present in quantity in secretory granules and cytoplasmic lipid bodies. Cisterns of smooth endoplasmic reticulum and ribosome clusters, closely associated with lipid bodies, contained the factor as did the nuclear matrix. Factor-positive lipid bodies were adjacent to nuclear pores and often indented perinuclear cisternae. Altered secretory granules with reduced density, characteristic of secretion by piecemeal degranulation in mast cells, showed reduced gold label for basic fibroblast growth factor; small, electron-lucent (80-100 nm) transport vesicles near altered granules were labelled for the factor. Since these mature mast cells do not display extensive arrays of classical secretory organelles, such as rough endoplasmic reticulum and Golgi structures, these new subcellular localizations for basic fibroblast growth factor suggest several possible alternative release routes for a cytokine devoid of a signal sequence characteristic of regulated secretory proteins.

Ultrastructural cytochemical, immunocytochemical and in situ hybridization methods with polyuridine probes detect mRNA in human mast cell granules.

Mature human mast cells are classical secretory cells that are filled with secretory-storage granules but are poorly endowed with visible free or membrane-bound cytoplasmic ribosomes. We recently reported close associations of ribosomes and various components essential to RNA metabolism in and close to human mast cell granules using multiple ultrastructural imaging methods. In view of these findings and an increased awareness of RNA sorting and localization to specific subcellular sites and organelles, we used human mast cells purified from non-tumour portions of lung samples resected at surgery for carcinoma and ultrastructural methods to investigate this further. Poly(U) probes were used to detect direct en grid binding, and radiolabelled as well as non-radiolabelled poly(U) probes were used in in situ hybridization protocols to detect poly(A)-positive pre-mRNA and mRNA in nuclear, cytoplasmic and granular compartments of mature human mast cells. Negative controls verified specificity of label; expected nuclear and cytoplasmic locations of poly(A)-positive RNA served as positive controls for each sample. These findings lend support to the hypothesis that site-specific synthesis in secretory-storage granules may occur in secretory cells.

Ultrastructural autoradiographic analysis of RNA in isolated human lung mast cells during secretion and recovery from secretion.

BACKGROUND: Previous work has implicated isolated, control human lung mast cell granules in RNA metabolism using multiple methods of high-magnification imaging based on different mechanistic principles. These methods have demonstrated ribosomes, RNA, U1snRNP and uridine in, around and attached to secretory granules. METHODS: Here, we have extended these studies using ultrastructural autoradiography of radiolabeled uridine incorporation in degranulating and recovering mast cells. RESULTS: We found that control cells incorporated uridine into granules, with values that decreased dramatically in conjunction with stimulated histamine secretion and granule extrusion, and that granule stores of tritiated uridine increased together with the reconstitution of secretory granules in recovering mast cells. CONCLUSION: These findings support a possible new role for secretory granules in RNA metabolism in mast cell biology.

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.

RNA is closely associated with human mast cell secretory granules, suggesting a role(s) for granules in synthetic processes.

The distribution of ribosomes in mature human mast cells, a major granulated secretory cell, does not resemble that in other secretory cells, such as pancreatic acinar cells and plasma cells. By routine ultrastructural analysis, ribosomes in human mast cells are often close to, attached to, or even appear to be within secretory granules. To document better these relationships, we used multiple electron microscopic imaging methods, based on different principles, to define RNA, ribosome, and granule relationships in mature human mast cells. These methods included EDTA regressive staining, RNase digestion, immunogold labeling of ribonucleoproteins or uridine, direct binding or binding after ultrastructural in situ hybridization of various polyuridine probes to polyadenine in mRNA, and ultrastructural autoradiographic localization of [3H]-uridine incorporated into cultured human mast cells. These different labeling methods demonstrated ribosomes, RNA, U1SnRNP (a small nuclear RNP specific for alternative splicing of mRNA), mRNA, and uridine to be associated with secretory granules in human mast cells, implicating granules in a larger synthetic role in mast cell biology.

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 (< or =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.

Different pathways of macromolecule extravasation from hyperpermeable tumor vessels.

Tumor microvessels are hyperpermeable to plasma proteins, a consequence of tumor cell-secreted vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). However, the pathways by which macromolecules extravasate from tumor vessels have been little investigated. To characterize tumor vessels more precisely and to elucidate the pathways by which macromolecules extravasated from them, we studied two well-defined, VPF/VEGF-secreting murine carcinomas, MOT and TA3/St. Whether grown in ascites or solid form, MOT tumors induced large, pericyte-poor "mother" vessels whose lining endothelium developed fenestrae that involved 1.8-5.6% of the surface. Fenestrae developed in parallel with markedly reduced endothelial cell vesiculo-vacuolar organelles (VVOs). TA3/St tumors, which secreted more VPF/VEGF than MOT tumors, elicited mother vessels with unchanged VVOs and without fenestrae. In both tumors, a plasma protein tracer, ferritin, extravasated through VVOs and in MOT tumors ferritin also extravasated through fenestrae. Endothelial gaps were not observed in either tumor. Thus, not all VPF/VEGF-secreting tumors induce fenestrated endothelium. Also, VVOs provide an internal store of membrane that can be transferred to the endothelial cell surface to provide the substantial increase in plasma membrane necessary for mother vessel formation in MOT tumors. Such transfer was apparently unnecessary in TA3/St tumors in which extensive early endothelial cell division provided the increased plasma membrane necessary for forming mother vessels.

Ultrastructural immunogold cytochemistry with autoimmune human sera and an antibody to uridine implicate human mast cell granules in RNA biology.

Human mast cells are professional secretory cells that store synthetic products in large granules filling their cytoplasm. Unlike many secretory cells, the principal synthetic organelle, ribosome-rich endoplasmic reticulum, is a minor component of their cytoplasm. Sightings of nonmembrane-bound ribosomes in and near their secretory granules stimulated detailed ultrastructural studies of various RNA species to implicate secretory-storage granules in RNA biology. In the work reported here, postembedding immunogold ultrastructural cytochemistry indicates that human mast cells contain uridine, an integral ingredient of RNA, and ribonucleoproteins, known to associate with small nuclear RNAs important for splicing RNA precursors, several ribonucleoproteins with possible functions in other aspects of RNA biology and ribonucleoproteins known to associate with ribosomes. These findings should catalyse future work toward establishing the full functional repertoire of secretory-storage granules.

Ribonuclease-gold ultrastructural localization of heparin in isolated human lung mast cells stimulated to undergo anaphylactic degranulation and recovery in vitro.

BACKGROUND: Human mast cells are a rich and unique source of heparin, which is stored in cytoplasmic secretory granules and accounts for metachromasia, a staining property used to identify mast cells by light microscopy. OBJECTIVE: The sub-cellular locations of heparin in secretory human mast cells and human mast cells recovering from secretion are not known. Acquisition of this knowledge requires ultrastructural imaging of well-preserved cells with a visible probe which binds to heparin. We sought to develop this knowledge regarding human mast cell secretion by using a labelling method for heparin that depends on the well-known property of ribonuclease inhibition by heparin. METHODS: Human lung mast cells were isolated, partially purified, either stimulated or not stimulated to secrete with anti-IgE, and recovered 20 min or 6 h later for routine electron microscopy. Histamine secretion was also determined. A previously developed post-embedding, enzyme-affinity-gold electron microscopic technique to image ribonucleic acid (RNA) with ribonuclease-gold (R-G), which also binds to the enzyme inhibitor, heparin, was employed to determine the sub-cellular locations of heparin in non-secretory and secretory mast cells as well as in mast cells recovered from short-term cultures after secretion. Specificity controls for the novel use of this method and quantification of granule labelling in these controls were performed. RESULTS: Heparin was labelled by R-G in electron-dense granules within non-secretory human lung mast cells (HLMCs), in electron-dense granules that persisted in secretory HLMCs at the maximum histamine secretion time (20 min), and in electron-dense granules within recovering HLMCs. Specificity controls showed that gold alone did not label HLMCs and that absorption with heparin significantly reduced or abrogated HLMC granule staining with R-G, but that RNA absorption did not. Heparin stores were absent in newly formed, electron-lucent intracytoplasmic degranulation channels in secretory HLMCs. Electron-dense granule matrices in the process of extrusion to the cell exterior still retained heparin at the instant of cellular secretion. Non-granule heparin stores bound R-G in recovering HLMCs. These locations included resolving degranulation channels, as newly emergent granules partitioned and condensed within them, and electron-dense content-containing vesicles and progranules within synthetic mast cells. Ultimately, all known ultrastructural patterns of HLMC granules developed in recovering cells, and each of them contained heparin. CONCLUSION: Heparin was secreted from HLMCs which were stimulated by anti-IgE, and heparin was recovered by a combination of conservative and synthetic mechanisms in HLMCs after a secretory event.