Background levels of neomorphic 2-hydroxyglutarate facilitate proliferation of primary fibroblasts.

Each cell types or tissues contain certain "physiological" levels of R-2-hydroxyglutarate (2HG), as well as enzymes for its synthesis and degradation. 2HG accumulates in certain tumors, possessing heterozygous point mutations of isocitrate dehydrogenases IDH1 (cytosolic) or IDH2 (mitochondrial) and contributes to strengthening their malignancy by inhibiting 2-oxoglutarate-dependent dioxygenases. By blocking histone de-methylation and 5-methyl-cytosine hydroxylation, 2HG maintains cancer cells de-differentiated and promotes their proliferation. However, physiological 2HG formation and formation by non-mutant IDH1/2 in cancer cells were neglected. Consequently, low levels of 2HG might play certain physiological roles. We aimed to elucidate this issue and found that compared to highest 2HG levels in hepatocellular carcinoma HepG2 cells and moderate levels in neuroblastoma SH-SY5Y cells, rat primary fibroblast contained low basal 2HG levels at early passages. These levels increased at late passage and likewise 2HG/2OG ratios dropped without growth factors and enormously increased at hypoxia, reaching levels compared to cancer HepG2 cells. Responses in SH-SY5Y cells were opposite. Moreover, external 2HG supplementation enhanced fibroblast growth. Hence, we conclude that low 2HG levels facilitate cell proliferation in primary fibroblasts, acting via hypoxia-induced factor regulations and epigenetic changes.

Methylenetetrahydrofolate reductase genetic polymorphisms and toxicity to 5-FU-based chemoradiation in rectal cancer.

BACKGROUND: There is a large degree of variation in tumour response and host toxicities associated with neoadjuvant chemoradiation for rectal cancer patients. We performed a complimentary pharmacogenetic study to investigate germline polymorphisms of genes involved in 5-fluorouracil (5-FU) and irinotecan pathways and their potential association with clinical outcomes and toxicities from neoadjuvant chemoradiation in patients with rectal cancer treated in a prospective genotype-directed study. METHODS: The germline DNA of 131 patients was genotyped for 10 variants in TYMS, MTHFR, DPYD, UGT1A1, ABCC1 and SLCO1B1 genes. Ninety-six patients were treated with 5-FU/radiotherapy (RT) and 35 received 5-FU/RT/irinotecan. Relationships between genetic variants and adverse events, tumour response, overall and disease-free survivals were assessed. RESULTS: MTHFR 1298A>C and MTHFR diplotypes (for 677C>T and 1298A>C) were associated with chemoradiation-related toxicity when 5-FU was used alone. MTHFR haplotypes (677C-1298C) and diplotypes (CA-TA and TA-TA) showed, respectively, a protective and a negative effect on the incidence of severe diarrhoea or mucositis. No association was observed between genetic markers and drug response. CONCLUSION: MTHFR polymorphisms can potentially predict toxicity in patients treated with 5-FU as a single chemotherapeutic drug.

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.

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 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 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.

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.

Hyperfibrinolysis in a case of myelodysplastic syndrome with leukemic spread of mast cells.

Mast cells (MC) are multipotent hemopoietic effector cells producing diverse mediators like histamine, heparin, or tissue type plasminogen activator. We report a 75-year-old male patient with myelodysplastic syndrome (MDS) of recent onset (3 months' history) associated with a massive leukemic spread of immature tryptase+ MC (tentative term: myelomastocytic leukemia). The patient presented with pancytopenia, bleeding, hypofibrinogenemia, and an increased cellular tryptase level. Moreover, an excessive elevation of plasmin-antiplasmin complexes (9,200 ng/ml; normal range: 10-150), an elevated D-dimer, and an increase in thrombin-antithrombin III complexes were found. The identity of the circulating MC was confirmed by immunophenotyping (CD117/c-kit+, CD123/IL-3R alpha-, CD11b/C3biR-), biochemical analysis (cellular ratio [ng:ng] of tryptase to histamine >1), and electron microscopy. Bone marrow (bm) examination showed trilineage dysplasia (17% blasts), 30% diffusely scattered MC, and a complex karyotype. No dense, compact MC infiltrates (mastocytosis) were detectable in bm sections. Despite hyperfibrinolysis and mediator syndrome (flushing, headache), the patient received remission induction polychemotherapy (DAV) followed by two cycles of consolidation with intermediate dose ARA-C (2 x 1 g/m2/day on days 1, 3, and 5). He entered complete remission after the first chemotherapy cycle without evidence of recurring MDS. Moreover, in response to chemotherapy, the hyperfibrinolysis and mediator syndrome resolved, and the circulating c-kit+ MC disappeared. We suggest consideration of polychemotherapy as a therapeutic option in patients with high-risk MDS of recent onset, even in the case of MC lineage involvement.

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.

Caveolae and vesiculo-vacuolar organelles in bovine capillary endothelial cells cultured with VPF/VEGF on floating Matrigel-collagen gels.

In situ vascular endothelium is characterized by many cytoplasmic vesicles (caveolae) and vacuoles. In venules these are organized into prominent clusters called vesiculo-vacuolar organelles or VVOs. VVOs provide an important pathway for plasma protein extravasation in response to vasoactive mediators. In contrast, cultured endothelial cells isolated from many sources lack VVOs and generally have few caveolae. Our goal was to preserve VVOs in cultured endothelium. Bovine adrenal microvascular endothelial cells (BCEs) cultured on floating Matrigel-collagen Type I gels with vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exhibited typical VVOs by electron microscopy. Both in vivo and in culture VVOs were caveolin-positive by immunoelectron microscopy. On the basis of caveolin immunostaining, VVOs could also be detected by light (confocal) microscopy. When BCEs were cultured without VPF/VEGF, caveolin staining was finely punctate and electron microscopy confirmed the near absence of VVOs. BCE VVOs were sensitive to N-ethylmaleimide. Other types of endothelium cultured on Matrigel-collagen gels with or without VPF/VEGF exhibited few caveolae and no VVOs. Therefore, preservation of VVOs in cultured endothelium required a specific combination of endothelial cells (BCEs), surface matrix (Matrigel-collagen), and growth factor (VPF/VEGF). These endothelial cells should be useful for in vitro studies of trans-endothelial transport.

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.

Ultrastructural analysis of human skin biopsy specimens from patients receiving recombinant human stem cell factor: subcutaneous injection of rhSCF induces dermal mast cell degranulation and granulocyte recruitment at the injection site.

We performed an ultrastructural analysis of 10 skin biopsy specimens that had been obtained from three women who were undergoing daily subcutaneous dosing with recombinant methionyl-human stem cell factor (rhSCF) as part of a phase I clinical trial. The biopsy specimens were obtained at sites of subcutaneous administration of rhSCF, within approximately 1 to 2 hours of rhSCF injection, and, at the same time, at contralateral control sites that had not been directly injected with rhSCF. We previously reported that subcutaneous dosing with rhSCF in these subjects induced the local development of a wheal and flare response, which was associated with evidence of mast cell degranulation, as well as a systemic increase in numbers of cutaneous mast cells. The present electron microscopic analysis revealed that all biopsies of swollen, erythematous rhSCF-injected sites exhibited anaphylactic degranulation of both mature and immature mast cells, an acute inflammatory response characterized by the migration of neutrophils, basophils (some of which exhibited evidence of piecemeal degranulation), and eosinophils through blood vessel walls into the perivascular and extravascular spaces, and edema and fibrin deposition within the interstitium. By contrast, the control biopsies contained no evidence of mast cell degranulation or acute inflammation. However, both control and rhSCF-injected sites exhibited mast cells that were undergoing granule building and maturation. Thus at the doses tested in these subjects, subcutaneous injection of rhSCF induced anaphylactic-type degranulation of dermal mast cells at the injection site, with an acute inflammatory response that was associated with the recruitment of granulocytes. By contrast, mast cells at sites distant from those directly injected with rhSCF exhibited no evidence of enhanced secretion.

Co-compartmentalization of MAP kinases and cytosolic phospholipase A2 at cytoplasmic arachidonate-rich lipid bodies.

Lipid bodies are inducible lipid domains abundantly present in leukocytes engaged in inflammation. They are rich in esterified arachidonate and are also potential sites for eicosanoid-forming enzyme localization. It is therefore of interest to know whether arachidonate-releasing cytosolic phospholipase A2 (cPLA2) localizes at lipid bodies. Here, we present evidence that cPLA2 and its activating protein kinases, mitogen-activated protein (MAP) kinases, co-localize at lipid bodies. U937 cells express high levels of cPLA2 and contain numerous cytoplasmic lipid bodies. Using double-labeling immunocytochemistry we demonstrated punctate cytoplasmic localizations of both cPLA2 and MAP kinases in U937 cells that were perfectly concordant with fluorescent fatty-acid-labeled lipid bodies. The co-localization of cPLA2 and MAP kinases at lipid bodies was confirmed by subcellular fractionation and immunoblot. Lipid body fractions free of cytosol and other organelles contained significant amounts of [14C]arachidonate-labeled phosphatidylcholine and cPLA2 enzymatic activities. Immunoblotting with specific antibodies identified cPLA2 as well as MAP kinases, including ERK1, ERK2, p85, and p38, in lipid bodies. The co-compartmentalization within arachidonate-rich lipid bodies of cPLA2 and its potentially activating protein kinases suggests that lipid bodies may be structurally distinct intracellular sites active in extracellular ligand-induced arachidonate release and eicosanoid formation.