Vascular labelling with monastral blue B

Vascular labelling is an established technique of experimental pathology whereby leaky vessels can be identified in vivo. A suspension of a suitable colloidal pigment is injected intravenously; the pigment is then trapped in the wall of the leaky vessels. The colloidal preparation of carbon black, which has been used for many years for this purpose, is no longer commercially available. This communication introduces a substitute: Monastral blue B which gives beautiful preparations in whole mounts, is readily visible in paraffin and plastic embedded histologic sections, has a distinctive appearance in electron micrographs, and is nontoxic in the required dosage.     

The role of perfusion washout in limb revascularization procedures

Amputated rat hindlimbs were subjected to either normothermic (26 degrees C) or hypothermic (4 degrees C) ischemia. Experimental limbs had their microcirculation washed out (either before or after the ischemic insult) with a physiologic acellular plasma substitute previously reported to enhance flap survival following extended periods of warm ischemia. Control limbs were not washed out; i.e., stagnant blood remained in these limbs. Following the ischemic interval, amputated limbs were replanted. Monastral blue B, a colloidal pigment capable of labeling leaky blood vessels, was administered systemically to all rats just prior to vascular declamping. Limb biopsies of skin and muscle were harvested 30 minutes following revascularization in order to assess Monastral labeling and, therefore, the functional integrity of the microcirculation. Results confirm that stagnant blood under conditions of warm ischemia is detrimental to the functionality of the microcirculation in both skin (p less than 0.03) and muscle (p less than 0.007). Accordingly, perfusion washout, when performed prior to the ischemic period, enhances limb survival following 6 hours of warm ischemia (p less than 0.01). Hypothermia protects against the detrimental effects of stagnant blood; perfusion offers no benefit if hypothermic conditions prevail. Physiologic mechanisms responsible for these findings are discussed.     

Vessel abnormalization: another hallmark of cancer? Molecular mechanisms and therapeutic implications

As a result of excessive production of angiogenic molecules, tumor vessels become abnormal in structure and function. By impairing oxygen delivery, abnormal vessels fuel a vicious cycle of non-productive angiogenesis, which creates a hostile microenvironment from where tumor cells escape through leaky vessels and which renders tumors less responsive to chemoradiation. While anti-angiogenic strategies focused on inhibiting new vessel growth and destroying pre-existing vessels, clinical studies showed modest anti-tumor effects. For many solid tumors, anti-VEGF treatment offers greater clinical benefit when combined with chemotherapy. This is partly due to a normalization of the tumor vasculature, which improves cytotoxic drug delivery and efficacy and offers unprecedented opportunities for anti-cancer treatment. Here, we overview key novel molecular players that induce vessel normalization.     

Blood-brain barrier disruption in the hypothalamus of young adult spontaneously hypertensive rats

Vascular permeability and endothelial glycocalyx were examined in young adult spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and Wistar Kyoto rats (WKY) as a control, in order to determine earlier changes in the blood-brain barrier (BBB) in the hypothalamus in chronic hypertension. These rats were injected with horseradish peroxidase (HRP) as an indicator of vascular permeability. Brain slices were developed with a chromogen and further examined with cationized ferritin, a marker for evaluating glycocalyx. Staining for HRP was seen around vessels in the hypothalamus of SHR and SHRSP, but was scarce in WKY. The reaction product of HRP appeared in the abluminal pits of endothelial cells and within the basal lamina of arterioles, showing increased vascular permeability in the hypothalamus of SHR and SHRSP, whereas there were no leaky vessels in the frontal cortex of SHR and SHRSP, or in both areas of WKY. The number of cationized ferritin particles binding to the capillary endothelial cells was decreased in the hypothalamus of SHR and SHRSP, while the number decreased in the frontal cortex of SHRSP, compared with those in WKY. Cationized ferritin binding was preserved in some leaky arterioles, while it was scarce or disappeared in other leaky vessels. These findings suggest that BBB disruption occurs in the hypothalamus of 3-month-old SHR and SHRSP, and that endothelial glycocalyx is markedly damaged there without a close relationship to the early changes in the BBB.     

Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina

The distributions of occludin and claudin-1, two tight junction–associated integral membrane proteins were investigated by immunohistochemical analysis of whole-mount preparations of the blood vessels in the myelinated streak of the rabbit retina. Light microscopy revealed that occludin and claudin-1 immunoreactivities were abundant along the interface of adjacent endothelial cells of all blood vessels. Electron microscopy revealed that both proteins were distributed in a regular pattern (at regular intervals of approximately 80 nm) along the length of tight junctions, probably in the regions of tight junction strands. No other structures or cell types expressed either of these two proteins in the myelinated streak. Whereas occludin immunoreactivity was concentrated only at the tight junction interface, claudin-1 immunoreactivity also extended into the cytoplasm of the endothelial cells, suggesting a different structural role for claudin-1 than for occludin at tight junctions. Retinal pigment epithelial cells expressed occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid. Also consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was expressed at only a low level in, and showed an irregular distribution along, the vessels of the choroid, a vascular bed that lacks blood-barrier properties. Further, the distribution of occludin was examined during formation and remodelling of the rat retinal vasculature. Occludin expression was evident at the leading edge of vessel formation and was found on all vessels in both the inner and outer vascular plexus. Numerous vascular segments at the early stage of vascular formation and regression lost occludin expression. The biological significance of this transient loss of occludin expression in terms of barrier function remains to be elucidated.     

2-Methoxycinnamaldehyde inhibits tumor angiogenesis by suppressing Tie2 activation

Blood vessels are mainly composed of intraluminal endothelial cells (ECs) and mural cells adhering to the ECs on their basal side. Immature blood vessels lacking mural cells are leaky; thus, the process of mural cell adhesion to ECs is indispensable for stability of the vessels during physiological angiogenesis. However, in the tumor microenvironment, although some blood vessels are well-matured, the majority is immature. Because mural cell adhesion to ECs also has a marked anti-apoptotic effect, angiogenesis inhibitors that destroy immature blood vessels may not affect mature vessels showing more resistance to apoptosis. Activation of Tie2 receptor tyrosine kinase expressed in ECs mediates pro-angiogenic effects via the induction of EC migration but also facilitates vessel maturation via the promotion of cell adhesion between mural cells and ECs. Therefore, inhibition of Tie2 has the advantage of completely inhibiting angiogenesis. Here, we isolated a novel small molecule Tie2 kinase inhibitor, identified as 2-methoxycinnamaldehyde (2-MCA). We found that 2-MCA inhibits both sprouting angiogenesis and maturation of blood vessels, resulting in inhibition of tumor growth. Our results suggest a potent clinical benefit of disrupting these two using Tie2 inhibitors.     

On the functions of the pore cells in the connective tissue of terrestrial pulmonate molluscs

Summary The fine structure of the pore cells in connective tissue in the kidney of Achatina achatina and the skin of the slug Arion hortensis is described and evidence is presented which shows that these cells, in the latter species, are involved in the synthesis of the respiratory blood pigment, haemocyanin. The involvement of these cells in phagocytosis of colloidal particles was demonstrated following introduction of ferritin and colloidal gold into the blood. The extracellular coat which surrounds the cells is permeable to ferritin, but is impermeable to colloidal gold. Following penetration of the extracellular coat the ferritin enters the sub-surface cisternae and is taken into the cells where it crystallises within membrane-bound vesicles.     

Lipopigment fine structure in human seminal vesicle and prostate gland epithelia

Tissue cultures of hepatocytes were studied for the formation of lipid pigments as related to aging of the cultures and of the addition to them of: 1) colloidal iron; 2) L-ascorbic acid; and 3) tyrosine. The presence of lipid pigments was gauged by their sudanophilia after a light solvent extraction and yellow autofluorescence. It has been found that all the three agents enhanced pigmentogenesis. It has been concluded that both pro-oxidants and antioxidants may enhance lipid pigment formation: the first by enhancing lipid oxidation, the second by enhancing copolymerization which terminates the oxidative chain.     

The blood system of the flabelligerid polychaete Flabelliderma commensalis (Moore)

The blood system of the flabelligerid polychaete, Flabelliderma commensalis has been explored by dissection, light and electron microscopy and absorption spectrophotometry. The main longitudinal vessels are the dorsal, ventral, perineural, sub-oesophageal, supra-oesophageal and heart. Each segment has a segmental vessel which communicates with the dorsal vessel in thoracic setigers and the gut sinus in abdominal setigers. Branches of the segmental vessels in setigers 2–9 supply the gonads. A blood sinus envelopes most of the gut. Circulation is maintained by the pumping of the heart which immediately supplies blood to the supra-oesophageal ganglion, the branchiae and the palps. These are paralleled by a system of collecting vessels. The sinus of the supra-oesophageal ganglion receives a number of different axonal endings, some of which may be neurosecretory. The retroperitoneal vessels in their most developed form are composed of an intima, longitudinal and circular muscles and a peritoneum. The heart vessel contains a cardiac body whose cells appear to contain vacuoles of blood pigment. The blood pigment exhibits the absorption characteristics of a chlorocruorin with maxima at 438, 558 and 606 nm.     

Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.     

Fas ligand (CD95 ligand) controls angiogenesis beneath the retina

A principal cause of blindness is subretinal neovascularization associated with age-related macular degeneration. Excised neovascular membranes from patients with age-related macular degeneration demonstrated a pattern of Fas+ new vessels in the center of the vascular complex, surrounded by FasL+ retinal pigment epithelial cells. In a murine model, Fas (CD95)-deficient (Ipr) and FasL-defective (gld) mice had a significantly increased incidence of neovascularization compared with normal mice. Furthermore, in gld mice there is massive subretinal neovascularization with uncontrolled growth of vessels. We found that cultured choroidal endothelial cells were induced to undergo apoptosis by retinal pigment epithelial cells through a Fas-FasL interaction. In addition, antibody against Fas prevented vascular tube formation of choroidal endothelial cells derived from the eye in a three-dimensional in vitro assay. Thus, FasL expressed on retinal pigment epithelial cells may control the growth and development of new subretinal vessels that can damage vision.     

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.     

Endocytosis and degradation of interstitial retinol-binding protein: differential capabilities of cells that border the interphotoreceptor matrix

Between the pigment epithelium and the outer limiting membrane of the retina is an extracellular compartment filled with the interphotoreceptor matrix (IPM). A prominent component of the IPM is a glycoprotein known as interstitial retinol-binding protein (IRBP). Using in vitro techniques, we compared the ability of the cells that border this compartment to internalize colloidal gold (CG) coated with either IRBP or ovalbumin, a glycoprotein not found in the IPM. Neither IRBP-CG nor ovalbumin-CG was internalized by the Muller's cells. Both rod and cone photoreceptors take up IRBP-CG, which is observed in small vesicles and multivesicular bodies. Neither photoreceptor type takes up ovalbumin-CG. Acid phosphatase cytochemistry indicates that acid phosphatase reaction product in the multivesicular bodies co-localizes with IRBP-CG, which suggests that this molecule is degraded by rod and cone photoreceptors and is not recycled. The pigment epithelium internalizes IRBP-CG and ovalbumin-CG, both of which remain in small cytoplasmic vesicles near the apical plasma membrane. There is no indication that vesicles that contain either IRBP-CG or ovalbumin-CG fuse with the lysosomal system in the pigment epithelial cells during the incubation.     

Leaky vessels as a potential source of stromal acidification in tumours

Malignant tumours are characterised by higher rates of acid production and a lower extracellular pH than normal tissues. Previous mathematical modelling has indicated that the tumour-derived production of acid leads to a gradient of low pH in the interior of the tumour extending to a normal pH in the peritumoural tissue. This paper uses mathematical modelling to examine the potential of leaky vessels as an additional source of stromal acidification in tumours. We explore whether and to what extent increasing vascular permeability in vessels can lead to the breakdown of the acid gradient from the core of the tumour to the normal tissue, and a progressive acidification of the peritumoural stroma. We compare our mathematical simulations to experimental results found in vivo with a tumour implanted in the mammary fat pad of a mouse in a window chamber construct. We find that leaky vasculature can cause a net acidification of the normal tissue away from the tumour boundary, though not a progressive acidification over time as seen in the experiments. Only through progressively increasing the leakiness can the model qualitatively reproduce the experimental results. Furthermore, the extent of the acidification predicted by the mathematical model is less than as seen in the window chamber, indicating that although vessel leakiness might be acting as a source of acid, it is not the only factor contributing to this phenomenon. Nevertheless, tumour destruction of vasculature could result in enhanced stromal acidification and invasion, hence current therapies aimed at buffering tumour pH should also examine the possibility of preventing vessel disruption.     

Cancer-stroma targeting therapy by cytotoxic immunoconjugate bound to the collagen 4 network in the tumor tissue

Some cytotoxic immunoconjugates have been approved for malignant lymphoma, a representative of hypervascular and stroma-poor tumors. However, many human solid tumors possess abundant intercellular stromata that prevent diffusion of cancer cell-specific monoclonal antibodies (mAb) and become a barrier preventing immunoconjugates from directly attacking cancer cells. Here we show the successful development of a new strategy that overcomes this drawback and achieves a highly localized concentration of a topoisomerase I inhibitor, SN 38, by conjugating it via an ester bond to a mAb targeted against collagen 4, a plentiful component of the tumor stroma. Poly(ethylene glycol) (PEG) was utilized as a spacer, close to each bond, to maintain stability in the blood. Immunoconjugates selectively extravasated from leaky tumor vessels and minimally from normal vessels because the immunoconjugates are too large to pass through normal vessel walls. Stroma-targeting immunoconjugates bound to the stroma to create a scaffold, from which sustained release of cytotoxic agent occurred and the agent subsequently diffused throughout the tumor tissue to damage both tumor cells and vessels. Cancer-stroma-targeting immunoconjugate therapy was thus validated as a new modality of oncological therapy, especially for refractory, stromal-rich cancers.     

PEDF: anti-angiogenic guardian of ocular function

Sight-threatening eye diseases can be caused and exacerbated by the aberrant growth of new blood vessels. Recent work indicates that this neovascularization not only is a response to a rise in the local concentration of molecules that induce such angiogenesis but also requires a fall in the levels of endogenous molecules that inhibit angiogenesis. One of the most potent of these endogenous inhibitors is pigment epithelium-derived factor (PEDF), which serves as a survival factor for neuronal components of the eye as well as an essential inhibitor of the growth of ocular blood vessels. Its anti-angiogenic activity is selective in that it is effective against newly forming vessels but spares existing ones, and it is reversible. The molecular basis for this delicate control of endothelial cells is beginning to be understood and strategies to test the ability of PEDF to ameliorate or prevent vessel damage in the eye are developing rapidly.     

Labelling of regenerating retinal pigment epithelium by colloidal iron oxide and ferritin conjugated to wheat germ agglutinin

Summary In order to determine if there are biochemical changes in plasma-membrane oligosaccharides of regenerating retinal pigment epithelium, the binding of colloidal iron oxide at low pH and ferritin-conjugated wheat germ agglutinin — probes of sialic acid and N-acetylglucosamine on the cell surface — was examined electron-microscopically. An animal model of retinal pigment epithelium regeneration — rabbits with sodium iodate induced retinopathy — was used. In this model, large expanses of regenerating pigment epithelium are present for comparison with zones of spared pgiment epithelium in the same animals. In thin sections examined by transmission electron microscopy, ferritin-conjugated wheat germ agglutinin appeared to bind more intensely to the exposed plasma membrane of regenerating retinal pigment epithelium than to spared pigment epithelium, or that of normal rabbits. Morphometry verified this. Colloidal iron oxide intensely labelled the plasma membranes of regenerating, spared, and normal pigment epithelium, and was visibly reduced after exposure of tissue to neuraminidase. The observations indicate that the plasma membrane of regenerating retinal pigment epithelium bears sialic acid and N-acetylglucosamine residues as in normal retinal pigment epithelium. However, the amount of plasma membrane bearing exposed N-acetylglucosamine increases during regeneration.     

Essential role of the coxsackie- and adenovirus receptor (CAR) in development of the lymphatic system in mice

The coxsackie- and adenovirus receptor (CAR) is a cell adhesion molecule predominantly associated with epithelial tight junctions in adult tissues. CAR is also expressed in cardiomyocytes and essential for heart development up to embryonic day 11.5, but not thereafter. CAR is not expressed in vascular endothelial cells but was recently detected in neonatal lymphatic vessels, suggesting that CAR could play a role in the development of the lymphatic system. To address this, we generated mice carrying a conditional deletion of the CAR gene (Cxadr) and knocked out CAR in the mouse embryo at different time points during post-cardiac development. Deletion of Cxadr from E12.5, but not from E13.5, resulted in subcutaneous edema, hemorrhage and embryonic death. Subcutaneous lymphatic vessels were dilated and structurally abnormal with gaps and holes present at lymphatic endothelial cell-cell junctions. Furthermore, lymphatic vessels were filled with erythrocytes showing a defect in the separation between the blood and lymphatic systems. Regionally, erythrocytes leaked out into the interstitium from leaky lymphatic vessels explaining the hemorrhage detected in CAR-deficient mouse embryos. The results show that CAR plays an essential role in development of the lymphatic vasculature in the mouse embryo by promoting appropriate formation of lymphatic endothelial cell-cell junctions.     

Immediate pigment darkening: its evolutionary roles may include protection against folate photosensitization

The evolution of dark human skin colors in tropical areas is possibly related to photoprotection of folates. However, natural folates absorb mainly UVB radiation, and too little UVB can penetrate down to folates in dermal vessels to cause serious damage. However, endogenous photosensitizers, like riboflavin and uroporphyrin, absorbing UVA and visible light, can cause photosensitization of folates. Immediate pigment darkening (IPD), generated by UVA, has an absorption spectrum covering those of the endogenous photosensitizers. IPD is most prominent for darker skin types, which were typical for populations living under tropical solar fluences. We here propose that the biological role of IPD is protection of folates against photodegradation, which would be of large evolutionary importance for early hominids.     

Mapping of mutations affecting pyoverdine production in Pseudomonas aeruginosa

Abstract Pyoverdine, the yellow-green fluorescent pigment produced by Pseudomonas aeruginosa , is a highly efficient siderophore. Pyoverdine-deficient ( pvd ) mutants of P. aeruginosa PAO isolated after mutagenesis were non-fluorescent and unable to grow in the presence of 2.8 mM ethylenediamine-di-( o -hydroxyphenylacetate) (EDDHA). Addition of purified pyoverdine to media containing EDDHA restored growth of pvd mutants. 6 pvd mutations were mapped between catA and mtu -9002 (at 65–70 min on the chromosome map) by R68.45-mediated conjugation. 2 slightly leaky pvd mutations were localised between argC and strA (at 35 min) by transduction. Thus, we have identified at least 2 genes or gene clusters required for pyoverdine production in P. aeruginosa .