Similar endothelial glycocalyx structures in microvessels from a range of mammalian tissues: evidence for a common filtering mechanism?

The glycocalyx or endocapillary layer on the luminal surface of microvessels has a major role in the exclusion of macromolecules from the underlying endothelial cells. Current structural evidence in the capillaries of frog mesentery indicates a regularity in the structure of the glycocalyx, with a center-to-center fiber spacing of 20 nm and a fiber width of 12 nm, which might explain the observed macromolecular filtering properties. In this study, we used electron micrographs of tissues prepared using perfusion fixation and tannic acid treatment. The digitized images were analyzed using autocorrelation to find common spacings and to establish whether similar structures, hence mechanisms, are present in the microvessel glycocalyces of a variety of mammalian tissues. Continuous glycocalyx layers in mammalian microvessels of choroid, renal tubules, glomerulus, and psoas muscle all showed similar lateral spacings at ∼19.5 nm (possibly in a quasitetragonal lattice) and longer spacings above 100 nm. Individual glycocalyx tufts above fenestrations in the first three of these tissues and also in stomach fundus and jejunum showed evidence for similar short-range structural regularity, but with more disorder. The fiber diameter was estimated as 18.8 (± 0.2) nm, but we believe this is an overestimate because of the staining method used. The implications of these findings are discussed.

Impaired vascular permeability regulation caused by the VEGF165b splice variant in pre-eclampsia.

OBJECTIVE: Pre-eclampsia is diagnosed by hypertension and proteinuria, probably caused by endothelial dysfunction, resulting in symptoms including oedema, inflammation and altered metabolism. Vascular endothelial growth factor A (VEGF-A) is detected at higher concentrations in plasma from patients with pre-eclampsia than in plasma from normotensive pregnant patients when determined by radioimmunoassay. This study tested the hypothesis that circulating VEGF-A in pre-eclamptic plasma is biologically active in vivo, and aimed to identify specific isoforms responsible for this activity. DESIGN: Plasma from pre-eclamptic (n = 17) and normotensive (n = 10) pregnant women was perfused into Rana mesenteric microvessels, and the subsequent change in microvascular permeability was measured using a single-vessel perfusion micro-occlusion technique. RESULTS: Pre-eclamptic but not normotensive plasma resulted in a 5.25 ± 0.8-fold acute increase in vascular permeability (P = 0.0003). This increase could be blocked by the incubation of plasma with bevacizumab, an antibody to VEGF-A (n = 7; P = 0012), and by VEGF-A receptor inhibition by SU5416 at doses specific to VEGF-A receptor-1 (VEGFR1), but not by the VEGF-A receptor-2 inhibitor, ZM323881. Although VEGF(165) b levels were not significantly altered in the PET samples, the increase in permeability was also inhibited by incubation of pre-eclamptic plasma with an inhibitory monoclonal antibody specific for VEGF165b (n=6; P<0.01), or by the addition of placental growth factor 1 (PlGF-1; n = 3; P < 0.001). PlGF-1 was detected at lower concentrations in pre-eclamptic plasma than in normotensive plasma. CONCLUSIONS: These findings suggest that circulating VEGF-A levels in pre-eclampsia are biologically active because of a loss of repression of VEGFR1 signalling by PlGF-1, and VEGF165b may be involved in the increased vascular permeability of pre-eclampsia.

Hydrocortisone administration for the treatment of refractory hypotension in critically ill newborns.

OBJECTIVE: The purpose of this observation was to evaluate the safety and efficacy of hydrocortisone (HC) for the treatment of refractory hypotension in term and preterm infants. A secondary purpose was to determine the utility of serum cortisol concentrations in predicting the response to treatment. STUDY DESIGN: This is a retrospective observational study of 117 infants treated with a standardized HC protocol for refractory hypotension. Refractory hypotension was defined as a mean arterial pressure (MAP) less than the gestational age (GA) despite a total inotrope dose of 20 microg per kg per min. Baseline serum cortisol concentrations were determined prior to treatment with stress dose HC. RESULT: Treatment with HC increased the MAP at 2, 6, 12 and 24 h after initiation, decreased the total inotrope dose at 6, 12 and 24 h, and was associated with resolution of oliguria. There was no correlation between the pretreatment baseline cortisol concentration and GA, birth weight or the response to treatment. The incidence of grades III to IV intraventricular hemorrhage, periventricular leukomalacia, bacterial or fungal sepsis and spontaneous intestinal perforation (SIP) after HC treatment was similar to institutional historic controls prior to institution of this standardized HC protocol. CONCLUSION: HC treatment was associated with a rapid resolution of cardiovascular compromise. The incidence of significant side effects was similar to that in previously published reports, including a comparable incidence of SIP. On the basis of our results, measuring baseline serum cortisol concentration to guide the management of refractory hypotension is unwarranted.

Influence of ionic strength, electrolyte type, and NOM on As(V) adsorption onto TiO2.

As(V) adsorption onto a commercially available TiO2 (Degussa P25) in NaCl or NaClO4 at various concentrations (0.001-0.1 M) was investigated. The effect of natural organic matter (NOM) on As(V) removal through the adsorption by TiO2 was also examined. In either electrolyte, As(V) adsorption onto TiO2 increased with the increase of ionic strength under alkaline conditions (pH 7.0-11.0). Under acidic conditions (pH 4.0-6.0), the adsorption of As(V) onto TiO2 was insensitive to ionic strength in NaClO4 electrolyte but decreased with increasing ionic strength in NaCl electrolyte. The presence of 2-15 mg/L NOM as C significantly decreased the fraction of As(V) adsorbed onto TiO2 at pH 6.0 regardless of the initial As(V) concentration (1-15 microM). The measurement of zeta potential of TiO2 with and without As(V) suggests that the presence of As(V) can shift the point of zero charge (pH(pzc)) of TiO2 to a lower pH value. The overall data presented in this study suggest that As(V) can form both inner-sphere and outer-sphere complexes on TiO2 surface, and NOM is an important factor controlling As(V) adsorption onto TiO2.

Neodymium isotope evidence for a chondritic composition of the Moon.

Samarium-neodymium isotope data for six lunar basalts show that the bulk Moon has a 142Nd/144Nd ratio that is indistinguishable from that of chondritic meteorites but is 20 parts per million less than most samples from Earth. The Sm/Nd formation interval of the lunar mantle from these data is 215(-21)(+23) million years after the onset of solar system condensation. Because both Earth and the Moon likely formed in the same region of the solar nebula, Earth should also have a chondritic bulk composition. In order to mass balance the Nd budget, these constraints require that a complementary reservoir with a lower 142Nd/144Nd value resides in Earth's mantle.

Dysregulation of the fibroblast growth factor system in major depression.

In this report we describe findings that imply dysregulation of several fibroblast growth factor (FGF) system transcripts in frontal cortical regions of brains from human subjects with major depressive disorder (MDD). This altered gene expression was discovered by microarray analysis of frontal cortical tissue from MDD, bipolar, and nonpsychiatric control subjects and was verified by quantitative real-time PCR analysis and, importantly, in a separate cohort of MDD subjects. Furthermore, we show, through a separate analysis of specific serotonin reuptake inhibitor (SSRI)-treated and non-SSRI-treated MDD subjects that the observed changes in expression of FGF transcripts are not secondary to drug treatment. Rather, changes in specific FGF transcripts are attenuated by SSRIs and may thus be partially responsible for the mechanism of action of these drugs. We also make available the gene-expression profile of all of the other growth factors and growth factor receptors detected in these postmortem samples.

Toxic cure: Hyperfractionated radiotherapy with concurrent cisplatin and fluorouracil for Stage III and IVA head-and-neck cancer in the community.

PURPOSE: To evaluate efficacy and toxicity of the Duke University chemoirradiation regimen for locally advanced head-and-neck cancer in a regional community cancer center. METHODS AND MATERIALS: Between June 1998 and June 2002, 50 patients with Stage III or IVA squamous cell carcinoma of the head and neck were treated definitively with concurrent combined modality therapy (CMT). Patients received accelerated, hyperfractionated radiotherapy (AFRT), 1.2-1.25 Gy b.i.d., to a median prescribed dose of 70 Gy. Chemotherapy consisted of cisplatin 12 mg and fluorouracil 600 mg/m(2) daily for 5 consecutive days during Weeks 1 and 6, followed by two cycles after AFRT. Patients with N2-N3 neck disease (n = 21; 42%) were considered for neck dissection depending on their response to AFRT and chemotherapy. Twenty-nine patients with Stage III and IVA disease treated between 1991 and 1997 with definitive RT alone served as historical controls. RESULTS: Forty-nine patients (98%) in the CMT group completed the prescribed AFRT and 38 (76%) completed four cycles of chemotherapy. Three of 8 patients who underwent neck dissection had a pathologically complete response. The median follow-up for all patients was 23 months. The actuarial progression-free survival rate at 2 years was 75% for the CMT group vs. 40% (p <0.01) for the RT group. The overall survival rate was 80% and 43% (p <0.01), respectively, for the CMT and RT groups. Acute Radiation Therapy Oncology Group Grade 3 toxicities for the CMT group were mucosal (n = 50; 100%), skin (n = 9; 18%), and hematologic (n = 3; 6%). Late Grade 3-4 toxicities consisted of pharyngeal stricture (n = 7; 14%), laryngeal chondritis (n = 3; 6%), osteoradionecrosis (n = 2; 4%), and peripheral neuropathy (n = 1; 2%). CONCLUSION: This aggressive regimen of AFRT with concurrent cisplatin and fluorouracil with or without neck dissection is feasible in the community setting for patients with Stage III and IVA head-and-neck cancer. Early results indicated excellent survival, albeit with universal acute mucosal, and considerable, although acceptable, late toxicity.

Measurement of hydraulic conductivity of single perfused Rana mesenteric microvessels between periods of controlled shear stress.

A new method for the determination of hydraulic conductivity in individually perfused microvessels in vivo is described. A vessel is cannulated at both ends with glass micropipettes and the fluid filtration rate across the vessel wall measured from the velocities of red cells when the pressure in the micropipettes is balanced. Hydraulic conductivity measured using this double-cannulation method (2.6 (+/- 0.9) x 10(-7) cm s(-1) cmH(2)O(-1)) was not significantly different from that measured using a previously described technique in the same vessel (2.4 (+/- 0.9) x 10(-7) cm s(-1) cmH(2)O(-1) using the Landis-Michel method). Shear stress on the vessel wall was controlled by changing the difference between the inflow and outflow pressures during periods of perfusion. The volume flow through the vessel, calculated from red cell velocity either in the vessel or in the pipette, was linearly proportional to this pressure difference. Higher flow rates could only be calculated from red cell velocities in the micropipette. There was no relationship between the imposed shear stress and intervening measurements of hydraulic conductivity (r = 0.029). This novel technique has advantages over the Landis-Michel method, which include the control of outflow resistance, the measurement of shear stress under conditions of controlled pressure, the elimination of compression damage to the vessel (since vessel occlusion is not necessary) and assessment of hydraulic conductivity over the same length of vessel throughout the experiment. The measurement of solute concentrations by indwelling micropipette electrodes and the collection of perfusate for analysis are other possibilities.

Regulation of microvascular permeability by vascular endothelial growth factors.

Generation of new blood vessels from pre-existing vasculature (angiogenesis) is accompanied in almost all states by increased vascular permeability. This is true in physiological as well as pathological angiogenesis, but is more marked during disease states. Physiological angiogenesis occurs during tissue growth and repair in adult tissues, as well as during development. Pathological angiogenesis is seen in a wide variety of diseases, which include all the major causes of mortality in the west: heart disease, cancer, stroke, vascular disease and diabetes. Angiogenesis is regulated by vascular growth factors, particularly the vascular endothelial growth factor family of proteins (VEGF). These act on two specific receptors in the vascular system (VEGF-R1 and 2) to stimulate new vessel growth. VEGFs also directly stimulate increased vascular permeability to water and large-molecular-weight proteins. We have shown that VEGFs increase vascular permeability in mesenteric microvessels by stimulation of tyrosine auto-phosphorylation of VEGF-R2 on endothelial cells, and subsequent activation of phospholipase C (PLC). This in turn causes increased production of diacylglycerol (DAG) that results in influx of calcium across the plasma membrane through store-independent cation channels. We have proposed that this influx is through DAG-mediated TRP channels. It is not known how this results in increased vascular permeability in endothelial cells in vivo. It has been shown, however, that VEGF can stimulate formation of a variety of pathways through the endothelial cell, including transcellular gaps, vesiculovacuolar organelle formation, and fenestrations. A hypothesis is outlined that suggests that these all may be part of the same process.

Openings in frog microvascular endothelium at different rates of increase in pressure and at different temperatures.

Experiments were carried out on single mesenteric capillaries and venules of pithed frogs to determine whether the rate of increase in intravascular pressure (dP/dt) influenced the critical pressure (P(B)) which increases wall permeability. Vessels, microperfused with frog Ringer solutions containing 0.1% bovine serum albumin and red cells, were occluded downstream before pressure was raised either as a ramp or in a series of 13.6 cmH2O steps. By varying step duration, the mean dP/dt could be matched to dP/dt applied as a steady ramp. P(B) was recorded as the pressure at which there was an abrupt increase in filtration with red cells passing to and through one or more sites in the vessel wall. In all vessels, increasing dP/dt raised P(B), with no differences between steps and ramps. The relation between P(B) and dP/dt was linear, consistent with a latent period, T (the slope), between a critical pressure being reached and the abrupt increase in permeability being observed. Direct observation confirmed this latent period. Between 12 and 20 (o)C, T was 8.5 +/- 0.47 s; between 0 and 5 degrees C, T was 11.5 +/- 0.97 s. Tissue cooling did not influence the time constant, tau, describing the rate of stretch of wall following a step increase in pressure and used to measure wall visco-elastic properties. Nor was the value of tau (1.15 +/- 0.06 s, n = 42) consistent with T being accounted for by visco-elasticity. It is suggested that the latent period may indicate an active response of the endothelium.

Expression of orphanin FQ and the opioid receptor-like (ORL1) receptor in the developing human and rat brain.

The orphanin peptide system, although structurally similar to the endogenous opioid family of peptides and receptors, has been established as a distinct neurochemical entity. The distribution of the opioid receptor-like (ORL1) receptor and its endogenous ligand orphanin FQ (OFQ) in the central nervous system of the adult rat has been recently reported, and although diffusely disseminated throughout the brain, this neuropeptide system is particularly expressed within stress and pain circuitry. Little is known concerning the normal expression of the orphanin system during gestation, nor how opiate or stress exposure may influence its development. Using in situ hybridization techniques, the present study was undertaken to determine the normal pattern of expression of ORL1 mRNA in the human and rat brain at various developmental stages. Rat embryos, postnatal rat brains and postmortem human brains were collected, frozen and cut into 15 microm coronal sections. In situ hybridization was performed using riboprobes generated from cDNA containing representative human and rat ORL1 and OFQ sequences. Both ORL1 and OFQ mRNA is detected as early as E12 in the cortical plate, basal forebrain, brainstem and spinal cord. Expression for both ORL1 and OFQ is strongest during the early postnatal period, remaining strong in the spinal cord, brainstem, ventral forebrain, and neocortex into the adult. Human ORL1 and OFQ expression is observed at 16 weeks gestation, remaining relatively unchanged up to 36 weeks. The influence of early orphanin expression on maturation of stress and pain circuitry in the developing brain remains unknown.

Implications of platinum-group element accumulation along U.S. roads from catalytic-converter attrition.

Automobile catalytic converters are dispersing platinum-group elements (PGEs) Rh, Pt, and Pd into the environment (1-3). This paper represents the first detailed study to assess the PGE content of soils and grasses from U.S. roadsides. These soils were analyzed using cation exchange pretreatment and ultrasonic nebulizer-ICP-MS (4). Highway and several urban sites showed Pt abundances of 64-73 ng/g immediately adjacent to the roadside, with corresponding Pd and Rh abundances of 18-31 ng/g and 3-7 ng/g, respectively. All Pt and most Pd and Rh abundances are statistically above local background soil values. Platinum, Rd, and Rh show positive correlations with traffic-related elements (Ni, Cu, Zn, and Pb) but no correlations with nontraffic-related elements (Y, Ga). Iridium and Ru show no correlations with any of these trace elements. These PGE abundances are comparable to European studies (5-7) and are approaching concentrations that would be economically viable to recover. This study also demonstrates transport of Pt statistically above background more than 50 m from the roadside. Further study is necessary to see how mobile the PGEs are in roadside environments, but these initial data indicate only Pt is taken up by plants.

Biomass byproducts for the remediation of wastewaters contaminated with toxic metals.

Pollution of the environment with toxic metals is widespread and often involves large volumes of wastewater. Remediation strategies must be designed to support high throughput while keeping costs to a minimum. Biosorption is presented as an alternative to traditional physicochemical means for removing toxic metals from wastewater. We have investigated the metal binding qualities of two biomass byproducts that are commercially available in quantity and at low cost, namely "spillage", a dried yeast and plant mixture from the production of ethanol from corn, and ground corn cobs used in animal feeds. The biomass materials effectively removed toxic metals, such as Cu, Cs, Mo, Ni, Pb, and Zn, even in the presence of competing metals likely to be found in sulfide mine tailing ponds. The effectiveness of these biosorbents was demonstrated using samples from the Berkeley Pit in Montana. Investigations included column chromatography and slurry systems, and linear distribution coefficients are presented. X-ray spectroscopy was used to identify the binding sites for metals adsorbed to the spillage material. The results of our experiments demonstrate that the biosorption of metals from wastewaters using biomass byproducts is a viable and cost-effective technology that should be included in process evaluations.

Effects of temperature on the wall strength and compliance of frog mesenteric microvessels.

In single perfused mesenteric microvessels of pithed frogs, we assessed wall strength from the critical pressure, PB, which has to be applied within the vessel in order to induce openings in the walls through which fluid and cells can extravasate. PB was determined in capillaries and venules of tissues at 12-20 The P(B) (mean +/- S.E.M.) in 22 vessels between 12 and 20 degrees C, P(B) was 92.0 +/- 7.40 cm H2O which was significantly higher than at room temperatures (P<0.001). The compliance of the vessel wall was estimated using both the red cell method and the oil meniscus technique. There was no measurable effect of temperature on wall compliance. The compliance of vessels from which the cells had been removed by previous perfusion with detergent solutions was very similar to that of intact vessels between 12 and 20 degrees C and between 0 and 5 degrees C. The negligible effects of temperature upon compliance suggest that microvessel walls have to be distended to a greater extent in cold tissue before P(B) is reached. This, together with their rapid closure, is consistent with the hypothesis that pressure-induced openings in microvascular walls are dependent on an active response of the endothelium rather than being the result of stress failure of the basement membrane.

Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding.

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the micro, kappa, and delta opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with (125)I-[(14)Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes.

Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat.

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor-like receptor (ORL-1). It and its precursor, prepro-OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro-OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes.

Openings through endothelial cells associated with increased microvascular permeability.

Rapid increases in microvascular permeability are associated with the appearance of openings in microvascular endothelium, which are believed to develop between the endothelial cells of venules. Reconstruction of these openings, from electron micrographs of serial sections of the endothelium reveal that many pass through the periphery of the endothelial cells close to intact intercellular junctions. Transcellular pathways are the principal type of opening induced in microvascular endothelium by the ionophore A23187, by VEGF, and by high transmural pressures. Some mediators induce the fusion of vacuoles with the luminal and abluminal surfaces of the endothelium, and it is suggested that the transcellular openings may develop from vacuolar channels.

Transcellular openings through frog microvascular endothelium.

Reconstructions from serial ultrathin sections of microvascular endothelium suggest that gaps, which are induced by a range of stimuli, may pass through endothelial cells as well as between them. To address the possibility that the transcellular gaps are not artefacts of aldehyde fixation, we have reconstructed fourteen gaps, induced by the ionophore A23187, in frog mesenteric microvessels where the primary fixative was osmium tetroxide. All fourteen gaps were transcellular. The different actions of osmium tetroxide and glutaraldehyde lead us to consider that it is highly unlikely that transcellular gaps are fixation artefacts.

Endothelial gaps: time course of formation and closure in inflamed venules of rats.

In the rat trachea, substance P causes rapid but transient plasma leakage. We sought to determine how closely the number, morphology, and size of endothelial gaps correspond to the time course of this leakage. Endothelial gaps were examined by scanning electron microscopy (EM), by transmission EM, or by light microscopy after silver nitrate staining. Substance P-induced leakage of the particulate tracer Monastral blue peaked at 1 min but decreased with a half-life of 0.3 min. The number of silver-stained gaps also peaked at 1 min then decreased significantly more slowly (half-life 1.9 min) than the leakage. Scanning EM revealed two types of endothelial gaps, designated vertical gaps and oblique slits. Vertical gaps predominated at peak leakage, whereas oblique slits became more common as the leakage diminished. Measurements of the mean diameter of vertical gaps made by light microscopy, scanning EM, and transmission EM were all in the range of 0.36-0.47 micron. Fingerlike endothelial cell processes that appeared during gap formation became shorter as the leakage diminished (mean length: 1.44 microns at 1 min compared with 1.06 microns at 3 min after substance P), suggesting a role in gap closure. We conclude that the plasma leakage occurring immediately after an inflammatory stimulus results from the rapid formation of endothelial gaps. Multiple factors, including alterations in gap morphology, gap closure, and changes in driving force, are likely to participate in the rapid decrease in the leakage.