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Excessive vascular endothelial growth factor-A (VEGF-A) signaling induces vascular leakage and angiogenesis in diseases. VEGFR2 trafficking to the cell surface, mediated by kinesin-3 family protein KIF13B, is essential to respond to VEGF-A when inducing angiogenesis. However, the precise mechanism of how KIF13B regulates VEGF-induced signaling and its effects on endothelial permeability is largely unknown. Here we show that KIF13B-mediated recycling of internalized VEGFR2 through Rab11-positive recycling vesicle regulates endothelial permeability. Phosphorylated VEGFR2 at the cell-cell junction was internalized and associated with KIF13B in Rab5-positive early endosomes. KIF13B mediated VEGFR2 recycling through Rab11-positive recycling vesicle. Inhibition of the function of KIF13B attenuated phosphorylation of VEGFR2 at Y951, SRC at Y416, and VE-cadherin at Y685, which are necessary for endothelial permeability. Failure of VEGFR2 trafficking to the cell surface induced accumulation and degradation of VEGFR2 in lysosomes. Furthermore, in the animal model of the blinding eye disease wet age-related macular degeneration (AMD), inhibition of KIF13B-mediated VEGFR2 trafficking also mitigated vascular leakage. Thus, the present results identify the fundamental role of VEGFR2 recycling to the cell surface in mediating vascular permeability, which suggests a promising strategy for mitigating vascular leakage associated with inflammatory diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Central nervous system (CNS) dysfunction induced by sepsis and pathogenic microbial infections is reported to be closely associated with increased permeability of the blood-brain barrier (BBB), which is mainly mediated by the stimulation of lipopolysaccharide (LPS) on inflammatory signaling. Midazolam is a novel sedative acting on the benzodiazepine receptor, which is recently reported to exert a neuroprotective effect by inhibiting inflammation. The present study will explore the potential repair capacity of Midazolam on LPS-induced damage to the BBB. The in vivo mice model was established by intraperitoneal injection of LPS, while the in vitro model was constructed by stimulating endothelial cells utilizing LPS. We found that the increased malondialdehyde (MDA) level and reduced superoxide dismutase (SOD) activity in the brain cortices, promoted serum concentration of inflammatory factors, and elevated BBB permeability were found in the LPS group, all of which were dramatically reversed by 1 mg/kg and 2 mg/kg Midazolam. Interestingly, Midazolam increased the expression of the tight junction protein zonula occludens-1 (ZO-1). In LPS-challenged in vitro human brain microvascular endothelial cells (HBMECs), the increased concentration of inflammatory factors, reduced trans-endothelial electrical resistance (TEER) level, elevated relative value of trans-endothelial permeability, and downregulated ZO-1 were observed, all of which were pronouncedly alleviated by Midazolam, accompanied by the inhibition on the Ras homolog family member A/ Rho-kinase 2 (RhoA/ROCK-2) pathway. Furthermore, the regulatory effects of Midazolam on ZO-1 expression and the endothelial monolayer permeability in LPS-challenged HBMECs were abolished by the overexpression of RhoA. Collectively, our data imply that Midazolam ameliorated the impairment of the BBB against LPS by regulating the RhoA/ROCK2 pathway., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Changes in pulmonary microhemodynamics during modelling of pulmonary thromboembolism against the background of nebivolol and mirabegron pretreatment were studied in isolated perfused rabbit lungs. In both cases, the pulmonary artery pressure and precapillary and pulmonary vascular resistance increased to a greater extent than in control animals, but the increase in capillary hydrostatic pressure was less pronounced. The postcapillary resistance did not change in pulmonary embolism against the background of nebivolol administration and increased in case of mirabegron pretreatment; capillary filtration coefficient after nebivolol pretreatment increased less markedly than after mirabegron administration. The increase in capillary filtration coefficient after activation of β 3 -adrenoceptors with the specified drugs depended on the ratio of constriction of pulmonary veins, capillary hydrostatic pressure, and endothelial permeability.

Stroke is the leading cause of disability among adults as well as the 2nd leading cause of death globally. Ischemic stroke accounts for about 85% of strokes, and currently, tissue plasminogen activator (tPA), whose therapeutic window is limited to up to 4.5 h for the appropriate population, is the only FDA approved drug in practice and medicine. After a stroke, a cascade of pathophysiological events results in the opening of the blood-brain barrier (BBB) through which further complications, disabilities, and mortality are likely to threaten the patient's health. Strikingly, tPA administration in eligible patients might cause hemorrhagic transformation and sustained damage to BBB integrity. One must, therefore, delineate upon stroke onset which cellular and molecular factors mediate BBB permeability as well as what key roles BBB rupture plays in the pathophysiology of stroke. In this review article, given our past findings of mechanisms underlying BBB opening in stroke animal models, we elucidate cellular, subcellular, and molecular factors involved in BBB permeability after ischemic stroke. The contribution of each factor to stroke severity and outcome is further discussed. Determinant factors in BBB permeability and stroke include mitochondria, miRNAs, matrix metalloproteinases (MMPs), immune cells, cytokines, chemokines, and adhesion proteins. Once these factors are interrogated and their roles in the pathophysiology of stroke are determined, novel targets for drug discovery and development can be uncovered in addition to novel therapeutic avenues for human stroke management.

Enterovirus A71 (EV-A71) is the major cause of severe hand-foot-and-mouth diseases (HFMD), especially encephalitis and other nervous system diseases. EV-A71 capsid protein VP1 mediates virus attachment and is the important virulence factor in the EV-A71pathogenesis. In this study, we explored the roles of VP1 in the permeability of blood-brain barrier (BBB). Sera albumin, Evans blue, and dextran leaked into brain parenchyma of the 1-week-old C57BL/6J mice intracranially injected with VP1 recombinant protein. VP1 also increased the permeability of the brain endothelial cells monolayer, an in vitro BBB model. Tight junction protein claudin-5 was reduced in the brain tissues or brain endothelial cells treated with VP1. In contrast, VP1 increased the expression of virus receptor vimentin, which could be blocked with VP1 neutralization antibody. Vimentin expression in the VP1-treated brain endothelial cells was regulated by TGF-β/Smad-3 and NF-κB signal pathways. Moreover, vimentin over-expression was accompanied with compromised BBB. From these studies, we conclude that EV-A71 virus capsid protein VP1 disrupted BBB and increased virus receptor vimentin, which both may contribute to the virus entrance into brain and EV-A71 CNS infection.

Intracerebral hemorrhage (ICH) causes blood-brain barrier (BBB) damage along with altered element levels in the brain. BBB permeability was quantified at 3, 7, and 14 days with Evans Blue dye after collagenase-induced ICH in rat. At peak permeability (day 3), a gadolinium (Gd)-based contrast agent was injected to further characterize BBB disruption, and X-ray fluorescence imaging (XFI) was used to map Gd, Fe, Cl, and other elements. XFI revealed that Ca, Cl, Gd, and Fe concentrations were significantly elevated, whereas K was significantly decreased. Therefore, using Gd-XFI, we co-determined BBB dysfunction with alterations in the metallome, including those that contribute to cell death and functional outcome. Warfarin was administered 3 days post-ICH to investigate whether additional or new bleeding occurs during peak BBB dysfunction, and hematoma volume was assessed on day 4. Warfarin administration prolonged bleeding time after a peripheral cut-induced bleed, but warfarin did not worsen hematoma volume. Accordingly, extensive BBB leakage occurred after ICH, but did not appear to affect total hematoma size.

This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria-host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.

The utility of a circulatory three-compartment model for the assessment of tissue uptake kinetics is tested by comparison with the respective distributed models using pharmacokinetic data of rocuronium in patients These minimal physiologically based models have a common structure consisting of two subsystems representing the lung and the lumped systemic circulation, with two regions, the vascular and tissue space. The distributed models are based on either diffusion-limited tissue distribution, permeability-limited tissue uptake or the assumption of an empirical transit time density function. With a deviation in the estimate of the permeability-surface area product (PS) of about 18 %, the compartmental approach appears as a useful alternative on condition that a priori knowledge of cardiac output is included. It is also shown that the distribution clearance calculated from the parameters of a mammillary compartment model changes proportional to PS and can be used as an indirect measure of permeability-limited tissue uptake of drugs.

The blood-spinal cord barrier (BSCB) plays important roles in the recovery of spinal cord injury (SCI), and caveolin-1 is essential for the integrity and permeability of barriers. Basic fibroblast growth factor (bFGF) is an important neuroprotective protein and contributes to the survival of neuronal cells. This study was designed to investigate whether bFGF is beneficial for the maintenance of junction proteins and the integrity of the BSCB to identify the relations with caveolin-1 regulation. We examined the integrity of the BSCB with Evans blue dye and fluorescein isothiocyanate-dextran extravasation, measured the junction proteins and matrix metalloproteinases, and evaluated the locomotor function recovery. Our data indicated that bFGF treatment improved the recovery of BSCB and functional locomotion in contusive SCI model rats, reduced the expression and activation of matrix metalloproteinase-9, and increased the expressions of caveolin-1 and junction proteins, including occludin, claudin-5, p120-catenin, and β-catenin. In the brain, in microvascular endothelial cells, bFGF treatment increased the levels of junction proteins, caveolin-1 small interfering RNA abolished the protective effect of bFGF under oxygen-glucose deprivation conditions, and the expression of fibroblast growth factor receptor 1 and co-localization with caveolin-1 decreased significantly, which could not be reversed by bFGF treatment. These findings provide a novel mechanism underlying the beneficial effects of bFGF on the BSCB and recovery of SCI, especially the regulation of caveolin-1., Competing Interests: The authors declare no conflict of interest.

Traumatic brain injury is a serious cause of morbidity and mortality worldwide. After traumatic brain injury, the blood-brain barrier, the protective barrier between the brain and the intravascular compartment, becomes dysfunctional, leading to leakage of proteins, fluid, and transmigration of immune cells. As this leakage has profound clinical implications, including edema formation, elevated intracranial pressure and decreased perfusion pressure, much interest has been paid to better understanding the mechanisms responsible for these events. Various molecular pathways and numerous mediators have been found to be involved in the intricate process of regulating blood-brain barrier permeability following traumatic brain injury. This review provides an update to the existing knowledge about the various pathophysiological pathways and advancements in the field of blood-brain barrier dysfunction and hyperpermeability following traumatic brain injury, including the role of various tight junction proteins involved in blood-brain barrier integrity and regulation. We also address pitfalls of existing systems and propose strategies to improve the various debilitating functional deficits caused by this progressive epidemic.

Hypoglycemia is a common and serious problem among patients with type 1 diabetes receiving treatment with insulin. Clinical studies have demonstrated that hypoglycemic edema is involved in the initiation of hypoglycemic brain damage. However, the mechanisms of this edema are poorly understood. Vascular endothelial growth factor (VEGF), a potent regulator of blood vessel function, has been observed an important candidate hormone induced by hypoglycemia to protect neurons by restoring plasma glucose. Whether VEGF has a protective effect against hypoglycemia-induced damage in brain endothelial cells is still unknown. To investigate the effects of hypoglycemia on cerebral microvascular endothelial cells and assess the protective effect of exogenous VEGF on endothelial cells during hypoglycemia, confluent monolayers of the brain endothelial cell line bEnd.3 were treated with normal (5.5 mM glucose), hypoglycemic (0, 0.5, 1 mM glucose) medium or hypoglycemic medium in the presence of VEGF. The results clearly showed that hypoglycemia significantly downregulated the expression of claudin-5 in bEnd.3 cells, without affecting ZO-1 and occludin expression and distribution. Besides, transendothelial permeability significantly increased under hypoglycemic conditions compared to that under control conditions. Moreover, the hypoglycemic medium in presence of VEGF decreased endothelial permeability via the inhibition of claudin-5 degradation and improved hypoglycemia-induced cell toxicity. Furthermore, Glucose transporter-1 (Glut-1) and apoptosis regulator Bcl-2 expression were significantly upregulated. Taken together, hypoglycemia can significantly increase paraendocellular permeability by downregulating claudin-5 expression. We further showed that VEGF protected brain endothelial cells against hypoglycemia by enhancing glucose passage, reducing endothelial cell death, and ameliorating paraendocellular permeability.

Minimal physiologically based pharmacokinetic (mPBPK) models provide a simple and sensible approach that incorporates physiological elements into pharmacokinetic (PK) analysis when only plasma data are available. With this modeling concept, a second-generation mPBPK model was further developed with specific accommodations for the unique PK properties of monoclonal antibodies (mAb). This study applied this model to extensively survey mAb PK in man in order to seek general perspectives on mAb distributional and elimination features. Profiles for 72 antibodies were successfully analyzed with this model. The model results provide assessment regarding: (1) predominant clearance site, in plasma or interstitial fluid (ISF); (2) mAb ISF concentrations in two groups of lumped tissues with continuous (V(tight)) or fenestrated (V(leaky)) vascular endothelium; (3) Transcapillary escape rate (TER), an indicator of systemic vascular permeability. For 93% of surveyed mAbs, the model assuming clearance from plasma (CL) produced better or at least equivalent model performance than the model with clearance from ISF and yielded most consistent values of vascular reflection coefficients (σ1 and σ2) among all antibodies. The average mAb ISF concentration in V(tight) and V(leaky) at equilibrium was predicted to be about 6.8 and 37.9% of that in plasma. A positive correlation was detected between plasma clearance and TER among most mAbs, which could be interpreted as both parameters having common determinants related to ISF tissue distribution in this model context. The mAbs with relative higher plasma clearance (>0.035 L/h/70 kg) did not reveal such positive correlation between clearance and TER, implying that the factors contributing to high clearance may not necessarily increase tissue distribution and penetration. In conclusion, this mPBPK model offers a more mechanistic approach for analyzing plasma mAb PK than compartment models and generates parameters providing useful intrinsic distributional and elimination insights for a large number of mAbs that were examined in man.

Rapid first pass uptake of drugs is necessary to increase tissue deposition after intraarterial (IA) injection. Here we tested whether brain tissue deposition of a nanoparticulate liposomal carrier could be enhanced by coordinated manipulation of liposome surface charge and physiological parameters, such as IA injection during transient cerebral hypoperfusion (TCH). Different degrees of blood-brain barrier disruption were induced by focused ultrasound in three sets of Sprague-Dawley rats. Brain tissue retention was then compared for anionic, cationic, and charge-neutral liposomes after IA injection combined with TCH. The liposomes contained a non-exchangeable carbocyanine membrane optical label that could be quantified using diffuse reflectance spectroscopy (DRS) or visualized by multispectral imaging. Real-time concentration-time curves in brain were obtained after each liposomal injection. Having observed greater tissue retention of cationic liposomes compared to other liposomes in all three groups, we tested uptake of cationic liposomes in C6 tumor bearing rats. DRS and multispectral imaging of postmortem sections revealed increased liposomal uptake by the C6 brain tumor as compared to non-tumor contralateral hemisphere. We conclude that regional deposition of liposomes can be enhanced without BBB disruption using IA injection of cationic liposomal formulations in healthy and C6 tumor bearing rats.

Blood brain barrier (BBB) disruption is used (pre)clinically as a measure for brain tumor malignancy and grading. During treatment it is one of the parameters followed rigorously to assess therapeutic efficacy. In animal models, both invasive and non-invasive methods are used to determine BBB disruption, among them Evans blue injection prior to sacrifice and T1-weighted magnetic resonance imaging (MRI) post contrast injection. In this study, we have assessed the BBB integrity with the methods mentioned above in two experimental high grade glioma models, namely the GL261 mouse glioblastoma model and the Hs683 human oligodendroglioma model. The GL261 model showed clear BBB integrity loss with both, contrast-enhanced (CE) MRI and Evans blue staining. In contrast, the Hs683 model only displayed BBB disruption with CE-MRI, which was not evident on Evans blue staining, indicating a limited BBB disruption. These results clearly indicate the importance of assessing the BBB integrity status using appropriate methods. Especially when using large therapeutic molecules that have difficulties crossing the BBB, care should be taken with the appropriate BBB disruption assessment studies.

Vascular integrity is a critical parameter in normal growth and development. Loss of appropriate vascular barrier function is present in various immune- and injury-mediated pathological conditions. CD44 is an adhesion molecule expressed by multiple cell types, including endothelial cells (EC). The goal of the present study was to examine how loss of CD44 affected vascular permeability. Using C57BL/6 WT and CD44-KO mice, we found no significant permeability to Evan's Blue in either strain at baseline. However, there was significantly increased histamine-induced permeability in CD44-deficient mice compared to WT counterparts. Similar results were observed in vitro, where CD44-deficient endothelial monolayers were also impermeable to 40kD-FITC dextran in the absence of vasoactive challenge, but exhibited enhanced and prolonged permeability following histamine. However, CD44-KO monolayers have reduced baseline barrier strength by electrical resistance, which correlated with increased permeability, at baseline, to smaller molecular weight 4-kD FITC-dextran, suggesting weakly formed endothelial junctions. The CD44-KO EC displayed several characteristics consistent with impaired barrier function/dysfunctional EC junctions, including differential expression, phosphorylation, and localization of endothelial junction proteins, increased matrix metalloprotease expression, and altered cellular morphology. Reduced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression by CD44-KO EC in vivo and in vitro was also observed. Reconstitution of murine CD44 or PECAM-1 restored these defects to near WT status, suggesting CD44 regulates vascular permeability and integrity through a PECAM-1 dependent mechanism.

Endometriosis is an angiogenesis-dependent disease. Many studies demonstrated inhibition of angiogenesis leads to inhibition of endometriotic growth, however underlying mechanism is still not fully understood. Our previous study suggested vascular endothelial growth factor C (VEGF-C) as a target of anti-angiogenesis therapy for endometriosis. In this study, VEGF-C in endometrium and its role in angiogenesis of endometriosis were studied. Human endometrium were obtained from women with and without endometriosis for molecular studies. VEGF-A, VEGF-B, VEGF-C and VEGF-D mRNA and proteins in eutopic and ectopic endometrium were measured. Human endothelial cells were transfected with VEGF-C siRNA in vitro, effects of VEGF-C on endothelial cell migration, invasion and tube formation were investigated in vitro. Angiogenesis was inhibited in wild type mice, vascular permeability in dermal skin was determined in vivo. Transplanted endometrium were inhibited by VEGF-C siRNA in immunocompromised mice, development, growth and angiogenesis of the experimental endometriosis were compared in vivo. The results showed that VEGF-C mRNA and protein were increased in eutopic and ectopic endometrium of endometriosis patients. VEGF-C siRNA significantly inhibited endothelial cell migration and tube formation. VEGF-C siRNA significantly inhibited development and angiogenesis of the experimental endometriotic lesions in mice. Supplementation and over-expression of VEGF-C significantly reversed the inhibitory effects on the endothelial functions, vascular permeability and endometriotic growth. In conclusion, VEGF-C is increased in endometrium and it promotes endothelial functions, vascular permeability and development of experimental endometriosis. VEGF-C is important for angiogenesis in endometriosis.

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease models.

Experimental data and advanced imaging techniques are increasingly enabling the extraction of detailed vascular anatomy from biological tissues. Incorporation of anatomical data within perfusion models is non-trivial, due to heterogeneous vessel density and disparate radii scales. Furthermore, previous idealised networks have assumed a spatially repeating motif or periodic canonical cell, thereby allowing for a flow solution via homogenisation. However, such periodicity is not observed throughout anatomical networks. In this study, we apply various spatial averaging methods to discrete vascular geometries in order to parameterise a continuum model of perfusion. Specifically, a multi-compartment Darcy model was used to provide vascular scale separation for the fluid flow. Permeability tensor fields were derived from both synthetic and anatomically realistic networks using (1) porosity-scaled isotropic, (2) Huyghe and Van Campen, and (3) projected-PCA methods. The Darcy pressure fields were compared via a root-mean-square error metric to an averaged Poiseuille pressure solution over the same domain. The method of Huyghe and Van Campen performed better than the other two methods in all simulations, even for relatively coarse networks. Furthermore, inter-compartment volumetric flux fields, determined using the spatially averaged discrete flux per unit pressure difference, were shown to be accurate across a range of pressure boundary conditions. This work justifies the application of continuum flow models to characterise perfusion resulting from flow in an underlying vascular network.

The present study evaluates, both functionally and biochemically, brain tumor-induced alterations in brain capillary endothelial cells. Brain tumors were induced in Balb/c mice via intracranial injection of Lewis Lung carcinoma cells into the right hemisphere of the mouse brain using stereotaxic apparatus. Blood-brain barrier (BBB) permeability was assessed at various stages of tumor development, using both radiolabeled tracer permeability and magnetic resonance imaging with gadolinium diethylene-triamine-pentaacetate contrast enhancement (Gad-DTPA). The expression of the drug efflux transporter, P-glycoprotein (P-gp), in the BBB at various stages of tumor development was also evaluated by Western blot and immunohistochemistry. Median mouse survival following tumor cell injection was 17 days. The permeability of the BBB to (3)H-mannitol was similar in both brain hemispheres at 7 and 10 days post-injection. By day 15, there was a twofold increase in (3)H-mannitol permeability in the tumor bearing hemispheres compared to the non-tumor hemispheres. Examination of BBB permeability with Gad-DTPA contrast enhanced MRI indicated cerebral vascular permeability changes were confined to the tumor area. The permeability increase observed at the later stages of tumor development correlated with an increase in cerebral vascular volume suggesting angiogenesis within the tumor bearing hemisphere. Furthermore, the Gad-DPTA enhancement observed within the tumor area was significantly less than Gad-DPTA enhancement within the circumventricular organs not protected by the BBB. Expression of P-gp in both the tumor bearing and non-tumor bearing portions of the brain appeared similar at all time points examined. These studies suggest that although BBB integrity is altered within the tumor site at later stages of development, the BBB is still functional and limiting in terms of solute and drug permeability in and around the tumor.

18β-Glycyrrhetinic acid (GA) is a major metabolite of glycyrrhizin (GL), which is one of the components of glycyrrhiza root, a constituent herb of the traditional Japanese medicine yokukansan. It is well known that most GL is metabolized to GA in the intestine by bacteria. A previous in vitro study using cultured rat cortical astrocytes suggested that GA activates glutamate transport, which is a putative mechanism of the psychotropic effect of yokukansan. To activate the glutamate transport in the brain, GA must be absorbed into the blood after oral administration of yokukansan and then cross the blood-brain barrier (BBB) to reach the brain. However, there is no data on the BBB permeability of GA derived from yokukansan. In the present study, the BBB permeability of GA was investigated in both in vivo and in vitro studies. In the in vivo study, GA was detected in the plasma, brain, and cerebrospinal fluid of rats orally administered yokukansan. In the in vitro study using a BBB model composed of co-culture of endothelial cells, pericytes, and astrocytes, the permeability rate and apparent permeability coefficient of GA were found to be 13.3 ± 0.5 % and 16.5 ± 0.7 × 10(-6) cm/s. These in vivo and in vitro results suggest that GL in orally administered yokukansan is absorbed into the blood as GA, and then reaches the brain through the BBB. This evidence further supports the possibility that GA is an active component in the psychotropic effect of yokukansan.

A nonlinear, coupled biphasic-mass transport model that includes transvascular fluid exchange is proposed for flow-controlled infusions in brain tissue. The model accounts for geometric and material nonlinearities, a hydraulic conductivity dependent on deformation, and transvascular fluid exchange according to Starling's law. The governing equations were implemented in a custom-written code assuming spherical symmetry and using an updated Lagrangian finite-element algorithm. Results of the model indicate that, using normal physiological values of vascular permeability, transvascular fluid exchange has negligible effects on tissue deformation, fluid pressure, and transport of the infused agent. As vascular permeability may be increased artificially through methods such as administering nitric oxide, a parametric study was conducted to determine how increased vascular permeability affects flow-controlled infusion. Increased vascular permeability reduced both tissue deformation and fluid pressure, possibly reducing damage to tissue adjacent to the infusion catheter. Furthermore, the loss of fluid to the vasculature resulted in a significantly increased interstitial fluid concentration but a modestly increased tissue concentration. From a clinical point of view, this increase in concentration could be beneficial if limited to levels below which toxicity would not occur. However, the modestly increased tissue concentration may make the increase in interstitial fluid concentration difficult to assess in vivo using co-infused radiolabeled agents.

We have previously shown that activation of RhoA by bradykinin (BK) is associated with cytoskeleton rearrangement, tight junction (TJ) protein disassembly, and an increase in blood-tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). Subsequently, we investigated whether Rho-kinases (ROCKs), a family of downstream effectors of activated RhoA known to stimulate F-actin rearrangement, play a key role in the above-mentioned processes in RBMECs. Our study uses primary RBMECs as an in vitro BTB model and a specific ROCK inhibitor (Y-27632) and ROCK II small interfering RNA (siRNA) to establish whether ROCK plays a role in the process of TJ opening by BK. Y-27632 and ROCK II siRNA could partially inhibit endothelial leakage and restored normal transendothelial electric resistance (TEER) values in RBMECs. A shift in occludin and claudin-5 distribution from insoluble to soluble fractions was prevented by Y-27632. Additionally, Y-27632 inhibited BK-induced relocation of occludin and claudin-5 from cellular borders into the cytoplasm as well as stress fiber formation in RBMECs. A time-dependent increase in phosphorylated myosin light chain (p-MLC) and phosphorylated cofilin (p-cofilin) by BK was observed, which was also inhibited by Y-27632. An increase in ROCK activity by BK was inhibited by Y-27632. ROCK's contribution to BK-induced stress fiber formation is associated with TJ disassembly and an increase in BTB permeability.

Vascular remodeling plays a pivotal role in a variety of pathophysiological conditions where hypoxia and inflammation are prominent features. Intravascular ATP, ADP and adenosine are known as important regulators of vascular tone, permeability and homeostasis, however contribution of purinergic signalling to endothelial cell growth and angiogenesis remains poorly understood. By using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitia of control and chronically hypoxic neonatal calves, these studies were aimed to evaluate the effect of hypoxia on biochemical and functional properties of microvascular endothelial network at the sites of angiogenesis. In comparison with normoxic controls, VVEC from hypoxic animals are characterized by (1) drastically impaired nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5'-nucleotidase/CD73 activities with respective increases in basal extracellular ATP and ADP levels (2) higher proliferative responses to low micromolar concentrations of ATP and ADP; and (3) enhanced permeability and disordered adenosinergic control of vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran). Together, these results suggest that unique pattern of purine-mediated angiogenic activation and enhanced leakiness of VVEC from chronically hypoxic vessels may be defined by disordered endothelial nucleotide homeostasis at sites of active neovascularization.

This study was performed to determine whether low frequency ultrasound (LFU) irradiation, Papaverine (PA) infusion and combination LFU irradiation with PA infusion opened the blood-tumor barrier (BTB) by affecting tight junctions (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin and caludin-5. In a rat brain glioma model, we found that the mRNA and protein expression levels of ZO-1, occludin and claudin-5 were decreased by LFU irradiation and PA infusion. LFU-induced and PA-induced decrease of ZO-1, occludin and claudin-5 was further decreased after combining LFU irradiation with PA infusion. Immunohistochemistry assay showed that the decreased expression of ZO-1, occludin and claudin-5 was the most obvious in the tumor capillaries. Meanwhile, Evans blue assay showed that the permeability of BTB was increased, and transmission electron microscopy (TEM) indicated that TJ was opened. This led to the conclusion that LFU irradiation and PA infusion together can open the BTB by paracellular pathway. Significantly down-regulated expression levels of ZO-1, occludin and claudin-5 might be one of the molecular mechanisms of combining LFU and PA enhancing the permeability of BTB.

Objective: This study aimed to examine (1) whether polarity protein partitioning defective-3 (PARD-3) was expressed in endothelial cells (ECs) and contributed to endothelial barrier integrity and (2) whether altered PARD-3 expression and distribution were associated with disturbed endothelial junction protein VE-cadherin expression induced by factors derived from preeclamptic (PE) placentas., Methods: PARD-3 and VE-cadherin expressions were examined by immunofluorescent staining and Western blot in confluent ECs and in ECs treated with normal and PE placental conditioned medium (CM). Protein-protein interactions between PARD-3/VE-cadherin, PARD-3/ atypical protein kinase C (aPKCλ), and VE-cadherin/aPKCλ were examined by immuno-precipitation and immunobloting., Results: Similar to VE-cadherin, PARD-3 is localized at the cell contacts in control ECs. Both PARD-3 and VE-cadherin expressions were markedly reduced in cells treated with PE-CM for 2h, but not in cells treated with normal-CM compared to non-treated controls. Cytosol staining of VE-cadherin and PARD-3 was pronounced in cells after 24h treatment with PE-CM. PARD-3/VE-cadherin and PARD-3/aPKCλ complexes were detected in PE-CM treated cells, but not in untreated control cells and in cells after recovery. In contrast, VE-cadherin/aPKCλ complex was detected in control cells and in cells after recovery, but not in PE-CM treated cells., Conclusions: Polarity protein PARD-3 is localized at cell contacts. Factors-derived from PE placentas not only interrupt junction protein VE-cadherin distribution, but also perturb polarity protein PARD-3 expression and distribution in ECs. The results of PARD-3/VE-cadherin and PARD-3/aPKCλ complexes formation in cells treated with placental CM suggest that factors-derived from placenta could interfere both junction protein and polarity protein functions in ECs.

The development of hypercholesterolemia is a multifactorial process in which elevated plasma cholesterol levels play a central role. This study analyzed the variability of the expression and activity of myosin light chain kinase (MLCK) and endothelial permeability in the artery wall of rabbits after feeding the animals with a normal or a high-cholesterol diet. Hypercholesterolemia was induced by a high-cholesterol diet for 4 weeks. Aortas were removed and analyzed for endothelial permeability and MLCK expression. Samples of the arterial media were analyzed for MLCK activity and expression. A selective MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7) were used in hypercholesterolemia rabbit (1 mg/kg body weight). The aortas of high-cholesterol diet rabbits showed an increase in MLCK expression and activity (nearly threefold compare with control) as well as endothelial permeability. ML7 inhibit MLC phosphorylation and MLCK activity (nearly twofold compare with control) and endothelial permeability stimulated by cholesterol. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and activity through which endothelial permeability is increased.

During the early stages of angiogenesis, following stimulation of endothelial cells by vascular endothelial growth factor (VEGF), the vascular wall is breached, allowing high molecular weight proteins to leak from the vessels to the interstitial space. This hallmark of angiogenesis results in deposition of a provisional matrix, elevation of the interstitial pressure and induction of interstitial convection. Albumin, the major plasma protein appears to be an innocent bystander that is significantly affected by these changes, and thus can be used as a biomarker for vascular permeability associated with angiogenesis. Traditionally, albumin leak in superficial organs was followed by colorimetry or morphometry with the use of albumin binding vital dyes. Over the last years, the introduction of tagged-albumin that can be detected by various imaging methods, such as magnetic resonance imaging and positron emission tomography, opened new possibilities for quantitative three dimension dynamic analysis of permeability in any organ. Using these tools it is now possible to follow not only vascular permeability, but also interstitial convection and lymphatic drain. Active uptake of tagged albumin by caveolae-mediated endocytosis opens the possibility for using labeled albumin for vital staining of cells and cell tracking. This approach was used for monitoring recruitment of perivascular stroma fibroblasts associated with tumor angiogenesis.

Circulating protein C (PC) plays a vital role as an anti-coagulant and anti-inflammatory mediator. We show here that human endothelial cells produce PC that acts through novel mediators to enhance their own functional integrity. When endogenous PC or its receptor, endothelial protein C receptor (EPCR), was suppressed by small interfering (si) RNA, human umbilical cord endothelial cell (HUVEC) proliferation was decreased and apoptosis elevated. Interestingly, PC or EPCR siRNA significantly increased HUVEC permeability, which is likely via reduction of the angiopoietin (Ang)1/Ang2 ratio and inhibition of the peripheral localization of the tight junction protein, zona occludins-1. In addition, PC or EPCR siRNA inhibited type IV collagen and matrix metalloproteinase-2, providing the first evidence that PC contributes to vascular basement membrane formation. These newly described actions of endogenous PC act to stabilize endothelial cells and enhance barrier function, to potentially promote the functional integrity of blood vessels.

A physical theory explaining the anisotropic dispersion of water and solutes in biological tissues is introduced based on the phenomena of Taylor dispersion, in which highly diffusive solutes cycle between flowing and stagnant regions in the tissue, enhancing dispersion in the direction of microvascular flow. An effective diffusion equation is derived, for which the coefficient of dispersion in the axial direction (direction of capillary orientation) depends on the molecular diffusion coefficient, tissue perfusion, and vessel density. This analysis provides a homogenization that represents three-dimensional transport in capillary beds as an effectively one-dimensional phenomenon. The derived dispersion equation may be used to simulate the transport of solutes in tissues, such as in pharmacokinetic modeling. In addition, the analysis provides a physically based hypothesis for explaining dispersion anisotropy observed in diffusion-weighted imaging (DWI) and diffusion-tensor magnetic resonance imaging (DTMRI) and suggests the means of obtaining quantitative functional information on capillary vessel density from measurements of dispersion coefficients. It is shown that a failure to account for flow-mediated dispersion in vascular tissues may lead to misinterpretations of imaging data and significant overestimates of directional bias in molecular diffusivity in biological tissues. Measurement of the ratio of axial to transverse diffusivity may be combined with an independent measurement of perfusion to provide an estimate of capillary vessel density in the tissue.

The olfactory ensheathing (glial) cells (OECs) have been identified to be useful candidate cells to support regeneration after being transplanted into injured fiber tracts of the central nervous system. We investigated by means of immunocytochemistry and freeze-fracturing the morphology and molecular composition of OEC tight junctions in the rat olfactory system. In addition, we tested the hypothesis whether tight junctions and orthogonal arrays of particles (OAPs) which contain the water channel protein aquaporin-4 (AQP4), are mutually exclusive as suggested in previous studies. In OECs, we found neither OAPs nor AQP4, but tight junctions immunoreactive for ZO-1, occludin, and claudin-5, but immunonegative for ZO-2 and claudin-3. To shed more light on the function of OEC tight junctions, we tested the permeability and tight junction composition of blood vessels and fila olfactoria. We found them both, permeable for infused lanthanum nitrate, and to be immunopositive for ZO-1 and claudin-5. The tight junctions of the OECs are discussed to be responsible for micro-compartmentalization within the olfactory fiber tract providing a benefit for axonal growth.

From studies using macrovascular endothelium, it was concluded that Rho A activation generally leads to endothelial barrier breakdown. Here, we characterized the role of Rho GTPases in endothelial barrier regulation in four different cell lines, both microvascular and macrovascular. Rho A activation by cytotoxic necrotizing factor y (CNFy) induced stress fiber formation in all cell lines. This was paralleled by gap formation and barrier breakdown in microvascular mesenteric endothelial cells (MesEnd), human dermal microvascular endothelial cells (HDMEC) as well as in macrovascular pulmonary artery endothelial cells (PAEC) but not in microvascular myocardial endothelial cells (MyEnd). In MyEnd cells, activation of Rac 1 and Cdc42 by CNF-1 strengthened barrier properties whereas in MesEnd, HDMEC and PAEC all three GTPases were activated which increased permeability in PAEC but not in MesEnd and HDMEC. In PAEC, CNF-1-induced decrease of barrier properties was blocked by the Rho kinase inhibitor Y27632 indicating that co-activation of Rho A dominated the barrier response. Inactivation of Rac 1 by toxin B or by lethal toxin (LT) compromised barrier properties in all cell lines. Taken together, Rac 1 requirement for endothelial barrier maintenance but not the destabilizing role of Rho A seems to be ubiquitous.

The vascular system has the critical function of supplying tissues with nutrients and clearing waste products. To accomplish these goals, the vasculature must be sufficiently permeable to allow the free, bidirectional passage of small molecules and gases and, to a lesser extent, of plasma proteins. Physiologists and many vascular biologists differ as to the definition of vascular permeability and the proper methodology for its measurement. We review these conflicting views, finding that both provide useful but complementary information. Vascular permeability by any measure is dramatically increased in acute and chronic inflammation, cancer, and wound healing. This hyperpermeability is mediated by acute or chronic exposure to vascular permeabilizing agents, particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A). We demonstrate that three distinctly different types of vascular permeability can be distinguished, based on the different types of microvessels involved, the composition of the extravasate, and the anatomic pathways by which molecules of different size cross-vascular endothelium. These are the basal vascular permeability (BVP) of normal tissues, the acute vascular hyperpermeability (AVH) that occurs in response to a single, brief exposure to VEGF-A or other vascular permeabilizing agents, and the chronic vascular hyperpermeability (CVH) that characterizes pathological angiogenesis. Finally, we list the numerous (at least 25) gene products that different authors have found to affect vascular permeability in variously engineered mice and classify them with respect to their participation, as far as possible, in BVP, AVH and CVH. Further work will be required to elucidate the signaling pathways by which each of these molecules, and others likely to be discovered, mediate the different types of vascular permeability.

(1) The blood-brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.

Vascular permeability is closely linked with angiogenesis in a number of pathologies. In the retina, the normally well-developed blood-retinal barrier is altered in a host of eye diseases preceding or commensurate with angiogenesis. This review examines the literature regarding the tight junction complex that establishes the blood-retinal barrier focusing on the transmembrane proteins occludin and the claudin family and the membrane associated protein zonula occludens. The changes observed in these proteins associated with vascular and epithelial permeability is discussed. Finally, novel literature addressing the link between the tight junction complex and angiogenesis is considered.

Malignant brain tumors, such as glioblastoma, are characterized by extensive angiogenesis and permeability of the blood-brain barrier (BBB). The infiltration of glioma cells away from the primary tumor mass is a pathological characteristic of glial tumors. The infiltrating tumor cells represent a significant factor in tumor recurrence following surgical debulking, radiation, and chemotherapy treatments. Vascular endothelial growth factor (VEGF)-mediated vascular permeability (VP) has been associated with the progression of glioma tumor growth and infiltration into surrounding normal brain parenchyma. While VEGF induces a robust VP response in control mice (src+/+ or src+/-), the VP response is blocked in src-/- mice that demonstrate a 'leakage-resistant phenotype' in the brain. We used the Src-deficient mouse model to determine the role of Src in the maintenance of the BBB following orthotopic implantation and growth of glioma cells in the brain. Although solid tumor growth was the same in control and src-/- mice, the infiltrating component of glioma growth was reduced in src-/- mice. Characterization of the expression and localization of the extracellular matrix (ECM) protein fibrinogen was evaluated to determine the effect of a Src-mediated VP defect in the host compartment. These studies indicate that the reduced VP of host brain blood vessels of src-/- mice mediates a reduction in glioma cell invasion in a mouse brain tumor xenograft model.

We have demonstrated previously that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in mouse microvascular myocardial endothelial (MyEnd) cells in vitro as well as in rat mesenteric microvessels in vivo. In this study, we tested the hypothesis that specific activation of Rac-1 would stabilize microvascular endothelial barrier functions. For this purpose we used Escherichia coli Cytotoxic necrotizing factor (CNF-1) under conditions (300 ng/ml, 120 min) where it strongly activated Rac-1 and Cdc42 but not Rho A in MyEnd cells. Under these conditions, CNF-1 induced translocation of the actin-binding proteins cortactin and vasodilator-stimulated phosphoprotein (VASP) to cell junctions, increased the junction-associated actin filament belt, and reduced monolayer permeability. We also tested the effect of CNF-1 on endothelial barrier properties in vivo using single-perfused mesenteric microvessels. In contrast to cultured microvascular monolayers, CNF-1 did not reduce baseline barrier functions assayed as hydraulic conductivity (Lp). However, following 120 min pretreatment, CNF-1 significantly attenuated the peak Lp increase in response to platelet-activating factor (PAF, 10 nM) to 12.6+/-4 x 10(-7) cm/(s cmH(2)O) compared to 46.2+/-10 x 10(-7) cm/(s cmH(2)O) in experiments using PAF alone. These experiments indicate that activation of Rac-1 and Cdc42 stabilizes microvascular endothelial barrier functions in vitro and in vivo, likely by increasing the junction-associated actin cytoskeleton.

Neuropilin 1 (NRP1) is a co-receptor for vascular endothelial growth factor (VEGF(165)), an inducer of vascular permeability and angiogenesis. Numerous physiological factors enhance VEGF expression and function but only a few have been shown to be negative regulators. Previously, we have shown that the naturally occurring soluble form of NRP1 (sNRP1) inhibits binding of VEGF(165) to endothelial cells in vitro and impairs tumor growth in vivo. To investigate the role of sNRP1 in the regulation of vascular development and function, sNRP1 expression was targeted to the skin, where it is not normally expressed, using a keratin 14 (K14) promoter expression construct. K14-sNRP1 transgenic mice displayed normal skin architecture with a subtle abnormal vascular phenotype. While the overall number of skin blood vessels remained unchanged, the lumen size of smooth muscle-associated dermal vessels was reduced. K14-sNRP1 mice had reduced vascular permeability in response to VEGF(165), but also to VEGF(121) and platelet activating factor, suggesting that the lack of permeability was not solely due to the sequestration of VEGF. sNRP1 also reversed the increase in inflammation and edema induced by transgenic VEGF overexpression in cutaneous delayed-type hypersensitivity reactions. In summary, sNRP1 appears to primarily regulate vessel permeability while its effect on physiological angiogenesis is less evident in this model.

1. The aim of the present study was to reveal the effect of transient forebrain ischemia on the regional and temporal changes in the permeability of the blood-brain barrier (BBB) permeability for sodium fluorescein (MW: 376 Da) and Evan's blue-labeled albumin (MW: 67 kDa) in stroke-prone spontaneously hypertensive rats (SHRSP). 2. BBB permeability was significantly higher in the brain regions of 16-week-old control SHRSP than those in age-matched normotensive Wistar-Kyoto rats. 3. Transient forebrain ischemia evoked by 10-min bilateral carotid occlusion increased the permeability of the BBB for albumin, but not for sodium fluorescein, after 6 and 24 h of reperfusion in brain regions of SHRSP. 4. Extravasation of serum macromolecules may contribute to neuronal loss and development of hypertensive encephalopathy in SHRSP.

Unlabelled: Microcirculation viviperception and fluorescent tracer techniques were used and digital image analysis applied for quantitative measurement of fluorescein sodium (FiNa) permeability in microvessels of the cerebral pia mater. The diffusion permeation equations of FiNa within the blood vessels and through the vessel walls into the perivascular tissue of normal rats and ischemic rats were established with two vessels as study object, thus the permeation speed equations under different ischemic conditions were deduced. Based on analysis of the results, we deduced the relation between the permeability and the intersection angle of two vessels., Result: logarithm model showed a good fit of the experiment data. The permeation equation showed logarithmic distribution and then tended towards stability. FiNa could pass through microvessel walls with the highest speed in the one-hour ischemic rat group, and the permeation speed of FiNa in rats receiving reperfusion after twelve hours of ischemia was much faster than that in normal rats., Conclusion: the method can be useful for quantitative analysis of cerebral pia mater microvascular permeability

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.

We previously applied intravital lectin perfusion in mouse models to elucidate mechanisms underlying vascular permeability. The present work transfers this technique to human models, analysing vascular permeability in macro- and microvessels. Human vascular endothelial surface carbohydrate biochemistry differs significantly from its murine counterpart, lacking alpha-galactosyl epitopes and expressing the L-fucose moiety in the glycocalyx; the poly-N-lactosamine glycan backbone is common to all mammals. We examined extensively lectin binding specificities in sections and in vivo, and then applied the poly-N-lactosamine-specific lectin LEA and the L-fucose-specific lectin UEA-I in human intravital perfusions. Transendothelial transport differed in macrovessels and microvessels. In microvessels of adult human fat tissue, rectal wall and rectal carcinomas, slow transendothelial transport by vesicles was followed by significant retention at the subendothelial basement membrane; paracellular passage was not observed. Passage time exceeded 1 h. Thus we found barrier mechanisms resembling those we described previously in murine tissues. In both adult and fetal macrovessels, the vena saphena magna and the umbilical vein, respectively, rapid passage across the endothelial lining was observed, the tracer localising completely in the subendothelial tissues within 15 min; vesicular transport was more rapid than in microvessels, and retention at the subendothelial basement membrane briefer.

Clinical and basic research has increased our knowledge of the actions of the vasoactive hormone angiotensin II (Ang II), showing that it has multifunctional properties beyond its hemodynamic effects. It is commonly accepted that Ang II is a growth factor that participates in pathological settings, including renal diseases. However, a new aspect of this peptide is coming into focus: its potential role as a proinflammatory modulator. In this review, we summarize the apparently confusing information about the properties of Ang II and discuss its relations to the inflammatory process, as well as the potential mechanisms mediated by activation of nuclear transcription factors. Ang II seems to participate in the key events of the inflammatory response: First, it increases vascular permeability (via prostaglandins and vascular endothelial cell growth factor/vascular permeability factor) thus initiating the inflammatory process. Second, it participates in the recruitment of infiltrating cells into the tissues through direct activation of the inflammatory cells or by regulation of the expression of adhesion molecules and chemokines by resident cells. Finally, Ang II may contribute to tissue repair by regulation of cell growth and matrix synthesis. The renin-angiotensin system (RAS) thus appears to serve as a vascular inflammatory regulator and may even participate in immunologically-induced inflammation. However, more often activation of the RAS has an undesirable outcome, such as overhealing, because the inflammatory repair response itself involves a functionally imperfect system.

Use of contrast enhancement in conjunction with magnetic resonance (MR) imaging provides a means to evaluate tissue function, as well as morphology. Moreover, physiologic properties derived from kinetic analysis of dynamic contrast-enhanced data can improve the specificity of MR examinations. In this study, quantitative analysis of microvascular characteristics based on dynamic MR imaging were performed both for malignant and benign lesions using two types of contrast agents (CAs). A new MR macromolecular contrast medium (MMCM), 24 gadolinium-tetraazacyclododecanetetraacetic acid (DOTA)-dendrimer, was found to have a greater ability to distinguish benign from malignant lesions. When a blood pool agent was used, permeability differences in the two types of lesions were the most significant findings among all parameters considered.

Transection of the dorsal part of the spinal cord at the Th9 level disturbs permeability of blood vessels of thigh skin in rats. This effects is significantly inhibited by neurotoxic doses of capsaicin, which attests to the involvement of peptide-containing neurons. The data confirm the possibility of central modulation of the effector function of capsaicin-sensitive neurons.

1. The fluid homeostasis of the brain depends both on the endothelial blood-brain barrier and on the epithelial blood-cerebrospinal fluid (CSF) barrier located at the choroid plexuses and the outer arachnoid membrane. 2. The brain has two fluid environments: the brain interstitial fluid, which surrounds the neurons and glia, and the CSF, which fills the ventricles and external surfaces of the central nervous system. 3. CSF acts as a fluid cushion for the brain and as a drainage route for the waste products of cerebral metabolism. 4. Recent findings suggest that CSF may also act as a "third circulation" conveying substances secreted into the CSF rapidly to many brain regions.

Myofilament Ca2+ desensitization contributes to the contractile dysfunction of ischemic/reperfused ("stunned") myocardium. We examined the presence of reduced Ca2+ sensitivity of isometric force in chemically skinned fibers obtained from stunned myocardium using different modes of applying the detergent Triton X-100. Langendorff-perfused rat hearts underwent 20 min ischemia/20 min reperfusion, which caused a 35 +/- 3% decrease in left ventricular developed pressure, compared to continuously perfused control hearts. Stunned and control hearts were randomly allocated to two different permeabilization protocols: In group A, trabeculae were dissected and immersed in skinning solution containing 1% Triton X-100 for 20 min. Group B hearts remained fixed to the aortic cannula and skinning solution was infused retrogradely for 6 min prior to dissection of trabeculae. Extraction of cytosolic marker proteins was more complete in group-B than in group-A preparations. Group-A preparations from stunned hearts exhibited significant Ca2+ desensitization (pCa50 = 5.07 and 5.15 in stunned and control myocardium, respectively). In group B no such difference was observed, all preparations showing higher Ca2+ sensitivity and maximum force than group-A preparations (pCa50 = 5.32 in stunned versus 5.33 in control hearts). Prolonging group-A skinning to 150 min also abolished the difference in Ca2+ sensitivity between stunned and control myocardium. In conclusion, compared to a conventional protocol, skinning by perfusion results in more complete permeabilization and better preservation of myocardial contractile function. Ischemia/reperfusion at this moderate degree of contractile dysfunction induces Ca2+ desensitization at least partially by factors that can be extracted by thorough skinning.

Translocation of bacteria from the intestine causes local and systemic infection in severe acute pancreatitis. Increased intestinal permeability is considered a promoter of bacterial translocation. The mechanism leading to increased gut permeability may involve impaired intestinal capillary blood flow. The aim of this study was to evaluate and correlate early changes in capillary blood flow and permeability of the colon in acute rodent pancreatitis of graded severity. Edematous pancreatitis was induced by intravenous cerulein; necrotizing pancreatitis by intravenous cerulein and intraductal glycodeoxycholic acid. Six hours after induction of pancreatitis, the permeability of the ascending colon was assessed by the Ussing chamber technique; capillary perfusion of the pancreas and colon (mucosal and subserosal) was determined by intravital microscopy. In mild pancreatitis, pancreatic capillary perfusion remained unchanged (2.13 c 0.06 vs. 1.98 +/-0.04 nl x min(-1) x cap(-1) [control]; P = NS), whereas mucosal (1.59 +/-0.03 vs. 2.28 +/-0.03 nl x min(-1) x cap((-1))[control]; P <0.01) and subserosal (2.47 +/-0.04 vs. 3.74 +/-0.05 nl x min(-1) x cap((-1))[control]; P <0.01) colonic capillary blood flow was significantly reduced. Severe pancreatitis was associated with a marked reduction in both pancreatic (1.06 +/-0.03 vs. 1.98 +/-0.04 nl x min(-1) x cap(-1) [control]; P <0. 01) and colonic (mucosal: 0.59 +/-0.01 vs. 2.28 +/-0.03 nl x min(-1) x cap((-1))[control], P <0.01; subserosal: 1.96 +/-0.05 vs. 3.74 +/-0.05 nl x min(-1) x cap(-1) [control], P <0.01) capillary perfusion. Colon permeability tended to increase with the severity of the disease (control: 147 +/-19 nmol x thr(-1) x cm(-2); mild pancreatitis: 158 +/-23 nmol x hr(-1) x cm(-2); severe pancreatitis: 181 +/-33 nmol x hr(-1) x cm(-2); P = NS). Impairment of colonic capillary perfusion correlates with the severity of pancreatitis. A decrease in capillary blood flow in the colon, even in mild pancreatitis not associated with significant protease activation and acinar cell necrosis or impairment of pancreatic capillary perfusion, suggests that colonic microcirculation is especially susceptible to inflammatory injury. There was no significant change in intestinal permeability in the early stage of pancreatitis, suggesting a window of opportunity for therapeutic interventions to prevent the later-observed increase in gut permeability, which could result in improved intestinal microcirculation.

The applicability of H215O-positron emission tomographic (PET) imaging for the assessment of skeletal muscle perfusion during exercise was investigated in five healthy subjects performing intermittent isometric contractions on a calf ergometer. The workload of the left calf muscles was kept constant in all exercises, while that of the right calf muscles was varied. During exercise H215O distribution in the calf muscles was measured by PET. Radioactivity measured in the left calf muscles was used as a reference for the radioactivity measured in the right calf muscles. In all studies, muscles were delineated by uptake of radioactivity. Four subjects demonstrated high radioactivity in the gastrocnemius medialis muscle, in one subject high radioactivity was distributed over the triceps surae muscles. The observed muscles demonstrated also local foci of radioactivity indicating regionally enhanced tissue perfusion. The right-left ratio of radioactivity in the active muscles increased as a function of the load. We conclude that inter- and intramuscle perfusion differences can be measured during exercise by H215O-PET imaging.

Purpose: Our purpose was to review the available literature concerning the pathogenesis of ovarian hyperstimulation syndrome and, in light of the most recent information, to attempt to provide further insight on this iatrogenic complication associated with the induction of ovulation., Methods: Published studies related to this topic were identified through a computerized bibliographic search., Conclusions: The exact mechanism for the development of ovarian hyperstimulation syndrome is still obscure. It is well established that the syndrome is associated with the process of ovulation induced by either luteinizing hormone or human chorionic gonadotropin. Following ovulation, one or more substances produced by the ovary are liberated in excess, increasing capillary permeability, resulting in the clinical features of the syndrome. It may well be that the syndrome is not triggered by a single mechanism but by the production and secretion of several substances acting in concert. These may include prostaglandins, cytokines, the ovarian reninangiotensin system, vascular endothelial growth factor, and nitric oxide.