Changes In Vascular Permeability Research Articles

The Water Permeability Channels Aquaporins 1–4 Are Differentially Expressed in Granulosa and Theca Cells of the Preovulatory Follicle during Precise Stages of Human Ovulation

Changes in vascular permeability and expansion of the fluid-filled antrum are major events in the LH-induced ovulatory process. Our objective was to investigate the presence and expression levels of aquaporins (AQPs) in the granulosa and theca cell compartments of the follicle during defined phases of human ovulation. We conducted a prospective experimental study at the Department of Obstetrics and Gynaecology at a university hospital. Twenty-eight women underwent laparoscopic sterilization and at the same time follicle retrieval at four periovulatory phases. mRNA levels of AQP1-4 were measured in separated granulosa and theca cells from preovulatory phase, early ovulatory (EO) phase, late ovulatory phase, and postovulatory phase. Immunohistochemistry was done for AQP1-4 in intact human follicles. All four AQPs were expressed in both the theca and granulosa cells during ovulation. In granulosa cells, AQP1 levels increased in the late ovulatory and postovulatory phases. Expression of AQP2-3 followed a similar pattern with a marked increase in the EO phase, whereas AQP4 levels decreased from preovulatory to the EO phase. The presence of AQP1-4 in the human follicle was verified by immunohistochemistry. The results show for the first time the presence of AQP1-4 in human follicles during ovulation. The marked early rise in expression of AQP2 and AQP3 suggests a role during the process leading to follicular rupture, and the late rise of AQP1 suggests a role in corpus luteum formation.

Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo

AbstractThe endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin–binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1−/− compared with wild-type (WT) mice. Lsp1−/− animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.

Multi-Modal Proteomic Analysis of Retinal Protein Expression Alterations in a Rat Model of Diabetic Retinopathy

BackgroundAs a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.Methodology/Principal FindingsA multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated.Conclusions/SignificanceThese results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

Plasma soluble vascular endothelial growth factor receptor-1 levels predict outcomes of pneumonia-related septic shock patients: a prospective observational study

IntroductionDespite recent advances in the management of septic shock, mortality rates are still unacceptably high. Early identification of the high-mortality risk group for early intervention remains an issue under exploration. Vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor-1 (sVEGFR1) and urokinase plasminogen activator (uPA) have diverse effects in the pathogenesis of sepsis, which involve pro-inflammation, anti-inflammation, endothelial cell repair, and vascular permeability change. Their roles in predicting mortality and organ dysfunction remain to be clarified.MethodsPneumonia-related septic shock patients from medical intensive care units were enrolled for this prospective observational study. We also included 20 patients with pneumonia without organ dysfunction for comparison. Plasma levels of VEGF and sVEGFR1 and uPA activity within 24 hours of shock onset were measured. We compared plasma levels of these biomarkers with APACHE II scores between subgroups of patients, and evaluated their predictive value for 28-day mortality and organ dysfunction.ResultsA total of 101 patients, including 81 with pneumonia-related septic shock and 20 with pneumonia without organ dysfunction, were enrolled. Non-survivors of septic shock had significantly higher plasma sVEGFR1 levels (659.3 ± 1022.8 vs. 221.1 ± 268.9 pg/mL, respectively, P < 0.001) and uPA activity (47.2 ± 40.6 vs. 27.6 ± 17.2 units, respectively, P = 0.001) when compared with those of the survivors. Kaplan-Meier survival analysis demonstrated significantly higher mortality in patients with higher levels of sVEGFR1 (P < 0.001) and uPA activity (P = 0.031). In Cox regression analysis, plasma sVEGFR1 level was independently associated with, and best predicted, the 28-day mortality of septic shock (HR: 1.55, 95% CI: 1.05-2.30). Plasma sVEGFR1 level and uPA activity had good correlation with renal dysfunction, metabolic acidosis, and hematologic dysfunction; their levels significantly increased when the number of organ dysfunctions increased. In multivariate analysis, plasma sVEGFR1 level (HR: 2.82, 95% CI: 1.17-6.81) and uPA activity (HR: 2.75, 95% CI: 1.06-7.13) were independent predictors of the presence of concomitant multi-organ dysfunction. The predictive value of VEGF for mortality and organ dysfunction was limited in pneumonia-related septic shock patients.ConclusionsHigh plasma sVEGFR1 level in the early stage of pneumonia-related septic shock independently predicted 28-day mortality and multi-organ dysfunction.

Experimental Model of Zymosan‐Induced Arthritis in the Rat Temporomandibular Joint: Role of Nitric Oxide and Neutrophils

Aims. To establish a new model of zymosan-induced temporomandibular joint (TMJ) arthritis in the rat and to investigate the role of nitric oxide. Methods. Inflammation was induced by an intra-articular injection of zymosan into the left TMJ. Mechanical hypernociception, cell influx, vascular permeability, myeloperoxidase activity, nitrite levels, and histological changes were measured in TMJ lavages or tissues at selected time points. These parameters were also evaluated after treatment with the nitric oxide synthase (NOS) inhibitors L-NAME or 1400 W. Results. Zymosan-induced TMJ arthritis caused a time-dependent leucocyte migration, plasma extravasation, mechanical hypernociception, and neutrophil accumulation between 4 and 24 h. TMJ immunohistochemical analyses showed increased inducible NOS expression. Treatment with L-NAME or 1400 W inhibited these parameters. Conclusion. Zymosan-induced TMJ arthritis is a reproducible model that may be used to assess both the mechanisms underlying TMJ inflammation and the potential tools for therapies. Nitric oxide may participate in the inflammatory temporomandibular dysfunction mechanisms.

Assessing changes in vascular permeability in a hamster model of viral hemorrhagic fever

BackgroundA number of RNA viruses cause viral hemorrhagic fever (VHF), in which proinflammatory mediators released from infected cells induce increased permeability of the endothelial lining of blood vessels, leading to loss of plasma volume, hypotension, multi-organ failure, shock and death. The optimal treatment of VHF should therefore include both the use of antiviral drugs to inhibit viral replication and measures to prevent or correct changes in vascular function. Although rodent models have been used to evaluate treatments for increased vascular permeability (VP) in bacterial sepsis, such studies have not been performed for VHF.ResultsHere, we use an established model of Pichinde virus infection of hamsters to demonstrate how changes in VP can be detected by intravenous infusion of Evans blue dye (EBD), and compare those measurements to changes in hematocrit, serum albumin concentration and serum levels of proinflammatory mediators. We show that EBD injected into sick animals in the late stage of infection is rapidly sequestered in the viscera, while in healthy animals it remains within the plasma, causing the skin to turn a marked blue color. This test could be used in live animals to detect increased VP and to assess the ability of antiviral drugs and vasoactive compounds to prevent its onset. Finally, we describe a multiplexed assay to measure levels of serum factors during the course of Pichinde arenavirus infection and demonstrate that viremia and subsequent increase in white blood cell counts precede the elaboration of inflammatory mediators, which is followed by increased VP and death.ConclusionsThis level of model characterization is essential to the evaluation of novel interventions designed to control the effects of virus-induced hypercytokinemia on host vascular function in VHF, which could lead to improved survival.

A case of non-cardiogenic acute pulmonary edema in a patient with POEMS syndrome-associated pulmonary arterial hypertension

Dear Editor,POEMS syndrome is a monoclonal plasma cell disordercharacterized by polyneuropathy, organomegaly, endocrin-opathy, monoclonal protein, and skin changes [1]. It is alsoassociated with pulmonary arterial hypertension (PAH) orCastleman’s disease [1, 2]. However, there is no reportabout non-cardiogenic acute pulmonary edema (NCAPE).In this article, we describe a rare case of transient NCAPEin a patient with POEMS syndrome.A 55-year-old male with POEMS syndrome and Castle-man’s disease was referred to our institution because ofpulmonary hypertension (PH). Since no apparent causes ofPH were revealed, we reached the diagnosis of PAHconfirmed by right heart catheterization showing 49 mmHgof mean pulmonary artery pressure, 10 mmHg of meanpulmonary capillary wedge pressure, and 4.11 L/min ofcardiac output [3]. His complication included old myocar-dial infarction of distal part of left anterior descendingartery with mildly reduced left ventricular ejection fractionof 55%. Before the treatment of PAH, he acutely developeddyspnea without volume loading. Chest radiograph dem-onstrated bilateral alveolar infiltrates, and a ratio of arterialoxygen tension to fraction of inspired oxygen was89.8 mmHg. There were no changes in electrocardiographicand echocardiographic findings, and no elevations ofcardiac enzymes. Besides, no apparent causes of acuterespiratory distress syndrome such as bacteremia wereidentified [4]. We diagnosed that he developed NCAPE.He was treated with oxygen administration, nitroglycerin,and low-dose dobutamine as supportive measures. Hissymptoms diminished promptly over the next couple ofhours without ventilator support, accompanied by decreas-ing alveolar infiltrates.Since several cytokines such as vascular endothelialgrowth factor (VEGF) which act in conjunction withangiopoietin-1/2 are associated with exacerbations ofPOEMS syndrome, we evaluated these cytokines toelucidate possible mechanisms of NCAPE (Table 1)[1, 5,6]. There were acute increases in interleukin (IL)-6 andVEGF. However, no significant differences in othercytokines were revealed. Considering the Starling equation[7], we speculated that acute elevation of vascular perme-ability following VEGF elevation contributed to the onsetof NCAPE [4, 8]. It is also possible that elevated pre-capillary hydraulic pressure due to advanced PAH made thepatient susceptible to the change of vascular permeability[7]. We also speculated that acute elevation of IL-6provides essential stimulatory cues to acute VEGF eleva-tion, which may imply the risk of complication of Castle-man’s disease [8, 9].

Effect of N-acetylcysteine on markers of skeletal muscle injury after fatiguing contractile activity

The effects of N-Acetylcysteine (NAC), an unspecific antioxidant, on fatiguing contractile activity-induced injury were investigated. Twenty-four male Wistar rats were randomly assigned to two groups. The placebo group (N=12) received one injection of phosphate buffer (PBS) 1 h prior to contractile activity induced by electrical stimulation. The NAC group (NAC; N=12) received electrical stimulation for the same time period and NAC (500 mg/kg, i.p.) dissolved in PBS 1 h prior to electrical stimulation. The contralateral hindlimb was used as a control, except in the analysis of plasma enzyme activities, when a control group (rats placebo group not electrically stimulated and not treated) was included. The following parameters were measured: tetanic force, muscle fatigue, plasma activities of creatine kinase (CK) and lactate dehydrogenase (LDH), changes in muscle vascular permeability using Evans blue dye (EBD), muscle content of reactive oxygen species (ROS) and thiobarbituric acid-reactive substances (TBARS) and myeloperoxidase (MPO) activity. Muscle fatigue was delayed and tetanic force was preserved in NAC-treated rats. NAC treatment decreased plasma CK and LDH activities. The content of muscle-derived ROS, TBARS, EBD and MPO activity in both gastrocnemius and soleus muscles were also decreased by NAC pre-treatment. Thus, NAC has a protective effect against injury induced by fatiguing contractile activity in skeletal muscle.

MRI of the cat brain in the study of vascular permeability after intraarterial carotid injection of triolein

Triolein emulsion embolization into the carotid artery depicts reversible increased vascular permeability that can promote the effect of chemotherapy or can reduce the amount of chemotherapeutic drugs for equivalent effectiveness. To establish the minimum dosage of 0.5% triolein for studying vascular permeability changes in a triolein emulsion model. Sixty-six cats were divided into six groups based on the amount of emulsified triolein (0.5%) infused into the carotid artery: group 1 (n=12, 6 ml/kg), group 2 (n=12, 4.5 ml/kg), group 3 (n=12, 3 ml/kg), group 4 (n=10, 1.5 ml/kg), group 5 (n=10, 1 ml/kg), and group 6 (n=10, 3 ml/kg of saline (control group)). T1-weighted, T2-weighted, and post-contrast T1-weighted MRI was performed 2 h after the infusion of the triolein emulsion. Contrast enhancement ratios (CERs) were obtained with pre- and post-contrast T1-weighted images in the ipsilateral and contralateral hemispheres. Signal intensity ratios (SIRs) of the ipsilateral and contralateral hemispheres were evaluated on T2-weighted images. After removal of the brain tissues, edema ratios in the ipsilateral and contralateral hemispheres were obtained from wet versus dry brain weights. Data were statistically evaluated by analysis of variance, followed by the Tukey honestly significant difference test to compare the difference in the mean CER of the ipsilateral and contralateral hemispheres, mean SIR on T2-weighted image, and mean edema ratio between each group when overall significance was attained. In the ipsilateral hemispheres, the difference in the CER between the control group and groups 1 (P=0.004), 2 (P=0.043), and 3 (P=0.008) were statistically significant. The difference in the CERs between the triolein emulsion groups was not statistically significant (P>0.05). The T2-weighted SIRs were significantly different between the control group and groups 1 (P=0.027) and 2 (P=0.004). However, the edema ratios of all doses in the triolein emulsion groups showed no significant differences compared with the control group. The minimum dosage of 0.5% triolein emulsion to achieve increased vascular permeability in the hemisphere in cat brains appears to be 3 ml/kg. This minimum dosage of triolein emulsion can be useful for acquiring basic data in further studies of vascular permeability changes in a triolein emulsion model.

High-grade malignant glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

High-grade malignant glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up R. Stupp, J.-C. Tonn, M. Brada & G. Pentheroudakis On behalf of the ESMO Guidelines Working Group* Centre Hospitalier Universitaire Vaudois (CHUV) and University of Lausanne, Lausanne, and Department of Oncology-Hematology Riveria-Chablais, Vevey, Switzerland; Department of Neurosurgery, Universitatsklinikum Grosshadern, Ludwig-Maximilians-University Munich, Munich, Germany; Institute of Cancer Research and the Royal Marsden NHS Foundation Trust, Sutton, UK; Department of Medical Oncology, Ioannina University Hospital, Ioannina, Greece

Firefly luciferase-based dynamic bioluminescence imaging: a noninvasive technique to assess tumor angiogenesis.

Bioluminescence imaging (BLI) is emerging as a cost-effective, high-throughput, noninvasive, and sensitive imaging modality to monitor cell growth and trafficking. We describe the use of dynamic BLI as a noninvasive method of assessing vessel permeability during brain tumor growth. With the use of stereotactic technique, 10 firefly luciferase-transfected GL26 mouse glioblastoma multiforme cells were injected into the brains of C57BL/6 mice (n = 80). After intraperitoneal injection of D-luciferin (150 mg/kg), serial dynamic BLI was performed at 1-minute intervals (30 seconds exposure) every 2 to 3 days until death of the animals. The maximum intensity was used as an indirect measurement of tumor growth. The adjusted slope of initial intensity (I90/Im) was used as a proxy to monitor the flow rate of blood into the vascular tree. Using a modified Evans blue perfusion protocol, we calculated the relative permeability of the vascular tree at various time points. Daily maximum intensity correlated strongly with tumor volume. At postinjection day 23, histology and BLI demonstrated an exponential growth of the tumor mass. Slopes were calculated to reflect the flow in the vessels feeding the tumor (adjusted slope = I90/Im). The increase in BLI intensity was correlated with a decrease in adjusted slope, reflecting a decrease in the rate of blood flow as tumor volume increased (y = 93.8e-0.49, R2 = 0.63). Examination of calculated slopes revealed a peak in permeability around postinjection day 20 (n = 42, P < .02 by 1-way analysis of variance) and showed a downward trend in relation to both postinjection day and maximum intensity observed; as angiogenesis progressed, tumor vessel caliber increased dramatically, resulting in sluggish but increased flow. This trend was correlated with Evans blue histology, revealing an increase in Evans blue dye uptake into the tumor, as slope calculated by BLI increases. Dynamic BLI is a practical, noninvasive technique that can semiquantitatively monitor changes in vascular permeability and therefore facilitate the study of tumor angiogenesis in animal models of disease.

Phosphodiesterase IV inhibitor Rolipram attenuates ANP‐dependent intravascular fluid loss

Mice with deletion of the endothelial ANP receptor (guanylyl cyclase A receptor) (EC GC‐A KO mice) have expanded intravascular volumes and retain infused fluid in the vascular space (Schreier et al. Endocrinol 149: 4193, 2008). We have previously demonstrated in isoflurane anesthetized wild type C57BL/6 mice that pretreatment with Rolipram (8 mg/kg IP, 30 min), a phosphodiesterase IV inhibitor that raises intracellular cAMP and stabilizes the endothelial barrier, attenuates the ANP‐dependent increase in vascular permeability (FASEB J 23:950.12, 2009). To test the extent that ANP‐dependent changes in vascular permeability regulate vascular volume, we infused ANP (500 ng/kg·min) and measured a hematocrit increase from 41% to 47%, an 11% reduction in plasma volume. This infusion rate raises plasma ANP to physiological levels near those measured after an acute plasma volume expansion. Rolipram pretreatment completely blocks this ANP‐dependent fluid loss. EC GC‐A KO mice also show no increase in hematocrit with ANP infusion relative to their control littermates. Thus Rolipram appears to be as effective to attenuate ANP‐mediated fluid loss as EC GC‐A KO. We predict that mice similarly pretreated to stabilize the endothelial barrier will retain fluid in the vascular space when given plasma volume expansion. (Supported by pilot funds from Clinical and Translational Science Center, UC Davis UL1 RR024146; NCRR).

Heat shock protein 70 in the prevention of vascular calcification in renal failure

Vascular calcification (VC) is highly correlated with cardiovascular mortality in renal failure. Osteo/chondrocytic transformation of human aortic smooth muscle cells (HA‐SMCs) is important in the initiation and progression of VC. In vitro and in vivo studies have shown this process can be driven under hyperphosphatemic and hypercalcemic conditions seen in renal failure. Several transcription factors, including Cbfa1/Runx2 is essential for induction of osteo/chondrocytic transformation.We previously demonstrated that HSP70 prevents vascular permeability changes in certain acute inflammatory processes. Studies have shown significantly higher circulating HSP70 levels in individuals with no evidence of coronary artery disease (CAD), while lower levels in pre‐dialysis patients, and even lower levels in hemodialysis (HD) patients were found.Our results have shown for the first time that heat‐shock treatment induced expression of HSP70 and markedly reduced the extent of calcification in HA‐SMCs, in vitro. Cbfa1 was upregulated in calcified HA‐SMCs, while heat‐shock treatment down‐regulates its expression. Furthermore, HSP70 expression was found to be lower in arteries from renal failure patients compared to healthy controls, ex vivo. In conclusion, HSP70 reduces the development of calcification through the inhibition of smooth muscle cell transformation.

Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers

Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4-6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and beta-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.

A Bayesian image analysis of radiation induced changes in tumor vascular permeability

This work is motivated by a quantitative Magnetic Resonance Imaging study of the relative change in tumor vascular permeability during the course of radiation therapy. The differences in tumor and healthy brain tissue physiology and pathology constitute a notable feature of the image data-spatial heterogeneity with respect to its contrast uptake profile (a surrogate for permeability) and radiation induced changes in this profile. To account for these spatial aspects of the data, we employ a Gaussian hidden Markov random field (MRF) model. The model incorporates a latent set of discrete labels from the MRF governed by a spatial regularization parameter. We estimate the MRF regularization parameter and treat the number of MRF states as a random variable and estimate it via a reversible jump Markov chain Monte Carlo algorithm. We conduct simulation studies to examine the performance of the model and compare it with a recently proposed method using the Expectation-Maximization (EM) algorithm. Simulation results show that the Bayesian algorithm performs as well, if not slightly better than the EM based algorithm. Results on real data suggest that the tumor "core" vascular permeability increases relative to healthy tissue three weeks after starting radiotherapy, which may be an opportune time to initiate chemotherapy and warrants further investigation.

Pulling the Plug on Anthrax Research: Wild Horses and “The Doctor's Dilemma”

Pulling the Plug on Anthrax Research: Wild Horses and “The Doctor's Dilemma”

Changes in Vascular Permeability and Expression of Different Angiogenic Factors Following Anti-Angiogenic Treatment in Rat Glioma

BackgroundAnti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies.Methodologies and Principal FindingsSeven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (Ktrans), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased Ktrans at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1α, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors.ConclusionThese findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.

Local tissue distribution and cellular fate of vascular endothelial growth factor (VEGF) following intramuscular injection

Vascular endothelial growth factor (VEGF) is an extracellular matrix (ECM)-binding growth factor capable of driving neovascularization. VEGF can potentially be applied clinically via intramuscular (IM) injection to correct local ischemia associated with peripheral artery disease (PAD). As interactions with ECM elements and cognate receptors at the site of an IM injection define the local biology of VEGF and previous studies have only focused on systemic distribution measurements, we have established a method to monitor the local VEGF distribution and fate. Fluorescent-labeled VEGF was prepared that bound to ECM and activated a cognate receptor similarly to VEGF. Beginning by 2 h and becoming complete by 12 h following injection, fluorescence microscopy demonstrated the transition of labeled VEGF from an initial extensive interaction with ECM components to a focused labeling of vascular endothelial cells. Biochemical characterization verified the association of VEGF with ECM components and modification of endothelial cell function associated with vascular permeability changes known to accompany VEGF actions. Our data provide information concerning temporal and spatial VEGF fate and actions at the site of an IM injection that can help guide decisions regarding the identification of acceptable formulation strategies.

The mechanism of alleviation of paraquat induced vascular endothelial permeability by erythromycin through adenosine 3'5'-cyclic monophosphate pathway

To discuss the effects of erythromycin in protecting against paraquat induced vascular endothelial cell permeability and the possible molecular mechanism of the affect. The transwell was used to culture human umbilical vein endothelial cells (HUVEC) in vitro. The cultured endothelial cells were divided into three groups: control group (N group), paraquat group (P group), paraquat+ erythromycin group (P+E group). The variables were measured under different conditions at 6, 12, 36 and 48 hours by immunofluorescence and immunoradiometric assay to determine the changes in vascular endothelial permeability to macromolecules and the change in adenosine 3'5'-cyclic monophosphate (cAMP) concentration ([cAMP]). (1) Endothelial permeability was steady in N group. The permeability in P group was increased, and it was higher than N group at 36 hours and 48 hours with statistical significance (P<0.01 and P<0.05). The permeability of P+E group was lower than P group and was higher than N group at 36 hours (both P<0.01), and there was no difference between N group and P+E group at 48 hours. (2) The [cAMP] was stable in N group at different time points; [cAMP] was decreased in P group with statistical significance at 24 hours as compared with N group (P<0.05). [cAMP] was elevated in P+E group, and it was higher than that of P group after 24 hours (P<0.05 or P<0.01); moreover, [cAMP] of P+E group was higher than that of N group at 48 hours (P<0.01). Endothelial permeability and [cAMP] was significantly negatively correlated in both P group and P+E group ( r(1)=-0.913, r(2)=-0.648, both P<0.05). Erythromycin could improve the function of damaged vascular endothelial cell barrier after paraquat poisoning, and it was the result of lowering of cellular permeability due to elevation of endothelial cAMP.

Quantitative analysis of cytokine-induced vascular toxicity and vascular leak in the mouse brain

A storm of inflammatory cytokines is released during treatment with pro-inflammatory cytokines, such as interleukin-2 (IL-2), closely approximating changes initially observed during sepsis. These signals induce profound changes in neurologic function and cognition. Little is known about the mechanisms involved. We evaluated a number of experimental methods to quantify changes in brain blood vessel integrity in a well-characterized IL-2 treatment mouse model. Measurement of wet versus dry weight and direct measurement of small molecule accumulation (e.g. [ 3H]–H 2O, sodium fluorescein) were not sensitive or reliable enough to detect small changes in mouse brain vascular permeability. Estimation of brain water content using proton density magnetic resonance imaging (MRI) measurements using a 7 T mouse MRI system was sensitive to 1–2% changes in brain water content, but was difficult to reproduce in replicate experiments. Successful techniques included use of immunohistochemistry using specific endothelial markers to identify vasodilation in carefully matched regions of brain parenchyma and dynamic contrast enhanced (DCE) MRI. Both techniques indicated that IL-2 treatment induced vasodilation of the brain blood vessels. DCE MRI further showed a 2-fold increase in the brain blood vessel permeability to gadolinium in IL-2 treated mice compared to controls. Both immunohistochemistry and DCE MRI data suggested that IL-2 induced toxicity in the brain results from vasodilation of the brain blood vessels and increased microvascular permeability, resulting in perivascular edema. These experimental techniques provide us with the tools to further characterize the mechanism responsible for cytokine-induced neuropsychiatric toxicity.