Increased vascularpermeability causes pulmonary edema that impairs arterial oxygenation and thus contributes to morbidity and mortality associated with Acute Respiratory Distress Syndrome and sepsis. Although components of intercellular adhesive and tight junctions are critical for maintaining the endothelial barrier, there has been limited study of the roles of gap junctions and their component proteins (connexins). Since connexins can modulate inflammatory signaling in other systems, we hypothesized that connexins may also regulate pulmonary endothelial permeability. The relationships between connexins and the permeability response to inflammatory stimuli were studied in cultured human pulmonary endothelial cells. Prolonged treatment with thrombin, lipopolysaccharide, or pathological cyclic stretch increased levels of mRNA and protein for the major connexin, connexin43 (Cx43). Thrombin and lipopolysaccharide both increased intercellular communication assayed by transfer of microinjected Lucifer yellow. Although thrombin decreased transendothelial resistance in these cells, the response was attenuated by pretreatment with the connexin inhibitor carbenoxolone. Additionally, the decreases of transendothelial resistance produced by either thrombin or lipopolysaccharide were attenuated by reducing Cx43 expression by siRNA knockdown. Both carbenoxolone and Cx43 knockdown also abrogated thrombin-induced phosphorylation of myosin light chain. Taken together, these data suggest that increased lung vascularpermeability induced by inflammatory conditions may be amplified via increased expression of Cx43 and intercellular communication among pulmonary endothelial cells.
Figure 2. Mult VN activates Src and increases vascularpermeability. (A). HUVECs were pretreated with LM609 (LM) (25 mg/mL) for 30 min before mult or mono VN (10 mg/mL) treatment. Phosphorylation of Src (PY418) was determined by western blotting analysis(upper and middle panel); Mult VN stimulates tyrosine phosphorylation of VE-cadherin in HUVECs. Cells were incubated with mult VN (10 mg/mL) for 60 min or VEGF(50 ng/mL) for 30 min. Cell extracts were subjected to immunoprecipitation with antibody against VE-cadherin. Precipitated protein was analyzed by Western blot using anti-phosphotyrosine antibody. The same blot was then subsequently reprobed with antibody to VE-cadherin. (B). Mult VN, not mono VN increases vascularpermeability in vitro using the permeability assay. HUVEC monolayers were pretreated with LM609 (LM) (25 mg/mL) and su6656 (su) (1 mM) for 30 min before mult or mono VN (10 mg/mL) treatment. Permeability is represented by relative fluorescence units measured by the flux of FITC-Dextran across the monolayer of HUVECs. Immunoglobulin G (IgG) and dimethyl sulfoxide (DMSO) vehicle were used as the controls. (C). Knockdown of Src with siRNA blocked VN-induced vascularpermeability. The results are expressed as the mean 6 SEM. * p,0.05 vs. control.
BMSC treatment of stroke in previous experimental studies demonstrated differential thera- peutic effects on vascularpermeability in non-diabetic rats, where the treatment significantly reduced BBB leakage , and in T1DM rats, where the treatment increased BBB leakage . Thus, there is a need to clarify the therapeutic effects of BMSC on vascularpermeability in T2DM stroke rats. By employing CE-T1WI with gadopentetate, the present study demon- strates that, compared with the saline treatment, BMSC therapy administered at 3 days after stroke significantly (p<0.05) decreases BBB leakage in T2DM rats starting at 1 week after stroke, hence, leading to a significant (p<0.05) reduction of hemorrhagic spots starting from 3w after stroke and a marginally significant (p<0.08) reduction of hemorrhagic volume at 5w after stroke demarcated from SWI. The differences in vascular response of T1DM and T2DM stroke rats to BMSC treatment may be dependent on the time at which treatment is initiated, where cell administration were at 1 day and 3 days after stroke in T1DM and T2DM, respec- tively, and not necessarily on pathophysiological differences between T1DM and T2DM .
Progressive renal disease is characterized by tubulo-interstitial injury with ongoing inflammation and fibrosis. The Nlrp3 inflammasome contributes to these pathophysiological processes through its canonical effects in cytokine maturation. Nlrp3 may additionally exert inflammasome-independent effects following tissue injury. Hence, in this study we investigated potential non-canonical effects of Nlrp3 following progressive renal injury by subjecting WT and Nlrp3-deficient (2/2) mice to unilateral ureter obstruction (UUO). Our results revealed a progressive increase of renal Nlrp3 mRNA in WT mice following UUO. The absence of Nlrp3 resulted in enhanced tubular injury and dilatation and an elevated expression of injury biomarker NGAL after UUO. Moreover, interstitial edema was significantly elevated in Nlrp32/2 mice. This could be explained by increased intratubular pressure and an enhanced tubular and vascularpermeability. In accordance, renal vascular leakage was elevated in Nlrp32/2 mice that associated with reduced mRNA expression of intercellular junction components. The decreased epithelial barrier function in Nlrp32/2 mice was not associated with increased apoptosis and/ or proliferation of renal epithelial cells. Nlrp3 deficiency did not affect renal fibrosis or inflammation. Together, our data reveal a novel non-canonical effect of Nlrp3 in preserving renal integrity and protection against early tubular injury and interstitial edema following progressive renal injury.
increase of ICAM-1 expression on renal tissue from P. berghei infected mice at day 7 post infection. Interestingly, ex vivo adhesion assays using sections from renal tissue from infected mice at this time-point show increased iRBC adhesion. Taken together, these results suggest that P. berghei interaction with the renal tissue can occur via ICAM-1. Therefore, we assume that exposition of endothelial cells to products of oxidative stress and parasite load plays a crucial role to endothelial activation and microvascular dysfunction in infected kidneys, concomitant with a markedly up- regulation of ICAM-1 in renal tissue. The cytoadherence of infected erythrocytes as well recruitment of monocytes, neutrophils and polymorphonuclear leukocytes, during pathogenesis of malaria-associated AKI could potentially contribute to renal hypoxia. In addition, an up-regulation of hypoxia inducible factor-1a (HIF-1a) mRNA and decrease of angiogenic factors protein expression (VEGF) in renal tissue can further induce morphological modifications. Changes in vascularpermeability observed were quite expected, since microvascular dysfunction has been described before in the pathogenesis of ischemic-induced AKI. Recruitment of inflammatory cells during pathogenesis of malaria-associated AKI is in line with previous observations about involvement of infiltrating cells to increase vascularpermeability . This proinflammatory state also contributes to increase the occurrence of apoptotic events .
There were no differences between the groups treated with LED and LED, followed by LPT. In fact, it was expected to have smaller edema in these groups than in the group of halogen light; however, the LPT was supposed to improve the dental pulp response to the bleaching procedure. This result could be due to the fact that LEDs could have similar phototherapeutic effect on the dental pulp than the low intensity laser. Although, less evidence has been gathered on the bio- stimulative effect of noncoherent light (LED), some in vitro studies suggested macrophage activation as the first target of this kind of emission (Young et al. 1989). Recent studies have shown that it can exert a laser-like curative effect on skin and mucosal wounds (Whelan et al. 2001). Independently of the number of sessions used in the treatment, the dental bleaching with 35 % hydrogen peroxide agent and LED followed or not by LPT caused lesser increase in the dental pulp vascularpermeability than the conventional method using halogen light. These results and the observations of clinical trials of the absence of the sensitivity when dental bleaching was speed up by LEDs (Dederich and Bushick 2004; Stabholz et al. 2003) encouraged that for vital teeth, the dental bleaching using hydrogen peroxide should be done in association with non-exothermic light sources, especially the LEDs. Further studies should be carried out in order to clarify the processes involved in the dental pulp reaction to dental bleaching procedures.
Lung vascularpermeability was assessed using the Evans blue (EB, Sigma) dye extravasation procedure. After the last FA inhalation or last LLLT administration, EB dye was injected (20 mg/Kg, iv) and the rats were killed 15 min later. The lungs were perfused via the pulmonary artery with PBS, pH 7.0 containing 5 IU/ml heparin. Subsequently, fragments of lung paren- chyma, trachea and intrapulmonary bronchi were taken, weighed and incubated overnight in formamide (4 ml/g wet weight) at room temperature. The concentration of EB dye extracted to formamide was determined spectrophotometrically at 620 nm (Bio-Tek instruments) using a standard curve of EB in formamide medium (0.3–100 μg/ml). The extravasated dye was expressed as μg/g of dry tissue weight.
Adherens junction composed by homotypic adhesion of VE-cadherin plays prominent role in endothelial barrier function . Disruption of adherens junction leads to endothelial hyperpermeability [32,33]. Surface expression of VE-cadherin has been shown to be downre- gulated by various pro-inflammatory mediators such as VEGF, TNF-α and LPS [5,8,9]. Expres- sion of total VE-cadherin in HUVECs has also been shown to be downregulated by IL-1β via a Rac-dependent mechanism . Here we show p38 MAPK is required for various pro-inflam- matory factors to downregulate the expression of VE-cadherin. p38 MAPK is recognized as a key signaling pathway via which diverse pro-inflammatory mediators, environmental toxins, and oxidants disrupt the barrier function and increase vascularpermeability [18–20,35]. We show that 5-MTP does not affect basal phosphorylation levels of p38 MAPK; however, in response to inflammatory mediator stimulations, 5-MTP significantly reduces p38 MAPK phosphorylation. It is unclear how 5-MTP blocks p38 MAPK activation. It has been reported that p38 MAPK is endogenously controlled by phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase-2C, WIP-1 [36,37]. Induction of MKP-1 expression was reported to block TNF-α induced vascular hyperpermeability through inhibition of p38 MAPK activation [36,38]. The possibility that 5-MTP protects endothelial barrier function by inducing MKP-1 expression remains to be determined. Although Src family kinases are intimately involved in signaling VE-cadherin dysfunction and increased vascularpermeability induced by VEGF , recent reports show that Src-induced VE-cadherin phosphorylation is not suffi- cient for disrupting endothelial barrier function . Our finding of p38 MAPK as a key path- way employed not only by cytokines and LPS but also by VEGF to downregulate VE-cadherin expression is in keeping with this notion. It is unclear how p38 MAPK activation induced by diverse pro-inflammatory mediators leads to reduced expression of VE-cadherin. VE-cadherin expression is regulated at the transcriptional level by Wnt  and post-translationally by internalization and degradation, which depends on Src signaling pathway . p38 MAPK acti- vation has not been linked to suppression of VE-cadherin transcription or internalization. However, it was reported that p38 MAPK regulates opioid receptor endocytosis . Further studies will be needed to elucidate the mechanism by which p38 MAPK activation alters VE- cadherin expression.
The experimental models can be divided into two broad classes: (1) acute inﬂ ammatory models and (2) chronic inﬂ ammatory models. Acute models are designed to test drugs that modulate blood ﬂ ow (erythema), changes in vascularpermeability, leukocyte migration and chemotaxis, phagocytosis - PMNLs and other phagocytic cells, measurement of local pain, antipyretic activity, local analgesic action and rat paw edema. Chronic models are designed to ﬁ nd drugs that may modulate the disease process and these include sponge and pellet implants and granuloma pouches which deposit granulation tissue, adjuvant induced arthritis and rabbit monoarticular arthritis which have an immune etiology (Lewis, 1989). Experimental inﬂ ammation in whole animal is the usual starting point for anti-inﬂ ammatory testing. These experiments are varied and widely used, specially the rat paw edema test. It can be adapted in numerous ways using different inﬂ ammatory agents in attempt to mimic pathological inﬂ ammation and arthritis (Willianson, 1996).
Our previous studies suggest that during diabetes, a compromised BRB serves as a portal for bacteria to gain access to the eye from the bloodstream [28,29]. We reported that an increased incidence of K. pneumonaie and S. aureus EBE in a diabetic murine model correlated with the length of time following diabetes induction with streptozotocin (STZ) [28,29]. This increased EBE incidence also correlated with greater vascularpermeability in the eyes of STZ-induced diabetic mice [28,29]. Our results supported clinical reports that diabetes is a predisposing risk factor for the development of EBE [28,29]. However, diabetes progression results in a myriad of other host changes, including immunological deficits such as the inability of inflammatory cells to phagocytize K. pneumoniae and S. aureus [30,31]. To dissect the specific mechanisms that underlie EBE development, we sought to divorce BRB permeability from the immunologi- cal changes that occur during diabetes progression. Specifically, we hypothesized that dysfunc- tion of the RPE, a component of the outer BRB which is altered during the development of diabetes, facilitates the development of EBE. To test this hypothesis, we selectively induced RPE degeneration using sodium iodate (NaIO 3 ), an oxidizing agent that exerts toxicity specifi-
This assay was used to estimate alterations of vascularpermeability induced by intradermal injections of histamine (50 mg, histamine dihydrochloride, Fluka Ag, Buchs SG). The method used was described by Lykbe and Cummings and modified by Carvalho et al. (1999). This method consisted of the spectrophotometric determination of the amount of extravasated dye in the interstitial space induced by histamine. Groups of rats were treated orally with distilled water (0.5 mL, control group), Indomethacin (MSD Co., 10 mg/kg) or OEO (1226.8 mg/kg). Evans blue (25 mg/kg) was then injected intravenously. Histamine was given by intradermal injection into the animal’s back 10 min after the injection of the dye. Each animal received six injections of the same histamine solution in different locations. The animals were sacrificed 20 min after the last injection. To extract the dye, tissue close to the injection sites was removed (1.5 cm diameter), fragmented and placed in tubes containing 3 mL of formamide, then kept at 37 o C for 24 h. This material was
dengue research programs in Southeast Asia found that children were dying of a new clinical entity, the dengue vascularpermeability syndrome (DVPS; Fig. 1), character- ized by a complex of physiologic abnormalities affecting multiple organ systems including the liver, blood coagu- lation, complement, hematopoiesis, serum proteins, and the vascular system that reach a maximal expression at defervescence. 3,6---10 It soon was well understood that
agonist CGS21680 signiﬁcantly inhibited lung injury in LPS- treated mice. This treatment led to (i) signiﬁcantly decreased accumulation of PMNs (Figure 8A), (ii) reduced production of reactive oxygen metabolites (Figure 8A), (iii) less-pronounced increases in lung vascularpermeability (Figure 8B), and (iv) improved lung gas exchange (Figure 8B). Histological examination of CGS21680-treated mice revealed that ther- apeutic effects of the agonist were similar to those of exposure of mice to hypoxia. CGS21680 treatment resulted in inhibition of pulmonary PMN sequestration (Figure 8C), and—as shown for hypoxia above—was followed by a signiﬁcant reduction of lung tissue damage as assessed by the 4-fold decrease in the LIS (Figure 8C).
Acetic acid-induced vascularpermeability in mice The effect of OWB extract and CA on the vascularpermeability was tested in mice . Randomly selected Balb/C mice were divided into six groups (n = 6): Group I was served as vehicle control, groups II and III received 200 and 400 mg/kg of OWB extract, group IV and V received 20 and 40 mg/kg of CA, while group VI was administrated with indomethacin (10 mg/kg), orally. One hour after the treatment, 200 m l of 0.2% Evan’s blue in normal saline was injected through tail vein of each mouse (at 0.2 ml/20 g), and 30 min later each mouse was injected (i.p.) with 0.6% acetic acid in normal saline (0.2 ml). After 1h the animals were sacrificed, their abdomen was open to expose the entrails, and washed with normal saline (5 ml) to collect the content in a test tube. The content was then centrifuged, and the absorbance of the collected supernatant was measured in a spectrophotometer at 590 nm. The vascularpermeability effects were expressed as the absorbance (A) of the amount of dye leaked into the intraperito- neal cavity .
Furthermore, we demonstrated that the diabetic state increased the level of vascularpermeability in the cheek mucosa of the ulcerated an- imals and that this alteration appeared to be related to the diabetic state per se and not to the ulceration because a similar level was ob- served in the cheek mucosa of the normoglycemic ulcerated animals and the non-ulcerated control animals on the ﬁfth day post-wounding, a feature not observed in the diabetic animals. Moreover, in the diabetic state, the basal membrane of the capillaries in the microcirculation thickens and the capillary size decreases with the constant increase in hydrostatic pressure, which promotes inﬂammation of the microvascu- lar endothelium, which in turn maintains the increased level of vascularpermeability and tissue edema . Some of the mediators responsible for maintaining this increased vascularpermeability have been identi- ﬁed, including VEGF-A , prostaglandin E2  and cytokines (TNF- α and IL-1β). TNF-α and IL-1β have a synergistic effect on the capillary endothelium, leading to increased vascularpermeability during inﬂam- mation both in vivo and in vitro . Several studies have attributed the changes in vascularpermeability, particularly that occurring in the dia- betic state, to the overexpression of TNF-α [13,26]. This cytokine is known to induce reorganization of the endothelial cytoskeleton, which increases the level of vascularpermeability, causing leakage of ﬂuid and plasma proteins and thereby contributing to edema . Our study demonstrated that there were high levels of expression of IL-1β (at the 5th day post-wounding in the connective tissue and at the 10th day in the epithelium and connective tissue) and TNF-α (at the 5th day in the connective tissue) in the ulcers of the diabetic animals, coincident with the increase in vascularpermeability.
Determining the mechanisms by which aging leads to neurovascular decline and how exer- cise prevents this decline is important, as it could lead to the identification of therapeutic strate- gies that target similar processes. Here we suggest APOE as a strong candidate for mediating age-related neurovascular unit decline; Apoe expression decreases in the cortex and HP of aged mice, Apoe expression is preserved by exercise, and exercise has little to no effect on behavioral deficits, neurovascular dysfunction, and innate immune responses in aged Apoe-deficient mice. Given that mice deficient in Apoe show vascularpermeability, decreased CBF, synapse loss, and cognitive impairments [37,75,76], a decrease in Apoe expression in the aging brain would be predicted to impact the health of the neurovascular unit. Supporting this, a previous study shows an activation of a proinflammatory pathway in pericytes in Apoe-deficient mice leading to BM dysregulation and neurovascular breakdown . Interestingly, although exercise had no effect on most components of the neurovascular unit in Apoe-deficient mice, exercise did partially increase pericyte number, but not coverage, indicating exercise has at least some posi- tive effects on pericyte survival independent of APOE but they could be still dysfunctional. Another possibility is that intensity and duration of the exercise in this study was not sufficient for Apoe-deficient mice to reach the beneficial effects found in the wild-type mice. It is impor- tant to note that Apoe-deficient mice already had a dysfunctional neurovascular unit prior to running in contrast to the experiments using wild-type mice. Therefore, it is possible that exer- cise prevents neurovascular dysfunction but it has little to no effect on mice with an already dysfunctional neurovascular unit. This outcome would also be important, as neurovascular dysfunction from events such as brain injury or strokes could predispose to neurodegenerative diseases such as AD, and exercise may not be as beneficial in these circumstances.
This study demonstrated the reproducibility of DCE-MRI with CAs that have small and large molecular weights, and using standard MRI acquisition and analysis protocols in a breast xenograft tumor model. A major finding in this work was that the VIF was highly variable and needs to be calculated for each experimental setup. An automated VIF ROI selection method was developed that averaged time activity from voxels that were identified using pre-defined kinetic thresholds. Using these values, a low wCV of K trans were obtained. This indicates that this measure of tumor vascularpermeability can be measured with excellent reproducibility, and has higher ICC value than previously measured . Results of earlier studies have shown a range of wCV between 6% and 29% for K trans [15,25,28]. Evaluating the intra-subject ICC was required because only Figure 4. VIFs from Gd-DTPA (a) and P792 (c). VIFs were smoothed by temporal spline-fit function for Gd-DTPA (b) and P792 (d).
Animals were pretreated 1 h before of each phlogistic agent with vehicle or (+)-limonene epoxide (75 mg/kg). Vascularpermeability was evaluated 30 min after the ad- ministration of the compound 48/80 by the Evan’s blue test . It was injected 2.5% Evans blue (25 mg/kg) intrave- nously through retro orbital plexus 30 min before the com- pound 48/80. Paw was collected, weighed, and placed in glass tubes containing a solution of formamide (1 mL/paw) at 37 °C for 72 h to extract the dye. The amount of dye was measured at 630 nm using a standard curve of Evans blue. Afterwards, paw tissue was homogenized in potassi- um phosphate buffer containing 0.5% hexadecyltrimethyl ammonium bromide and centrifuged at 4500 rpm for 15 min at 4 °C. The pellet was resuspended, and activity of MPO was determined at 450 nm using o-dianisidine dihydrochloride and 1% hydrogen peroxide . One unit of MPO was defined as the amount of MPO capable of breaking 1 mmol of peroxide/min and data were reported in units per milligram of tissue (U/mg tissue).
Although schistosomiasis is related to an inflammatory condi- tion, and the major cytokines are well known, the knowledge about endothelial cell-leukocyte interaction in schistosomiasis is limited. Therefore, present work aimed to examine the influence of murine schistosomiasis on some endothelium-related events such as leukocyte adhesion, migration and vascularpermeability, and also the influence of the disease on the expression of the constitutive endothelial nitric oxide synthase (eNOS; EC 126.96.36.199), whose product (nitric oxide, NO) inhibits leukocyte traffic and vascularpermeability [15–17]. Our data suggest that murine schistosomi- asis enhances vascularpermeability and endothelial cell-leukocyte interactions in vivo and in vitro. These alterations relate, at least partially, to an endothelial cell phenotypic alteration characterized by decreased expression of eNOS and consequently its product NO, which are characteristics of endothelial dysfunction. In addition, our data strongly suggest that the disease primes murine endothelial cells in vivo for an increased leukocyte adhesion, which keep the information in culture.
High permeability pulmonary edema is induced by ACM injury and increased vascularpermeability; the path- ological basis is excessive systemic inflammatory response that induces pulmonary capillary permeability, interstitial pulmonary edema and alveolar ventilation/perfusion imbalance, leading to hypoxemia (mainly pulmonary dif- fusion dysfunction). In severe sepsis or early trauma, monocyte-macrophage system effector cells (especially macrophages) are activated. Many pro-inflammatory cy- tokines including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, histamine and leukot- rienes are released and are involved in the inflammatory reactions of ARDS; of these, TNF-α is a key part of the cytokine network and an initiating factor of inflammation, as well as one of the main molecules causing damage to pulmonary vascular endothelial cells. Studies have shown that serum TNF-α and IL-1β in patients with ARDS are significantly increased; IL-1β and IL-6 in bronchoalveolar lavage fluid are also significantly high. The IL-1β and IL-6 concentration in bronchial lavage fluid was closely related to the severity and prognosis of ARDS. 15,16