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Proper lymphatic function is necessary for the transport of fluids, macromolecules, antigens and immune cells out of the interstitium. The lymphatic endothelium plays important roles in the modulation of lymphatic contractile activity and lymph transport, but it’s role as a barrier between the lymph and interstitial compartments is less well understood. Alterations in lymphatic function have long been associated with edema and inflammation although the integrity of the lymphatic endothelial barrier during inflammation is not well-defined. In this paper we evaluated the integrity of the lymphatic barrier in response to inflammatory stimuli commonly associated with increased blood endothelial permeability. We utilized in vitro assays of lymphatic endothelial cell (LEC) monolayer barrier function after treatment with different inflammatory cytokines and signaling molecules including TNF-α, IL-6, IL-1β, IFN-γ and LPS. Moderate increases in an index of monolayer barrier dysfunction were noted with all treatments (20–60 % increase) except IFN-γ which caused a greater than 2.5-fold increase. Cytokine-induced barrier dysfunction was blocked or reduced by the addition of LNAME, except for IL-1β and LPS treatments, suggesting a regulatory role for nitric oxide. The decreased LEC barrier was associated with modulation of both intercellular adhesion and intracellular cytoskeletal activation. Cytokine treatments reduced the expression of VE-cadherin and increased scavenging of β-catenin in the LECs and this was partially reversed by LNAME. Likewise the phosphorylation of myosin light chain 20 at the regulatory serine 19 site, which accompanied the elevated monolayer barrier dysfunction in response to cytokine treatment, was also blunted by LNAME application. This suggests that the lymphatic barrier is regulated during inflammation and that certain inflammatory signals may induce large increases in permeability.

Background and Aim. Lipedema is a common painful SAT disorder characterized by enlargement of fat primarily in the legs of women. Case reports of lipedema tissue samples demonstrate fluid and fibrosis in the interstitial matrix, increased macrophages, and adipocyte hypertrophy. The aims of this project are to investigate blood vasculature, immune cells, and structure of lipedema tissue in a cohort of women. Methods. Forty-nine participants, 19 controls and 30 with lipedema, were divided into groups based on body mass index (BMI): Non-Obese (BMI 20 to <30 kg/m2) and Obese (BMI 30 to <40 kg/m2). Histological sections from thigh skin and fat were stained with H&E. Adipocyte area and blood vessel size and number were quantified using ImageJ software. Markers for macrophages (CD68), mast cells (CD117), T cells (CD3), endothelial cells (CD31), blood (SMA), and lymphatic (D2-40 and Lyve-1) vessels were investigated by IHC and IF. Results. Non-Obese Lipedema adipocyte area was larger than Non-Obese Controls (p=0.005) and similar to Obese Lipedema and Obese Controls. Macrophage numbers were significantly increased in Non-Obese (p<0.005) and Obese (p<0.05) Lipedema skin and fat compared to Control groups. No differences in T lymphocytes or mast cells were observed when comparing Lipedema to Control in both groups. SMA staining revealed increased dermal vessels in Non-Obese Lipedema patients (p<0.001) compared to Non-Obese Controls. Lyve-1 and D2-40 staining showed a significant increase in lymphatic vessel area but not in number or perimeter in Obese Lipedema participants (p<0.05) compared to Controls (Obese and Non-Obese). Areas of angiogenesis were found in the fat in 30% of lipedema participants but not controls. Conclusion. Hypertrophic adipocytes, increased numbers of macrophages and blood vessels, and dilation of capillaries in thigh tissue of non-obese women with lipedema suggest inflammation, and angiogenesis occurs independent of obesity and demonstrates a role of altered vasculature in the manifestation of the disease.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 μM L-NAME (N-Nitro-l-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of β-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure β-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 μM L-NAME attenuated (p < 0.05) the MPLEC. The β-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

Background Ulcerative colitis (UC) is the most common form of inflammatory bowel disease in the USA. A key component of UC is the increase in inflammatory angiogenesis of the colon during active disease. This increase is driven to a great extent by the over expression of VEGF-A. Recently, VEGF165 b (VEGF164 b in mouse), an anti-angiogenic form of VEGF-A was described and its regulation was determined to be disturbed in many pathologies such as cancer and pre-eclampsia. Results The aims of this study were to examine the role of this inhibitory VEGF by expressing this molecule in a model of intestinal inflammation, and to evaluate its expression as a potential new therapeutic approach for treating UC. A modified model of TNBS colitis was used to determine the effects of rVEGF164 b expression on colon inflammation. Expansion of the vascular system was assessed by immunhistochemical methods and macro- and microscopic measurements of inflammation in the colon were measured. Leukocyte invasion of the tissue was measured by myeloperoxidase assay and identification and counting of lymphoid follicles. Both angio- and lymphangiogenesis were reduced by expression of rVEGF164 b , which correlated with reduction in both gross and microscopic inflammatory scores. Leukocyte invasion of the tissue was also reduced by rVEGF164 b expression. Conclusions This is the first report using an endogenous inhibitory VEGF-A isoform for therapy in a model of experimental colitis. Inhibitory VEGF molecules play an important role in maintenance of gut homeostasis and may be dysregulated in UC. The results of this study suggest that restoration of rVEGF164 b expression has anti-inflammatory activity in a TNBS model and warrants further examination as a possible therapeutic for UC.

The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs).

Background Inflammatory Bowel Diseases (IBD) remain significant health problems in the US and worldwide. IBD is most often associated with eastern European ancestry, and is less frequently reported in other populations of African origin e.g. African Americans ('AAs'). Whether AAs represent an important population with IBD in the US remains unclear since few studies have investigated IBD in communities with a majority representation of AA patients. The Louisiana State University Health Sciences Center in Shreveport (LSUHSC-S) is a tertiary care medical center, with a patient base composed of 58% AA and 39% Caucasian (W), ideal for evaluating racial (AA vs. W) as well and gender (M vs. F) influences on IBD. Methods In this retrospective study, we evaluated 951 visits to LSUHSC-S for IBD (between 2000 to 2008) using non-identified patient information based on ICD-9 medical record coding (Crohn's disease 'CD'-555.0- 555.9 and ulcerative colitis 'UC'-556.0-556.9). Results Overall, there were more cases of CD seen than UC. UC and CD affected similar ratios of AA and Caucasian males (M) and females (F) with a rank order of WF > WM > AAF > AAM. Interestingly, in CD, we found that annual visits per person was the highest in AA M (10.7 ± 1.7); significantly higher (* -p < 0.05) than in WM (6.3 ± 1.0). Further, in CD, the female to male (F: M) ratio in AA was significantly higher (*- p < 0.05) (1.9 ± 0.2) than in Caucasians (F:M = 1.3 ± 0.1) suggesting a female dominance in AACD; no differences were seen in UC F: M ratios. Conclusion Although Caucasians still represent the greatest fraction of IBD (~64%), AAs with IBD made up >1/3 (36.4%) of annual IBD cases from 2000-2008 at LSUHSC-S. Further studies on genetic and environments risks for IBD risk in AAs are needed to understand differences in presentation and progression in AAs and other 'non-traditional' populations.

Lymphatic dysfunction has been linked to inflammation since the 1930s. Lymphatic function in the gut and mesentery is grossly underexplored in models of inflammatory bowel disease despite the use of lymphatic occlusion in early models of inflammatory bowel disease. Activation of the innate and adaptive immune system is a hallmark of TNBS-induced inflammation and is linked to disruption of the intrinsic lymph pump. Recent identification of crosstalk between lymphatic vessel resident immune cells and regulation of lymphatic vessel contractility underscore the importance of the timing of lymphatic dysfunction during tissue inflammation in response to TNBS.MethodsTo evaluate lymphatic function in TNBS induced inflammation, lymph was collected and flow measured from mesenteric lymphatics. Cellularity and cytokine profile of the lymph was also measured. Histopathology was performed to determine severity of injury and immunofluorescent staining of the mesentery was done to evaluate changes in the population of immune cells that reside near and on gastro-intestinal collecting lymphatics.ResultsLymph transport fell 24 hours after TNBS administration and began recovering at 72 hours. Significant reduction of lymph flow preceded significant increase in histopathological score and occurred simultaneously with increased myeloperoxidase activity. These changes were preceded by increased MHCII+ cells surrounding mesenteric lymphatics leading to an altered lymphatic environment that would favor dysfunction.ConclusionsAlterations in environmental factors that effect lymphatic function occur before the development of gross GI inflammation. Reduced lymphatic function in TNBS-mediated inflammation is likely an early factor in the development of injury and that recovery of function is associated with resolution of inflammation.

BackgroundThe pathophysiology of inflammatory bowel disease (IBD) includes leukocyte infiltration, blood and lymphatic remodeling, weight loss and protein enteropathy. The roles of angiopoietin-2 (Ang-2) in initiating gut inflammation, leukocyte infiltration and angiogenesis are not well understood.MethodsDisease activity index, histopathological scoring, myeloperoxidase assay, immunohistochemistry and sodium dodecyl sulphate- polyacrylamide gel electrophoretic methods were employed in the present study to addess the roles of Ang-2 in experimental colitis.ResultsSeveral important differences were seen in the development of experimental IBD in Ang-2−/− mice. Although weight change and disease activity differ only slightly in WT and Ang-2−/− + DSS treated mice, leukocyte infiltration, inflammation and blood and lymphatic vessel density is significantly attenuated compared to WT + DSS mice. Gut capillary fragility and water export (stool blood and form) appear significantly earlier in Ang-2−/− + DSS mice vs. WT. Colon lengths were also significantly reduced in Ang-2−/− and gut histopathology was less severe in Ang-2−/− compared to WT + DSS. Lastly, the decrease in serum protein content in WT + DSS was less severe in Ang-2−/− + DSS, thus protein losing enteropathy (PLE) a feature of IBD is relieved by Ang-2−/−.ConclusionThese data demonstrate that in DSS colitis, Ang-2 mediates inflammatory hemangiogenesis, lymphangiogenesis and neutrophil infiltration to reduce some, but not all clinical features of IBD. The implications for Ang-2 manipulation in the development of IBD and other inflammatory diseases and treatments involving Ang-2 are discussed. (Inflamm Bowel Dis 2009)