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Please cite this paper as: Cromer, Jennings, Odaka, Mathis and Alexander (2010). Murine rVEGF164b, an Inhibitory VEGF Reduces VEGF-A-Dependent Endothelial Proliferation and Barrier Dysfunction. Microcirculation 17(7), 536-547. Abstract Objective: To investigate the effects of the murine inhibitory vascular endothelial growth factor (VEGF, rVEGF164b), we generated an adenoviral vector encoding rVEGF164b, and examined its effects on endothelial barrier, growth, and structure. Method: Mouse vascular endothelial cells (MVEC) proliferation was determined by an MTT assay. Barrier of MVEC monolayers was measured by trans-endothelial electrical resistance (TEER). Reorganization of actin and zonula occludens-1 (ZO-1) were determined by fluorescent microscopy. Results: Mouse venous endothelial cells treated with murine VEGF-A (VEGF-A) (50 ng/mL) increased proliferation (60.7 ± 0.1%) within 24 hours ( p < 0.05) and rVEGF164b inhibited VEGF-A-induced proliferation. TEER was significantly decreased by VEGF-A (81.7 ± 6.2% of control). Treatment with rVEGF164b at 50 ng/mL transiently reduced MVEC barrier ( p < 0.05) at 30 minutes post-treatment (87.9 ± 1.7% of control TEER), and returned to control levels by 40 minutes post-treatment. Treatment with rVEGF164b prevented barrier changes by subsequent exposure to VEGF-A. Treatment of MVECS with VEGF-A reorganized F-actin and ZO-1, which was attenuated by rVEGF164b. Conclusions: VEGF-A may dysregulate endothelial barrier through junctional cytoskeleton processes, which can be attenuated by rVEGF164b. The VEGF-A stimulated MVEC proliferation, barrier dysregulation, and cytoskeletal rearrangement. However, rVEGF164b blocks these effects, therefore it may be useful for regulation studies of VEGF-A/VEGF-R signaling in many different models. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin E, a micronutrient (comprising α-, β-, γ- and δ-tocopherols, α-, β-, γ- and δ-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of α-, γ- and δ-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-α-induced VCAM-1 as in vitro models of inflammatory angiogenesis. α-, γ- and δ-tocopherols were not toxic to either cell type up to 40 μM. In BEC, confluent cell density was decreased by all concentrations of δ- and γ-tocopherol (10–40 μM) but not by α-tocopherol. LEC showed no change in cell density in response to tocopherols. δ-Tocopherol (40 μM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of γ-tocopherol, as well as the highest dose of α-tocopherol (40 μM), decreased cell invasiveness. δ-Tocopherol had no effect on LEC invasiveness at any molarity. δ-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; α- and γ-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 μM) concentrations of α-, γ- and δ-tocopherol, but showed no effects with smaller doses (10–20 μM) in BEC. γ-Tocopherol (10–20 μM) and α-tocopherol (10 μM), but not δ-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, α-, γ- and δ-tocopherol each dose-dependently reduced TNF-α-induced expression of VCAM-1. In LEC, there was no significant change to TNF-α-induced VCAM-1 expression with any concentration of α-, γ- or δ-tocopherol. These data demonstrate that physiological levels (0–40 μM) of α-, γ- and δ-tocopherols are nontoxic and dietary tocopherols, especially δ-tocopherol, can limit several BEC and LEC endothelial behaviors associated with angiogenesis. Tocopherols may therefore represent important nutrient-signals that limit cell behaviors related to inflammation/angiogenesis, which when deficient, may predispose individuals to risks associated with elevated angiogenesis such as inflammation and cancer; further differences seen from the tocopherols may be due to their blood or lymphatic cell origin. [Copyright &y& Elsevier]

Lymphangiogenesis is recognized as an important event in inflammation/resolution. Lymphangiogenesis is known to be regulated by the VEGFR-3/Flt-4 receptor, which binds VEGF-C and VEGF-D. Changes in the expression of VEGFR-3/Flt-4 might have a considerable effect on ability of cells like lymphatic endothelial cell's (LECs) to respond to these growth factors. Currently, it is not known how inflammatory cytokines affect the expression of VEGFR-3/Flt-4. We investigated the effects of TNF-α, IL-1β, and IFN-γ, on VEGFR-3/Flt-4 expression in LECs in vitro. Our data suggest that IL-1β downregulated VEGFR-3/Flt-4 expression, IFN-γ upregulated VEGFR-3/Flt-4 expression, while TNF-α had a minor effect. Compared to the controls without inflammatory cytokine stimulation, TNF-a (20ng/ml) had no effect on VEGR-3/Flt-4 expression (89 ± 10 % of control, not sig.), however, IL-1β (10ng/ml) appeared to downregulate VEGFR-3/Flt-4 expression (65 ± 12% of control, **p<0.01), while IFN-γ (1000 U/ml) appeared to upregulate VEGFR-3/Flt-4 expression (157 ± 8.1% of control, ***p<0.001). These data indicate that inflammatory cytokines produced by immune cells can regulate receptors involved in the control of lymphangiogenesis. [ABSTRACT FROM AUTHOR]

Peripheral artery disease (PAD) is a systemic vascular disease of the legs that results in a blockage of blood flow from the heart to the lower extremities. Now one of the most common causes of mortality in the U.S., the first line of therapy for PAD is to mechanically open the blockages using balloon angioplasty. Coating the balloons with antiproliferative agents can potentially reduce vessel re-narrowing, or restenosis after surgical intervention, but current drug-coated balloons releasing chemotherapy agents like paclitaxel have in some cases shown increased mortality long-term. Our aim was to design a novel drug-coated balloon using a polymeric nanodelivery system for a sustained release of polyphenols that reduce restenosis but with reduced toxicity compared to chemotherapy agents. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles with entrapped quercetin, a dimethoxy quercetin (rhamnazin), as well as quercetin covalently attached to PLGA, were developed. Balloon catheters were coated with polymeric nanoparticles using an ultrasonic method, and nanoparticle characteristics, drug loading, coating uniformity and drug release were determined. The adhesion of nanoparticles to vascular smooth muscle cells and the antiproliferative effect of nano-delivered polyphenols were also assessed. Of the nanoparticle systems tested, those with covalently attached quercetin provided the most sustained release over a 6-day period. Although these particles adhered to cells to a smaller extent compared to other nanoparticle formulations, their attachment was resistant to washing. These particles also exhibited the greatest anti-proliferative effect. In addition, their attachment was not altered when the cells were grown in calcifying conditions, and in PAD tissue calcification is typically a condition that impedes drug delivery. Moreover, the ultrasonic coating method generated a uniform balloon coating. The polymeric nanoparticle system with covalently attached quercetin developed herein is thus proposed as a promising platform to reduce restenosis post-angioplasty. [ABSTRACT FROM AUTHOR]

Abstract: Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91phox−/−) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN–HC and gp91phox−/−–HC mice. Recruitment of platelets derived from WT–HC mice in venules of SOD-TgN–HC or gp91phox−/−–HC recipients was comparable to ND levels. Adhesion of SOD-TgN–HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN–HC recipients, but not in WT–HC recipients. However, gp91phox−/−–HC platelets exhibited low levels of adhesion comparable to those of WT–ND in both hypercholesterolemic gp91phox−/− and WT recipients. Arteriolar dysfunction was evident in WT–HC mice, compared to WT–ND. Overexpression of SOD or, to a lesser extent, gp91phox deficiency restored arteriolar vasorelaxation responses toward WT–ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction. [Copyright &y& Elsevier]

Airborne particulate matter (PM) is associated with an increased risk for cardiovascular diseases. Although the goal of thermal remediation is to eliminate organic wastes through combustion, when incomplete combustion occurs, organics chemisorb to transition metals to generate PM-containing environmentally persistent free radicals (EPFRs). Similar EPFR species have been detected in PM found in diesel and gasoline exhaust, woodsmoke, and urban air. Prior in vivo studies demonstrated that EPFRs reduce cardiac function secondary to elevations in pulmonary arterial pressures. In vitro studies showed that EPFRs increase ROS and cytokines in pulmonary epithelial cells. We thus hypothesized that EPFR inhalation would promote lung inflammation and oxidative stress, leading to systemic inflammation, vascular endothelial injury, and a decline in vascular function. Mice were exposed to EPFRs for either 4 h or for 4 h/day for 10 days and lung and vascular function were assessed. After a 4-h exposure, plasma nitric oxide (NO) was reduced while endothelin-1 (ET-1) was increased, however lung function was not altered. After 10 day, plasma NO and ET-1 levels were again altered and lung tidal volume was reduced. These time course studies suggested the vasculature may be an early target of injury. To test this hypothesis, an intermediate time point of 3 days was selected. Though the mice exhibited no marked inflammation in either the lung or the blood, we did note significantly reduced endothelial function concurrent with a reduction in lung tidal volume and an elevation in annexin V protein levels in the lung. Although vascular dysfunction was not dependent upon inflammation, it may be associated with an injury at the air-blood interface. Gene expression analysis suggested roles for oxidative stress and aryl hydrocarbon receptor (Ahr) signaling. Studies probing the relationship between pulmonary oxidative stress and AhR signaling at the air-blood interface with vascular dysfunction seem warranted. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) triggers an intense inflammatory response in the neonatal gut associated with cytokine activation, altered nutrient status and intracellular O 2 -deprivation. Endothelial cell adhesion molecules (ECAMs) play critical roles in driving immune cell infiltration into inflamed gut. Currently, relationships between inflammation, metabolism and ECAM expression remain poorly understood in NEC. We studied the effects of metabolic depletion (aglycemia/ hypoxia) on TNF-α mediated ECAM expression including ICAM-1, MAdCAM-1, VCAM-1 and E-selectin, in vitro in intestinal microvascular endothelial cells (IMEC). To study the effects of TNF-α, aglycemia and hypoxia (alone or in combination) IMECs expression of adhesion molecules was studied using cell surface ELISA and immunoblotting. Total VCAM-1 expression was induced TNF-α and by hypoxia + TNF-α, cell surface expression was induced by hypoxia, TNF-α, TNF- α+hypoxia, and TNF- α+hypoxia and aglycemia. Total ICAM-1 increased following TNF- α, TNF- α+hypoxia, hypoxia + aglycemia, and TNF- α+hypoxia + aglycemia. Total MAdCAM-1 protein expression was significantly induced by a combination of TNF-α+hypoxia + aglycemia and cell surface expression induced by TNF- α+hypoxia. Surface expression of E-selectin was induced by TNF- α+aglycemia and TNF- α+hypoxia + aglycemia. Energy metabolism influences inflammation induced injury through mobilization of intestinal ECAMs, and may represent an important mechanism in NEC pathology. [ABSTRACT FROM AUTHOR]

Several groups have recently described the endothelial cells (EC) as an important target of pathological mediators in multiple sclerosis (MS). Despite the recognition of the EC as a significant target in MS and a possible beneficiary of Beta-interferon therapy, the structural changes which occur in the cerebrovascular endothelium and the effects of interferon-â1b on these changes have not been closely evaluated. Disruption or dysregulation of the blood brain barrier (BBB) in MS represents a loss of endothelial integrity, which may facilitate the transendothelial migration of activated leukocytes responsible for the development of demyelinating lesions of MS. We used proteomics (2-dimensional gel electrophoresis and MALDI-MS) to characterize the effects of serum from MS patients with active disease (with and without interferon-â1b therapy) on human cerebral endothelial cells. The results of this study revealed the up- and down-regulation of expression of several proteins related to vascular development, cell structure, and cell cycle control. Using this approach we identified 14-3-3 ε, metavinculin, myosin-9, plasminogen, reticulocalbin-2 and-3, ribonuclease/angiogenin inhibitor 1, annexin A1, tropomyosin and Ras-related protein Rap-1A as potential new markers of active MS disease. A better understanding of the alterations within the cerebrovascular endothelium in MS pathogenesis of MS may lead to development of more potent therapies in MS. β [ABSTRACT FROM AUTHOR]

Please cite this paper as: Chaitanya GV, Cromer W, Wells S, Jennings M, Mathis JM, Minagar A and Alexander JS. Metabolic Modulation of Cytokine-Induced Brain Endothelial Adhesion Molecule Expression. Microcirculation 19: 155-165, 2012. Abstract Objective: Cytokines contribute to cerebro-vascular inflammatory and immune responses by inducing ECAMs' expression. Ischemic insults can be separated into aglycemic and hypoxic components. However, whether aglycemia, hypoxia or OGD plays a major role in dysregulating BBB or promotes immune cell infiltration via ECAMs' expression is not clear. We investigated how expression of ICAM-1, VCAM-1, MAdCAM-1, PECAM-1, E- and P-selectin in response to TNF-α, IL-1β and IFN-γ was altered by aglycemia (A), hypoxia (H) or combined oxygen glucose deprivation (OGD). Methods: A cell surface enzyme linked immunoabsorbent assay (cell surface ELISA) was used to analyze ECAM expression. Results: We observed that ICAM-1 and PECAM-1 expressions were insensitive to hypoxia, aglycemia or OGD. Conversely, VCAM-1 and E-selectin were increased by hypoxia, but not by aglycemia. MAdCAM-1 and P-selectin were induced by hypoxia, and decreased by aglycemia. Patterns of cytokine-regulated ECAMs' expression were also modified by metabolic conditions. Conclusions: Our results indicate that patterns of inflammation-associated ECAMs represent cumulative influences from metabolic stressors, as well as cytokine activation. The expression of ECAMs following tissue injury reflects mechanistic interactions between metabolic disturbances, and alterations in tissue cytokines. Normalization of tissue metabolism, as well as cytokine profiles, may provide important targets for therapeutic treatment of inflammation. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized by increased leukocyte recruitment and subsequent tissue damage. An increase in the density of the microvasculature of the colon during IBD has been suggested, leading to the concept that angiogenesis may play a pathological role in IBD. Increased tissue and serum levels of the angiogenic cytokine VEGF-A have been reported in cases of active IBD. In this study, we examined the hypothesis that VEGF-A exerts a proinflammatory effect on colon microvascular endothelium that contributes to colonic inflammation. Leukocyte adhesion to VEGF-A-stimulated colon microvascular endothelial cells was examined using a parallel-plate hydrodynamic flow chamber. ICAM-1 adhesion molecule expression on colonic microvascular endothelium also was determined in response to VEGF-A stimulation, along with characterization of leukocyte adhesion molecule expression. High-dose VEGF-A (50 ng/ml) stimulation increased neutrophil and T cell adhesion to and decreased rolling velocities on activated endothelium, whereas low-dose VEGF-A (10 ng/ml) was without effect. Colonic endothelium constitutively expressed ICAM-1, which was significantly increased by treatment with 50 ng/ml VEGF-A or 10 ng/ml TNF-α but not 10 ng/ml VEGF-A. T cells expressed CD18 and CD1 la with no expression of CD11b, whereas neutrophils expressed CD18, CD11a, and CD11b. Finally, VEGF-A-dependent leukocyte adhesion was found to occur in a CD18-dependent manner. These results demonstrate that VEGF-A levels found in IBD exert a proinflammatory effect similar to other inflammatory agents and suggest that this cytokine may serve as an intermediary between angiogenic stimulation and cell-mediated immune responses. [ABSTRACT FROM AUTHOR]

Disruption of the blood-brain barrier (BBB) and transendothelial migration of inflammatory cells are crucial steps in the development of demyelinating lesions in multiple sclerosis (MS). Occludin and vascular endothelial-cadherin (VE-cadherin) are two major components of the tight junctions (TJs) in the brain microvasculature that help to create the BBB. In the present study, we investigated the effect of serum from MS patients on the expression of these two junctional markers and on the endothelial integrity. Serum from six MS patients in exacerbation, six in remission, and six normal controls (10% by volume) was incubated with cultured endothelial cells, and the expression of occludin and VE-cadherin was measured by immunoblotting. Serum from MS patients in exacerbation significantly reduced the expression of occludin and VE-cadherin compared with patients in remission and normal controls. This disintegrating effect was more pronounced for occludin than for VE-cadherin. We assume that the elevation in cytokines or other serum-soluble factors in MS patients in exacerbation likely provokes downregulation of occludin and VE-cadherin. This downregulation of TJs proteins may, therefore, contribute to the disruption of the BBB in this condition. [ABSTRACT FROM AUTHOR]

Background: MAdCAM-1 is an adhesion molecule expressed in Peyer's patches and lymphoid tissues which is mobilized by cytokines like TNF-α and is a major determinant of lymphocyte trafficking to the gut in human inflammatory bowel disease (IBD). It has been suggested that both reactive oxygen and nitrogen metabolites participate in regulating adhesion molecule expression in response to TNF-α. Methods: To examine how exogenous and endogenous sources of NO modulate MAdCAM-1 induction by TNF-α, we pre-treated mouse lymphatic endothelial cells with either long or short acting NO donors prior to TNF-α-stimulation, and measured MAdCAM-1 induction at 24 h. Results and Discussion: DETA-NO, a long-acting NO donor, and SperNO, a rapid releasing NO donor both inhibited TNF-α-stimulated MAdCAM-1 expression in a concentration dependent manner. Both NO donors also reduced a4b7-dependent lymphocyte endothelial adhesion. Inhibition of endogenous NO production by either L-NAME, a non-selective NOS inhibitor, or by 1400 w, a selective iNOS inhibitor failed to induce, or potentiate TNF-α regulated MAdCAM-1 expression. Conclusions: Exogenous NO donors may be beneficial in the treatment of IBD, while endogenous nitric oxide synthases may be less effective in controlling adhesion molecule expression in response to cytokines. [ABSTRACT FROM AUTHOR]