Endothelium-Dependent Vasorelaxant Effect of Butanolic Fraction from Caryocar brasiliense Camb. Leaves in Rat Thoracic Aorta

Caryocar brasiliense Camb. "pequi" is a native plant from the Cerrado region of Brazil that contains bioactive components reported to be antioxidant agents. Previous work has demonstrated that dietary supplementation with pequi decreased the arterial pressure of volunteer athletes. We found that the crude hydroalcoholic extract (CHE) of C. brasiliense leaves relaxed, in a concentration-dependent manner, rat aortic rings precontracted with phenylephrine, and that the butanolic fraction (BF) produced an effect similar to that of the CHE. Aortic relaxation induced by BF was abolished by endothelium removal, by incubation of the nitric oxide synthase inhibitor L-NAME, or the soluble guanylatecyclase inhibitor ODQ. However, incubation with atropine and pyrilamine had no effect on the BF-induced vasorelaxation. Moreover, this effect was not inhibited by indomethacin and tetraethylammonium. The concentration-response curve to calcium in denuded-endothelium rings was not modified after incubation with BF, and the vasorelaxation by BF in endothelium-intact rings precontracted with KCl was abolished after incubation with L-NAME. In addition, administration of BF in anesthetized rats resulted in a reversible hypotension. The results reveal that C. brasiliense possesses both in vivo and in vitro activities and that the vascular effect of BF involves stimulation of the nitric oxide/cyclic GMP pathway.

Alterations in vasoconstrictor responses to the endothelium-derived contracting factor uridine adenosine tetraphosphate are region specific in DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and alpha,beta-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-. UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The alpha,beta-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension. (C) 2011 Elsevier Ltd. All rights reserved.

Reduced endothelium-dependent relaxation to anandamide in mesenteric arteries from young obese Zucker rats

Impaired vascular function, manifested by an altered ability of the endothelium to release endothelium-derived relaxing factors and endothelium-derived contracting factors, is consistently reported in obesity. Considering that the endothelium plays a major role in the relaxant response to the cannabinoid agonist anandamide, the present study tested the hypothesis that vascular relaxation to anandamide is decreased in obese rats. Mechanisms contributing to decreased anandamide-induced vasodilation were determined. Resistance mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs) were used. Vascular reactivity was evaluated in a myograph for isometric tension recording. Protein expression and localization were analyzed by Western blotting and immunofluorescence, respectively. Vasorelaxation to anandamide, acetylcholine, and sodium nitroprusside, as well as to CB1, CB2, and TRPV1 agonists was decreased in endothelium-intact mesenteric arteries from OZRs. Incubation with an AMP-dependent protein kinase (AMPK) activator or a fatty acid amide hydrolase inhibitor restored anandamide-induced vascular relaxation in OZRs. CB1 and CB2 receptors protein expression was decreased in arteries from OZRs. Incubation of mesenteric arteries with anandamide evoked endothelial nitric oxide synthase (eNOS), AMPK and acetyl CoA carboxylase phosphorylation in LZRs, whereas it decreased phosphorylation of these proteins in OZRs. In conclusion, obesity decreases anandamide-induced relaxation in resistance arteries. Decreased cannabinoid receptors expression, increased anandamide degradation, decreased AMPK/eNOS activity as well as impairment of the response mediated by TRPV1 activation seem to contribute to reduce responses to cannabinoid agonists in obesity.

Untersuchung der zellulären Antwort von Tumor und Endothelium auf radioaktive Bestrahlung in vitro und in vivo

Tumore des Kopf-Hals Bereiches sprechen aufgrund schneller Resistenzbildung häufig schlecht auf die derzeit praktizierten Bestrahlungstherapien an. Der Erfolg dieser Behandlung wird dabei maßgeblich durch die Strahlenresistenz des malignen Gewebes limitiert. Das Verständnis der zugrunde liegenden zellulären und molekularen Mechanismen ist diesbezüglich unvollständig. Die Resistenzzunahme während der klinischen Behandlung könnte durch die Selektion strahlenresistenter Einzelzellen verursacht werden oder durch die Aktivierung von Resistenzmechanismen. Im Rahmen dieser Arbeit wurde die bestrahlungsvermittelte Freisetzung möglicherweise protektiv wirkender Faktoren durch Tumorzelllinien des Kopf-Hals Bereiches untersucht. Durch Bestrahlung erfolgte eine Induktion von VEGF (vascular endothelial growth factor) und FGF-2 (fibroblast growth factor 2), IL-8 (Interleukin-8) und PGE2 (Prostaglandin E2). Die Untersuchung von VEGF und FGF-2 zeigte weiterhin ein zytoprotektives Potential dieser Faktoren, d.h. die T

Espressione del vascular endothelium growth factor (VEGF) nella micosi fungoide

La micosi fungoide (MF) è un linfoma a cellule T primitivo della cute usualmente indolente negli stadi iniziali, ma con prognosi decisamente peggiore nelle fasi avanzate, ove attualmente non sono presenti strategie terapeutiche tali da indurre remissioni durature. Recenti osservazioni indicano che alti livelli di espressione del vascular endothelial growth factor (VEGF) nelle cellule della MF sembrano correlare con una prognosi peggiore. Nel presente studio, sono state vagliate le eventuali differenze di espressione di VEGF nella MF e nei linfociti T normali. In primo luogo sono stati raffrontati 63 casi di MF con 20 campioni corrispondenti alle diverse sottopopolazioni di linfociti T normali, per stabilire quale fra questi esprimesse maggiori livelli di VEGF. Tale esperimento ha dimostrato che il gene è notevolmente più espresso nella MF. Si è provveduto a stabilire se tale dato sia da correlarsi ad un fenomeno patologico o fisiologico. Quindi sono state eseguite indagini di gene expression profiling (GEP) allo scopo di vagliare i livelli di VEGF nella popolazione linfocitaria T normale (CD4+, CD8+, HLA-DR+ e HLA-DR-): da ciò è risultato che i linfociti T attivati esprimono maggiormente VEGF e che il loro GEP è globalmente paragonabile a quello della MF. Pertanto, i linfociti T attivati sono stati considerati la controparte normale delle cellule della MF. Successivamente si è confrontata l’espressione quantitativa di VEGF nei linfociti T attivati e nelle cellule della MF, evidenziando come questa sia maggiore nella popolazione neoplastica indipendentemente dallo stadio della malattia. Le indagini immunoistochimiche condotte su 18 casi di MF, hanno confermato quanto evidenziato attraverso il GEP. Concludendo, la ricerca ha dimostrato per la prima volta l’espressione di VEGF negli elementi della MF. Ciò porta a supporre che la de-regolazione genica della via di VEGF sia correlata nella patogenesi della MF e che tale molecola possa considerarsi un potenziale bersaglio terapeutico.

Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium

Endothelial ICAM-1 and ICAM-2 were shown to be essential for T cell diapedesis across the blood-brain barrier (BBB) in vitro under static conditions. Crawling of T cells prior to diapedesis was only recently revealed to occur preferentially against the direction of blood flow on the endothelial surface of inflamed brain microvessels in vivo. Using live cell-imaging techniques, we prove that Th1 memory/effector T cells predominantly crawl against the direction of flow on the surface of BBB endothelium in vitro. Analysis of T cell interaction with wild-type, ICAM-1-deficient, ICAM-2-deficient, or ICAM-1 and ICAM-2 double-deficient primary mouse brain microvascular endothelial cells under physiological flow conditions allowed us to dissect the individual contributions of endothelial ICAM-1, ICAM-2, and VCAM-1 to shear-resistant T cell arrest, polarization, and crawling. Although T cell arrest was mediated by endothelial ICAM-1 and VCAM-1, T cell polarization and crawling were mediated by endothelial ICAM-1 and ICAM-2 but not by endothelial VCAM-1. Therefore, our data delineate a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear-resistant T cell arrest, followed by endothelial ICAM-1 and ICAM-2 in mediating T cell crawling to sites permissive for diapedesis across BBB endothelium.

Excessive erythrocytosis compromises the blood-endothelium interface in erythropoietin-overexpressing mice

Elevated systemic haematocrit (Hct) increases risk of cardiovascular disorders, such as stroke and myocardial infarction. One possible pathophysiological mechanism could be a disturbance of the blood-endothelium interface. It has been shown that blood interacts with the endothelial surface via a thick hydrated macromolecular layer (the 'glycocalyx', or 'endothelial surface layer'--ESL), modulating various biological processes, including inflammation, permeability and atherosclerosis. However, the consequences of elevated Hct on the functional properties of this interface are incompletely understood. Thus, we combined intravital microscopy of an erythropoietin overexpressing transgenic mouse line (tg6) with excessive erythrocytosis (Hct 0.85), microviscometric analysis of haemodynamics, and a flow simulation model to assess the effects of elevated Hct on glycocalyx/ESL thickness and flow resistance. We show that the glycocalyx/ESL is nearly abolished in tg6 mice (thickness: wild-type control: 0.52 μm; tg6: 0.13 μm; P < 0.001). However, the corresponding reduction in network flow resistance contributes <20% to the maintenance of total peripheral resistance observed in tg6 mice. This suggests that the pathological effects of elevated Hct in these mice, and possibly also in polycythaemic humans, may relate to biological corollaries of a reduced ESL thickness and the consequent alteration in the blood-endothelium interface, rather than to an increase of flow resistance.

Additive effect of homocysteine- and cholesterol-lowering therapy on endothelium-dependent vasodilation in patients with cardiovascular disease

Endothelial dysfunction is a marker for development and progression of atherosclerosis. Statin therapy improves endothelial function in cardiovascular patients by reducing LDL-cholesterol and by pleiotropic effects. B-group vitamin supplementation restores endothelial function mainly by reducing homocysteine-induced oxidative stress. Thus, we evaluated the effect of rosuvastatin, B-group vitamins and their combination on endothelial function in high-risk cardiovascular patients.

Assessment of endothelium and inflammatory response at the onset of reperfusion injury in hand surgery

Background Activation of the endothelium, complement activation and generation of cytokines are known events during ischemia-reperfusion (I/R) that mediate tissue injury. Our aim was to elucidate their respective participation at the onset of the reperfusion phase. Tourniquet application in hand surgery causes short-term ischemia, followed by reperfusion and was therefore used as the model in this study. Methods Ten patients were included in the study after obtaining informed consent. A tourniquet was placed on the upper arm and inflated to 250 mmHg for 116 ± 16 min, during which the surgery was performed. Venous blood and tissue samples from the surgical area were taken at baseline as well as 0, 2, and 10 min after reperfusion and analyzed for the following parameters: Endothelial integrity and/or activation were analyzed by measuring heparan sulfate and syndecan-1 in serum, and vWF, heparan sulfate proteoglycan as well as CD31on tissue. Complement activation was determined by C3a and C4d levels in plasma, levels of C1-inhibitor in serum, and IgG, IgM, C3b/c, and C4b/c deposition on tissue. Cytokines and growth factors IL-5, IL-6, IL-7, IL-8, IL-10, IL-17, G-CSF, GM-CSF, MCP-1, TNFα, VEGF, and PDGF bb were measured in the serum. Finally, CK-MM levels were determined in plasma as a measure for muscle necrosis. Results Markers for endothelial activation and/or integrity as well as complement activation showed no significant changes until 10 min reperfusion. Among the measured cytokines, IL-6, IL-7, IL-17, TNFα, GM-CSF, VEGF, and PDGF bb were significantly increased at 10 min reperfusion with respect to baseline. CK-MM showed a rise from baseline at the onset of reperfusion (p < 0.001) and dropped again at 2 min (p < 0.01) reperfusion, suggesting ischemic muscle damage. Conclusions In this clinical model of I/R injury no damage to the endothelium, antibody deposition or complement activation were observed during early reperfusion. However, an increase of pro-inflammatory cytokines and growth factors was shown, suggesting a contribution of these molecules in the early stages of I/R injury.

Migration of immature mouse DC across resting endothelium is mediated by ICAM-2 but independent of beta2-integrins and murine DC-SIGN homologues

Immature dendritic cells (DC) reside in tissues where they initiate immune responses by taking up foreign antigens. Since DC have a limited tissue half-life, the DC pool in tissues has to be replenished constantly. This implies that precursor/immature DC must be able to cross non-activated endothelium using as yet unknown mechanisms. Here we show that immature, but not mature bone marrow-derived murine DC migrate across resting endothelial monolayers in vitro. We find that endothelial intercellular adhesion molecule-2 (ICAM-2) is a major player in transendothelial migration (TEM) of immature DC, accounting for at least 41% of TEM. Surprisingly, the ICAM-2-mediated TEM was independent of beta2-integrins, the known ICAM-2 ligands, since neither blocking of beta2-integrins with antibodies nor the use of CD18-deficient DC affected the ICAM-2-specific TEM. In humans, the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) was shown to interact with ICAM-2, suggesting a similar role in mice. However, we find that none of the murine DC-SIGN homologues mDC-SIGN, murine DC-SIGN-related molecule-1 (mSIGN-R1) and mSIGN-R3 is expressed on the surface of bone marrow-derived mouse DC. Taken together, this study shows that ICAM-2 strongly supports transmigration of immature DC across resting endothelium by interacting with ligands that are distinct from beta2-integrins and DC-SIGN homologues.

ABO histo-blood group antigen expression on the graft endothelium long term after ABO-compatible, non-identical heart transplantation

We recently reported a complete change in the endothelial ABO histo-blood group phenotype of a cardiac allograft long term after B to O mismatched transplantation. In the context of the current controversy on graft recolonization with recipient endothelial cells and its importance in the development of immunological unresponsiveness, we monitored the expression of endothelial ABH histo-blood group antigens of 10 ABO-compatible, non-identical cardiac allografts over an observation period of at least 30 months. ABH antigens as well as markers for endothelial cells, erythrocytes and thrombocytes were investigated retrospectively by immunohistochemistry using monoclonal antibodies on sections of formalin-fixed, paraffin-embedded biopsies and were evaluated semi-quantitatively by microscopy. In contrast to our earlier finding of the change in the endothelial ABO histo-blood group phenotype long term after ABO- mismatched transplantation, we could not confirm this change in 10 compatible but non-identical cases.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

T cell interaction with ICAM-1-deficient endothelium in vitro: essential role for ICAM-1 and ICAM-2 in transendothelial migration of T cells

Transendothelial migration is a crucial step in the complex process of lymphocyte extravasation during lymphocyte homing, immunosurveillance and inflammation. However, little is known about the precise role of cell adhesion molecules (CAM) involved in this particular event. To define the CAM involved in T cell adhesion versus transendothelial migration, we have previously established an in vitro transendothelial migration system using mouse T cells and mouse endothelioma cells. We demonstrate here that, using ICAM-1-deficient endothelioma cells derived from ICAM-1 mutant mice, transendothelial migration of T cells was inhibited to a much greater extent when compared to migration across wild-type cells treated with a blocking anti-ICAM-1 monoclonal antibody. This unexpected result was confirmed by a rescue experiment using retroviral transfer of wild-type ICAM-1 into ICAM-1-deficient endothelial cells. Additional experiments showed that, in the absence of functional ICAM-1, only ICAM-2 was involved in transendothelial migration, but not PECAM-1, VCAM-1, or E-selectin. Taking this novel approach, we show that ICAM-1 and ICAM-2 are essential for transendothelial migration of T cells.

Enhanced T cell transmigration across the murine liver sinusoidal endothelium is mediated by transcytosis and surface presentation of chemokines

Transmigration through the liver endothelium is a prerequisite for the homeostatic balance of intrahepatic T cells and a key regulator of inflammatory processes within the liver. Extravasation into the liver parenchyma is regulated by the distinct expression patterns of adhesion molecules and chemokines and their receptors on the lymphocyte and endothelial cell surface. In the present study, we investigated whether liver sinusoidal endothelial cells (LSEC) inhibit or support the chemokine-driven transmigration and differentially influence the transmigration of pro-inflammatory or anti-inflammatory CD4(+) T cells, indicating a mechanism of hepatic immunoregulation. Finally, the results shed light on the molecular mechanisms by which LSEC modulate chemokine-dependent transmigration. LSEC significantly enhanced the chemotactic effect of CXC-motif chemokine ligand 12 (CXCL12) and CXCL9, but not of CXCL16 or CCL20, on naive and memory CD4(+) T cells of a T helper 1, T helper 2, or interleukin-10-producing phenotype. In contrast, brain and lymphatic endothelioma cells and ex vivo isolated lung endothelia inhibited chemokine-driven transmigration. As for the molecular mechanisms, chemokine-induced activation of LSEC was excluded by blockage of G(i)-protein-coupled signaling and the use of knockout mice. After preincubation of CXCL12 to the basal side, LSEC took up CXCL12 and enhanced transmigration as efficiently as in the presence of the soluble chemokine. Blockage of transcytosis in LSEC significantly inhibited this effect, and this suggested that chemokines taken up from the basolateral side and presented on the luminal side of endothelial cells trigger T cell transmigration. CONCLUSION: Our findings demonstrate a unique capacity of LSEC to present chemokines to circulating lymphocytes and highlight the importance of endothelial cells for the in vivo effects of chemokines. Chemokine presentation by LSEC could provide a future therapeutic target for inhibiting lymphocyte immigration and suppressing hepatic inflammation.