Endothelial Intercellular Adhesion Molecule-1 Expression Research Articles

The Effect of Diammonium Glycyrrhizinate Lipid Ligand on Nonalcoholic Fatty Liver Disease Induced Leukocyte Infiltration in Rat Model

Objective: To investigate the effects of diammonium glycyrrhizinate lipid ligand (DGLL) on nonalcoholic fatty liver disease (NAFLD)-induced leukocyte infiltration in rat and underlying mechanisms. Methods: Sixty SD rats were randomly distributed into six groups, control group, NAFLD model group, DGLL (30, 60, 120 mg/kg) treatment group and positive control (biphenyl biester tablets (BDP), 200 mg/kg) group. The expression and activity of myeloperoxidase (MPO) in liver tissues were measured by immunohistochemistry and ELISA, respectively. The expressions of leukocyte adhesion molecules in whole blood were determined by flow cytometry. The intercellular adhesion molecule-1 (ICAM-1) expressions in liver tissues was detected by western blotting. Results: Compared with NAFLD model groups, the expression and activity of MPO in liver tissues of DGLL (30, 60, 120 mg/kg) treatment group were all significantly ameliorated by the administration of DGLL (P < 0.05). Meanwhile, the up-regulation of adhesion molecules expressed on monocytes and neutrophils in whole blood, as well as endothelial ICAM-1 expression in NAFLD model groups were eliminated by DGLL treatment (P < 0.05). Conclusion: DGLL ameliorated leukocyte activation and infiltration in rat liver with NAFLD, which might be related to suppression of adhesion molecules expression on leukocyte and endothelium.

Inhibition of Vascular Inflammation by Apolipoprotein A-IV.

BackgroundApolipoprotein (apo) A-IV, the third most abundant apolipoprotein in human high density lipoproteins (HDLs), inhibits intestinal and systemic inflammation. This study asks if apoA-IV also inhibits acute vascular inflammation.MethodsInflammation was induced in New Zealand White rabbits by placing a non-occlusive silastic collar around the common carotid artery. A single 1 mg/kg intravenous infusion of lipid-free apoA-IV or saline (control) was administered to the animals 24 h before collar insertion. The animals were euthanised 24 h post-collar insertion. Human coronary artery cells (HCAECs) were pre-incubated with reconstituted HDLs containing apoA-IV complexed with phosphatidylcholine, (A-IV)rHDLs, then activated by incubation with tumour necrosis factor (TNF)-α. Cell surface vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the TNF-α-activated HCAECs was quantified by flow cytometry. VCAM-1, ICAM-1 and 3β-hydroxysteroid-Δ24 reductase (DHCR24) mRNA levels were quantified by real time PCR.ResultsApolipoprotein ApoA-IV treatment significantly decreased collar-induced endothelial expression of VCAM-1, ICAM-1 and neutrophil infiltration into the arterial intima by 67.6 ± 9.9% (p < 0.01), 75.4 ± 6.9% (p < 0.01) and 74.4 ± 8.5% (p < 0.05), respectively. It also increased endothelial expression of DHCR24 by 2.6-fold (p < 0.05). Pre-incubation of HCAECs with (A-IV)rHDLs prior to stimulation with TNF-α inhibited VCAM-1 and ICAM-1 protein levels by 62.2 ± 12.1% and 33.7 ± 5.7%, respectively. VCAM-1 and ICAM-1 mRNA levels were decreased by 55.8 ± 7.2% and 49.6 ± 7.9%, respectively, while DHCR24 mRNA expression increased by threefold. Transfection of HCAECs with DHCR24 siRNA attenuated the anti-inflammatory effects of (A-IV)rHDLs. Pre-incubation of TNF-α-activated HCAECs with (A-IV)rHDLs also inhibited nuclear translocation of the p65 subunit of nuclear factor-κB (NF-κB), and decreased IκBα phosphorylation.ConclusionThese results indicate that apoA-IV inhibits vascular inflammation in vitro and in vivo by inhibiting NF-κB activation in a DHCR24-dependent manner.

Deep Sea Water-Dissolved Organic Matter Intake Improves Hyperlipidemia and Inhibits Thrombus Formation and Vascular Inflammation in High-Fat Diet Hamsters.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease caused by oxidative stress, inflammation and lipid deposition within liver cells, and is subsequently contributing to cardiovascular diseases such as atherosclerosis. Deep sea water (DSW) is characterized by its clearance and abundant nutrients with antioxidant and anti-inflammatory activity to confer therapeutic potential. We aimed to explore the therapeutic capability of our prepared multi-filtration DSW-dissolved organic matter (DSW-DOM) on high-fat diet-induced hyperlipidemia and endothelial dysfunction in hamsters. A high-fat/high-cholesterol diet led to increased oxidative stress, including blood reactive oxygen species (ROS), plasma malondialdehyde (MDA) and hepatic CYP2E1 expression; an increased hyperlipidemic profile and SREBP 1-mediated fatty liver; promoted NFκB p65-mediated hepatic inflammation; triggered PARP-mediated hepatic apoptosis; and enhanced endothelial intercellular adhesion molecule-1 (ICAM-1) and von Willebrand factor (VWF)-mediated atherosclerosis associated with the depressed hepatic antioxidant Paraoxonase 1 (PON1) expression. The DSW-DOM-enriched 1295 fraction, with strong H2O2 scavenging activity, efficiently reduced several oxidative stress parameters, the lipid profile, inflammation, and apoptosis, possibly through the PON1-mediated antioxidant capability. Furthermore, DSW-DOM treatment significantly decreased the endothelial ICAM-1 and VWF expression, subsequently leading to the elongation of time to occlusion of FeCl3-induced arterial thrombosis and to the inhibition of FeCl3-induced fluorescent platelet adhesion to mesentery arterioles in the high-fat diet. Based on the above results, our data suggest that DSW-DOM intake via antioxidant defense mechanisms confers protective effects against high-fat diet-enhanced, oxidative stress-mediated hyperlipidemia, and endothelial dysfunction evoked atherosclerosis by downregulating oxidative injury, lipogenesis, inflammation and apoptosis.

Adenoviral mediated expression of anti-inflammatory progranulin by placental explants modulates endothelial cell activation by decrease of ICAM-1 expression

IntroductionFunctional disorders of the villous trophoblast may result in preeclampsia through the release of endothelial activating substances. Progranulin is an anti-inflammatory, pro-angiogenic cytokine with TNF-α antagonizing activity. The trophoblastic expression of progranulin is increased during preeclampsia. The aim of the study was to investigate the impact of placental progranulin synthesis on endothelial cell activation. MethodsPlacental progranulin expression was modified by transduction of an adenoviral vector. Primary isolated human umbilical venous endothelial cells (HUVECs) were incubated with conditioned medium of first trimester placental explants. Functional studies on HUVECs included assays for proliferation, viability, cytotoxicity and analyzes of Intercellular adhesion molecule-1 (ICAM-1) and E-selectin expression. ResultsPlacental progranulin expression was more than 10-fold higher by using an adenoviral-mediated overexpression system (Ad.PGRN) compared to control vector (Ad.CTRL) and untreated controls. Incubation of HUVECs with conditioned placental medium revealed a dose-dependent increase of cytotoxicity, reduced cell proliferation and viability and resulted in an increase of ICAM-1 and E-selectin expression. Overexpression of progranulin (Ad.PGRN) antagonized the ICAM-1 expression induced by conditioned medium. However progranulin did not influence the effects on cell proliferation, viability, cytotoxicity and E-selectin expression in HUVECs. DiscussionRegulation of gene expression in human placental explants is possible by usage of an adenoviral vector system. The increase of endothelial ICAM-1 expression following the incubation with placental conditioned medium was partly reversed by overexpression of placental progranulin. It is suggested that up-regulation of the placental progranulin expression is an endogenous anti-inflammatory mechanism that partially antagonizes the endothelial cell activation during preeclampsia.

Long-term endothelial dysfunction in irradiated vessels: an immunohistochemical analysis.

Microvascular free flap reconstruction has become astandard technique in head and neck reconstructive surgery. Pre-operative radiotherapy is associated with ahigher incidence of free flap malperfusion and the need for operative revision. Irradiated vessels present characteristic histomorphological and structural changes. Alterations in endothelial cells of irradiated arteries remain incompletely investigated especially with regard to long-term changes in endothelial dysfunction supporting an intraluminal pro-thrombotic and pro-inflammatory milieu. Endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E‑ and P‑selectin, endothelial NO-synthase (eNOS), thrombomodulin and plasminogen activator inhibitor-1 (PAI-1) in irradiated and non-irradiated arteries was analysed using immunohistochemistry and Remmele scale grading. The average radiation dose was 58.7 ± 7.0 Gy; the time interval between end of radiation and tissue sampling was 106.0 ± 86.8months. Endothelial expression of ICAM-1, VCAM-1, E‑ and P‑selectin as well as PAI-1 was significantly increased in previously irradiated arteries compared with non-irradiated controls, whereas thrombomodulin and eNOS expression did not show any differences. However, when comparing non-irradiated free flap arteries with irradiated arteries from the head and neck area in respective individuals, eNOS expression was significantly lower in irradiated vessels whereas ICAM-1, VCAM-1, E‑/p-Selectin and PAI-1 showed significantly higher expression levels. There is ongoing endothelial dysfunction in terms of increased expression of pro-thrombotic and pro-inflammatory markers in irradiated arteries even years after radiotherapy. Treating this endothelial dysfunction might reduce the complication rates associated with microvascular free flap reconstructions in irradiated patients.

Effects of fermented Sorghum bicolor L. Moench extract on inflammation and thickness in a vascular cell and atherosclerotic mice model.

Atherosclerosis is a major cause of coronary heart disease. As a result of the development of atherosclerotic lesions, the walls of blood vessels become thicker and inhibit blood circulation. Atherosclerosis is caused by a high-fat diet and vascular injury. Chronic arterial inflammation plays an important role in the pathogenesis of atherosclerosis. In particular, secretion of the pro-atherogenic cytokine tumor necrosis factor-α induces expression of endothelial adhesion molecules including P-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1), which mediate attachment of circulating monocytes and lymphocytes. In this study, we examined the anti-atherosclerotic effect of sorghum, which is known to have anti-oxidant and anti-inflammatory activity. A 50% ethanol extract of Sorghum bicolor L. Moench fermented with Aspergillus oryzae NK (fSBE) was used for experiments. In vitro expression of endothelial adhesion molecules VCAM-1 and ICAM-1 and pro-inflammatory factor cyclooxygenase-2 was significantly decreased and that of the anti-atherogenic factor heme oxygenase-1 significantly increased by fSBE (P < 0.05). At the in vivo level, we examined fat droplets of liver tissue, and aortic thickness via histological analysis, and determined the blood lipid profile through chemical analysis. fSBE at a dose of 200mg/kg significantly improved blood and vascular health (P < 0.05). Taken together, these results demonstrate that fSBE has potential as a therapeutic anti-atherosclerotic agent.

RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis.

The objective of this study is to determine the role and underlying mechanisms of RGC-32 (response gene to complement 32 protein) in atherogenesis. RGC-32 was mainly expressed in endothelial cells of atherosclerotic lesions in both ApoE-/- (apolipoprotein E deficient) mice and human patients. Rgc-32 deficiency (Rgc32-/-) attenuated the high-fat diet-induced and spontaneously developed atherosclerotic lesions in ApoE-/- mice without affecting serum cholesterol concentration. Rgc32-/- seemed to decrease the macrophage content without altering collagen and smooth muscle contents or lesional macrophage proliferation in the lesions. Transplantation of WT (wild type) mouse bone marrow to lethally irradiated Rgc32-/- mice did not alter Rgc32-/--caused reduction of lesion formation and macrophage accumulation, suggesting that RGC-32 in resident vascular cells, but not the macrophages, plays a critical role in the atherogenesis. Of importance, Rgc32-/- decreased the expression of ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cells both in vivo and in vitro, resulting in a decrease in TNF-α (tumor necrosis factor-α)-induced monocyte-endothelial cell interaction. Mechanistically, RGC-32 mediated the ICAM-1 and VCAM-1 expression, at least partially, through NF (nuclear factor)-κB signaling pathway. RGC-32 directly interacted with NF-κB and facilitated its nuclear translocation and enhanced TNF-α-induced NF-κB binding to ICAM-1 and VCAM-1 promoters. RGC-32 mediates atherogenesis by facilitating monocyte-endothelial cell interaction via the induction of endothelial ICAM-1 and VCAM-1 expression, at least partially, through NF-κB signaling pathway.

Distinct Roles For ROCK1 and ROCK2 in the Regulation of Oxldl-Mediated Endothelial Dysfunction

Background/Aims: This study used Rho-associated protein kinase (ROCK) isoform-selective suppression or a ROCK inhibitor to analyze the roles of ROCK1 and ROCK2 in regulating endothelial dysfunction triggered by oxidized low-density lipoprotein (oxLDL). Methods: ROCK1 or ROCK2 expression in human umbilical vein endothelial cells (HUVECs) was suppressed by small interfering RNA (siRNA). HUVECs were pretreated with 30 μM Y27632 (pan ROCK inhibitor) for 30 min before exposure to 200 μg/mL oxLDL for an additional 24 h. Cell viability was determined by the MTT assay, and cell apoptosis was evaluated by the TUNEL assay. Protein expression and phosphorylation were assessed by Western blot analysis. The morphology of total and phosphorylated vimentin (p-vimentin) and the co-localization of vimentin with vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were detected by the immunofluorescence assay. The adhesion of promonocytic U937 cells to HUVECs was observed by light microscopy. Results: ROCK2 suppression or Y27632 treatment, rather than ROCK1 deletion, effectively reduced endothelial cell apoptosis and preserved cell survival. ROCK2 suppression exhibited improved vimentin and p-vimentin cytoskeleton stability and decreased vimentin cleavage by attenuating caspase-3 activity. In addition, increased p-vimentin expression induced by oxLDL was significantly inhibited by ROCK2 deletion or Y27632 treatment. In contrast, ROCK1 suppression showed no obvious effects on the vimentin cytoskeleton, but significantly regulated the expression of adhesion molecules. Endothelial ICAM-1 or VCAM-1 expression induced by oxLDL was obviously inhibited by ROCK1 suppression or Y27632 treatment. Moreover, the expression of ICAM-1 induced by oxLDL could also be reduced by ROCK2 suppression. Furthermore, ROCK2 deficiency or Y27632 treatment inhibited the redistribution of adhesion molecules and their co-localization with vimentin caused by oxLDL. These effects resulted in the significant inhibition of monocyte-endothelial adhesion induced by oxLDL. Conclusion: The results of this study support the novel concept that ROCK1 is involved in oxLDL-induced cell adhesion by regulating adhesion molecule expression, whereas ROCK2 is required for both endothelial apoptosis and adhesion by regulating both the vimentin cytoskeleton and adhesion molecules. Consequently, ROCK1 and ROCK2 have distinct roles in the regulation of oxLDL-mediated endothelial dysfunction.

Resveratrol mitigates trophoblast and endothelial dysfunction partly via activation of nuclear factor erythroid 2-related factor-2

IntroductionMaternal endothelial dysfunction underlying preeclampsia arises from excessive placental release of anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt1), soluble endoglin (sEng) and activin A. Resveratrol, an activator of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, mediates the gene expression of antioxidant and vasoprotective factors that may counter the endothelial damage imposed by these anti-angiogenic factors. The objective of this study was to assess whether resveratrol could reduce placental oxidative stress and production of anti-angiogenic factors in vitro and/or improve in vitro markers of endothelial dysfunction via Nrf2 activation. MethodWe used in vitro term placental explants to assess the effects of resveratrol on placental oxidative stress and production of sFlt1, sEng and activin A. Using human umbilical vein endothelial cells we investigated the effects of resveratrol on markers of in vitro endothelial dysfunction, including the expression of intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), E-selectin and endothelin-1, and endothelial permeability. To confirm that resveratrol mediated its effects via Nrf2, we examined the impact of resveratrol on the same in vitro markers of endothelial and placental dysfunction following Nrf2 knockdown. ResultsResveratrol significantly decreased placental oxidative stress and the production of sFlt1 and activin A. Resveratrol significantly mitigated tumor necrosis factor–α stimulated endothelial expression of ICAM1, VCAM1, E-selectin and endothelin-1 and prevented an increase in endothelial monolayer permeability. Nrf2 knockdown abolished some of the protective effects of resveratrol on endothelial cells, but not in primary trophoblast cells. ConclusionFeatures of placental and endothelial dysfunction characteristic of preeclampsia are improved by resveratrol in vitro, partially via the modulation of Nrf2.

Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation.

Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%]FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. The supernatants [10%-40%]FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%]FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of β-arrestin-1 with BLT2, protein kinase C (PKC)βI , and PKCδ and served as the first event in a two-event rodent model of ALI. Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients.

Abstract 211: Response Gene to Complement 32 Deficiency Protects Endothelial Cell From Inflammation and Attenuates Atheroslcerosis

Response gene to complement 32 (RGC-32) is involved in diet-induced obesity and hepatic steatosis. Therefore, we hypothesized that RGC-32 plays a role in atherosclerosis. Our current study showed that RGC-32 expression was dramatically induced in endothelial cells (ECs) of the atherosclerotic lesions from both apolipoprotein E-deficient (ApoE -/- ) mice and diseased human arteries. RGC-32 deficiency (Rgc32 -/- ) significantly attenuated the high-fat diet-induced and spontaneously developed atherogenesis in ApoE -/- mice without affecting the serum lipid profiles. In addition, Rgc32 -/- decreased the macrophage content without affecting fibrous cap size. Rgc32 -/- in bone marrow cells had no effect on atherosclerosis development, and Rgc32 -/- mice transplanted with wild-type (WT) bone marrow cells exhibited decreased atherosclerotic lesion area and macrophage infiltration, suggesting that RGC-32 in resident vascular cells plays a critical role in the atherogenesis. Rgc32 -/- decreased the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in ECs both in vivo and in vitro , resulting in a decreased monocyte-EC interaction. Mechanistically, RGC-32 regulated ICAM-1 and VCAM-1 transcription via activation of nuclear factor (NF)-κB. It appeared that RGC-32 interacted with NF-κB, which facilitated NF-κB binding to ICAM-1 and VCAM-1 promoters. Indeed, blockade of NF-κB activity by its selective inhibitor ammonium pyrrolidinedithiocarbamate blocked RGC-32-induced ICAM-1 and VCAM-1 expression. In conclusion, RGC-32 promoted atherogenesis by inducing monocyte-EC interaction through NF-κB-dependent induction of endothelial ICAM-1 and VCAM-1 expression.

Thrombin-activated platelet-derived exosomes regulate endothelial cell expression of ICAM-1 via microRNA-223 during the thrombosis-inflammation response

Platelet activation and endothelial damage play essential roles in atherosclerosis. The pathophysiology of this process is mediated by chemokines and exosomes, two critical players in cell communication. Thrombin-activated platelet-derived exosomes have protective effects on atherosclerosis and endothelial inflammation. To confirm these findings, we isolated exosomes using differential ultracentrifugation and transmission electron microscopy. The exosomes were identified using NanoSight-tracking analysis. Immunofluorescence staining and western blotting were performed to assess exosome uptake and intercellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVECs). We found that the levels of miR-223, miR-339 and miR-21 were elevated in thrombin-activated platelet exosomes. This finding was verified in our atherosclerosis mouse model. We also found that miR-223 transfection in HUVECs inhibited ICAM-1 expression under TNF-α stimulation. Furthermore, the miR-223 inhibitor blocked the downregulating effects of exosomes on ICAM-1 expression. We examined the key proteins of two classical signaling pathways, MAPK and NF-κB, and found that miR-223 inhibited the phosphorylation of p38, JNK and ERK and blocked the nuclear translocation of NF-κB p65. Our results suggest that thrombin-activated platelet-derived exosomes inhibit ICAM-1 expression during inflammation. MiR-223 may mediate this process via regulation of the NF-κB and MAPK pathways.

Polystyrene-Divinylbenzene-Based Adsorbents Reduce Endothelial Activation and Monocyte Adhesion Under Septic Conditions in a Pore Size-Dependent Manner

Endothelial activation with excessive recruitment and adhesion of immune cells plays a central role in the progression of sepsis. We established a microfluidic system to study the activation of human umbilical vein endothelial cells by conditioned medium containing plasma from lipopolysaccharide-stimulated whole blood or from septic blood and to investigate the effect of adsorption of inflammatory mediators on endothelial activation. Treatment of stimulated whole blood with polystyrene-divinylbenzene-based cytokine adsorbents (average pore sizes 15 or 30 nm) prior to passage over the endothelial layer resulted in significantly reduced endothelial cytokine and chemokine release, plasminogen activator inhibitor-1 secretion, adhesion molecule expression, and in diminished monocyte adhesion. Plasma samples from sepsis patients differed substantially in their potential to induce endothelial activation and monocyte adhesion despite their almost identical interleukin-6 and tumor necrosis factor-alpha levels. Pre-incubation of the plasma samples with a polystyrene-divinylbenzene-based adsorbent (30 nm average pore size) reduced endothelial intercellular adhesion molecule-1 expression to baseline levels, resulting in significantly diminished monocyte adhesion. Our data support the potential of porous polystyrene-divinylbenzene-based adsorbents to reduce endothelial activation under septic conditions by depletion of a broad range of inflammatory mediators.Electronic supplementary materialThe online version of this article (doi:10.1007/s10753-016-0408-1) contains supplementary material, which is available to authorized users.

Biomimetic channel modeling local vascular dynamics of pro-inflammatory endothelial changes.

Endothelial cells form the inner lining of blood vessels and are exposed to various factors like hemodynamic conditions (shear stress, laminar, and turbulent flow), biochemical signals (cytokines), and communication with other cell types (smooth muscle cells, monocytes, platelets, etc.). Blood vessel functions are regulated by interactions among these factors. The occurrence of a pathological condition would lead to localized upregulation of cell adhesion molecules on the endothelial lining of the blood vessel. This process is promoted by circulating cytokines such as tumor necrosis factor-alpha, which leads to expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell surface among other molecules. ICAM-1 is critical in regulating endothelial cell layer dynamic integrity and cytoskeletal remodeling and also mediates direct cell-cell interactions as part of inflammatory responses and wound healing. In this study, we developed a biomimetic blood vessel model by culturing confluent, flow aligned, endothelial cells in a microfluidic platform, and performed real time in situ characterization of flow mediated localized pro-inflammatory endothelial activation. The model mimics the physiological phenomenon of cytokine activation of endothelium from the tissue side and studies the heterogeneity in localized surface ICAM-1 expression and F-actin arrangement. Fluorescent antibody coated particles were used as imaging probes for identifying endothelial cell surface ICAM-1 expression. The binding properties of particles were evaluated under flow for two different particle sizes and antibody coating densities. This allowed the investigation of spatial resolution and accessibility of ICAM-1 molecules expressed on the endothelial cells, along with their sensitivity in receptor-ligand recognition and binding. This work has developed an in vitro blood vessel model that can integrate various heterogeneous factors to effectively mimic a complex endothelial microenvironment and can be potentially applied for relevant blood vessel mechanobiology studies.

Expression of Platelet Collagen Receptor Glycoprotein VI in Coronary Heart Disease: Interaction between Platelets and Endothelial Cells.

1) To investigate the expression of platelet collagen receptor glycoprotein VI (GPVI) on the surface of platelets in patients with coronary heart disease (CHD). 2) To investigate the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells after exposure to subjects' platelets. Platelets were extracted from blood samples of 97 subjects including 43 with acute coronary syndrome (ACS), 21 with stable angina pectoris (SAP), and 33 serving as controls in which CHD was excluded. Using flow cytometry, we measured the expression of GPVI and platelet degranulation markers CD62P and CD63. In addition we quantified expression of endothelial cell ICAM-1 and VCAM-1. A higher expression of platelet GPVI was observed in ACS compared with SAP and control groups. The expression level of platelet GPVI significantly correlated with CD62P and CD63 in the CHD population (r = 0.713 and 0.615, both p < 0.01). Significantly higher ICAM-1 expression was observed in endothelial cells after interaction with platelets from the ACS population. In the CHD subjects, the surface expression of platelet GPVI correlated with the level of ICAM-1 in endothelial cells (r = 0.371, p < 0.05). Platelet GPVI associated with platelet activation and endothelial inflammation is a vital index for predicting CHD occurrence and severity.

Bilirubin Inhibits the Transmigration of Monocytes Across Endothelial Cell Monolayers: Implications for Atherosclerosis Prevention

Monocyte infiltration into the vascular endothelium, a critical early step in the formation of atherosclerotic plaques, is mediated primarily by the binding of α and β integrins to endothelial cell adhesion molecules Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1). Our lab previously has shown that bilirubin, a potent, chain-breaking antioxidant, disrupts VCAM-1-dependent processes by scavenging reactive oxygen species (ROS) signaling intermediates. Therefore, we examined the effect of bilirubin on the transmigration of THP-1 human monocytic cells across TNF-α-activated human umbilical vein endothelial cell (HUVEC) monolayers. Bilirubin, at physiological concentrations (0 – 20 µM) was found to inhibit the movement of THP-1 cells across HUVEC monolayers in a dose-dependent manner. Bilirubin did not alter endothelial cell expression of VCAM-1 or ICAM-1 in response to TNF-α, nor did it inhibit the binding of THP-1 cells to HUVEC monolayers, supporting that bilirubin exerts its effect at the level of cellular transmigration. Studies employing blocking antibodies support that VCAM-1 is the principal adhesion molecule mediating monocyte transmigration. In light of epidemiological studies demonstrating an inverse association between serum bilirubin levels and the risk of cardiovascular disease, we speculate that bilirubin prevents plaque formation by inhibiting monocyte migration across the vascular endothelium through disruption of VCAM-1 signaling.

Vascular-resident CD169-positive monocytes and macrophages control neutrophil accumulation in the kidney with ischemia-reperfusion injury.

Monocytes and kidney-resident macrophages are considered to be involved in the pathogenesis of renal ischemia-reperfusion injury (IRI). Several subsets of monocytes and macrophages are localized in the injured tissue, but the pathologic roles of these cells are not fully understood. Here, we show that CD169(+) monocytes and macrophages have a critical role in preventing excessive inflammation in IRI by downregulating intercellular adhesion molecule-1 (ICAM-1) expression on vascular endothelial cells. Mice depleted of CD169(+) cells showed enhanced endothelial ICAM-1 expression and developed irreversible renal damage associated with infiltration of a large number of neutrophils. The perivascular localization of CD169(+) monocytes and macrophages indicated direct interaction with blood vessels, and coculture experiments showed that the direct interaction of CD169(+) cell-depleted peripheral blood leukocytes augments the expression levels of ICAM-1 on endothelial cells. Notably, the transfer of Ly6C(lo) monocytes into CD169(+) cell-depleted mice rescued the mice from lethal renal injury and normalized renal ICAM-1 expression levels, indicating that the Ly6C(lo) subset of CD169(+) monocytes has a major role in the regulation of inflammation. Our findings highlight the previously unknown role of CD169(+) monocytes and macrophages in the maintenance of vascular homeostasis and provide new approaches to the treatment of renal IRI.

Pentoxifylline attenuates leukocyte–endothelial interactions in a two-hit model of shock and sepsis

BackgroundThis study investigated the effects of pentoxifylline (PTX) combined with resuscitation fluids on microcirculatory dysfunctions in a two-hit model of shock and sepsis. Materials and methodsMale Wistar rats (250 g) were submitted to hemorrhagic shock and reperfusion followed by sepsis induced by cecal ligation and puncture. For the initial treatment of shock, rats were randomly divided into: sham, no injury, no treatment; hypertonic saline solution (HS) (7.5%, 4 mL/kg); lactated Ringer's solution (LR, 3 × shed blood volume); HS + PTX (4 mL/Kg + 25 mg/kg PTX); and LR + PTX (3 × shed blood volume + 25 mg/kg PTX). After 48 h of being exposed to the double injury, leukocyte–endothelial interactions were assessed by intravital microscopy of the mesentery. Endothelial expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) was evaluated by immunohistochemistry, as well as lung neutrophil infiltration by histology. ResultsLactated Ringer's solution induced marked increases (P < 0.001) in the number of rolling leukocytes per 10 min (two-fold), adherent leukocytes per 100 μm venule length (six-fold), migrated leukocytes per 5000 μm2 (eight-fold), P-selectin and ICAM-1 expression (four-fold), and lung neutrophil infiltration (three-fold) compared with sham. In contrast, PTX attenuated leukocyte–endothelial interactions, P-selectin and ICAM-1 expression at the mesentery when associated with either LR (P < 0.001) or HS (P < 0.05). Neutrophil migration into the lungs was similarly reduced by PTX (P < 0.05). ConclusionsData presented showed thatpentoxifylline attenuates microcirculatory disturbances at the mesenteric bed with significant minimization of lung inflammation after a double-injury model of hemorrhagicshock and reperfusion followed by sepsis.

Antiplatelet aggregation and endothelial protection of I4, a new synthetic anti-diabetes sulfonylurea compound

I4 is a new synthetic anti-diabetes sulfonylurea compound. The aim of present study was to investigate the preventive effects and primary action mechanisms of I4 on platelet-mediated arterial thrombosis. Platelet aggregation and 5-hydroxytryptamine (5-HT) secretion ex vivo was detected. The time-to-occlusion (TTO), thrombus weight and content of von Willebrand factor (vWF) in rat model of electrical- and ferric chloride-induced vessel occlusion were determined. Meanwhile, a rat model of type 2 diabetes mellitus (T2DM) was established to evaluate the effect of I4 on levels of plasma p-selectin, 6-keto-prostaglandin F1a (6-keto-PGF1a), thromboxane B2 (TXB2), tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1). NO synthesis, NOS activity, adhesion of platelet toward endothelial cell and intercellular adhesion molecule-1 (ICAM-1) expression were examined. Results showed that I4 exhibited a higher inhibitory potency than Glimepiride on ADP-induced platelet aggregation and 5-HT release ex vivo. In addition, I4 reduced the thrombus weight and content of vWF and markedly prolonged TTO. Oral administration of I4 (1 ∼ 10 mg/kg) inhibited p-selectin production, elevated the ratio of plasma 6-keto-PGF1a/TXB2 and t-PA/PAI-1 in T2DM rats. Furthermore, I4 significantly improved NO synthesis and NOS activity, lowered adhesion ratio of platelet toward endothelial cells and ICAM-1 expression on HUVECs. These observations suggest that I4 markedly improves platelet-mediated arterial thrombosis by inhibiting platelet activation and release reaction, ameliorating the endothelial dysfunction such as the suppression of vWF production and the reduction of the overexpression of ICAM-1, displayed its potential in alleviating diabetes-associated vascular complications.

Early Inflammatory Predictors of Cardiac Allograft Vasculopathy

<h3>Purpose</h3> To determine whether prediction of cardiac allograft vasculopathy (CAV) in heart transplant patients improves by adding inflammatory markers to an atherothrombotic (AT) risk-prediction model. <h3>Methods and Materials</h3> AT markers (myocardial fibrin, loss of vascular antithrombin and tissue plasminogen activator, arterial endothelial intercellular adhesion molecule-1 expression) and inflammatory markers interleukin-6 (IL-6) and C-reactive protein (CRP) were measured in serial heart biopsy and serum samples of 172 patients followed prospectively for 8.9±5.0 years for CAV. First stepwise logistic regression models were estimated using only AT markers obtained from (1) the first post-transplant biopsy only (median: 9 days), or (2) all biopsies obtained within 3 months. Then model improvement was evaluated after adding IL-6 and CRP to the AT models. Final multivariate models were adjusted for other covariates and cross-validated in 200 bootstrapped samples. <h3>Results</h3> Most AT markers univariately predicted CAV at 5- and 10-years. In multivariate AT models, antithrombin and fibrin were the only significant predictors of CAV. Adding the inflammatory marker CRP, but not IL-6, to the best AT models (Table 1) improved discriminatory power as indicated by the increase in ROC value. <h3>Conclusions</h3> AT markers significantly predict future CAV. Adding the inflammatory marker CRP, but not IL-6, improves the predictive power of the AT model. [figure 1] [figure 2]