Vascular Leukocyte Adhesion Research Articles

Soluble adhesion molecules and functional outcome after ischemic stroke: A Mendelian randomization study

ObjectivesWe employed Mendelian randomization to determine whether genetically predicted circulating levels of endothelial-derived adhesion molecules (soluble intercellular adhesion molecule-1 [sICAM-1]), soluble vascular-leukocyte adhesion molecule-1 [sVCAM-1], and soluble-endothelial-leukocyte adhesion molecule [sE-selectin]) were associated with functional outcome after ischemic stroke. MethodsIndependent genetic variants robustly associated with soluble adhesion molecules, located at or close to the coding gene (cis), were used as genetic instruments. The functional outcome was evaluated using the 3-month modified Rankin Scale (mRS) score after ischemic stroke. A poor functional outcome was defined as mRS ≥ 3 at 3 months. We extracted summary data for functional outcome after ischemic stroke from the Genetics of Ischaemic Stroke Functional Outcome network (n = 6,021). ResultsGenetically elevated sICAM-1 (OR 1.28, 95% CI 1.05-1.56) and sE-selectin (OR 2.69, 95% CI 1.23-5.86) levels were related with poor post-stroke outcome. However, we found no evidence that genetically elevated sVCAM-1 were associated with post-stroke outcome (OR 1.36, 95% CI 0.39-4.66). ConclusionsWe found that genetically elevated higher sICAM-1 and sE-selectin levels are associated with poor post-stroke outcome. Further studies are warranted to evaluate the potential of ICAM-1 and E-selectin to be drug targets for post-stroke recovery.

Reelin Depletion Protects Against Atherosclerosis by Decreasing Vascular Adhesion of Leukocytes.

[Figure: see text].

Reelin depletion protects against autoimmune encephalomyelitis by decreasing vascular adhesion of leukocytes.

Neuroinflammation as a result of immune cell recruitment into the central nervous system (CNS) is a key pathogenic mechanism of multiple sclerosis (MS). However, current anti-inflammatory interventions depleting immune cells or directly targeting their trafficking into the CNS can have serious side effects, highlighting a need for better immunomodulatory strategies. We detected increased Reelin concentrations in the serum of patients with MS, resulting in increased endothelial permeability to leukocytes through increased nuclear factor κB-mediated expression of vascular adhesion molecules. We thus investigated the prophylactic and therapeutic potential of Reelin immunodepletion in experimental autoimmune encephalomyelitis (EAE) and further validated the results in Reelin knockout mice. Removal of plasma Reelin by either approach protected against neuroinflammation and largely abolished the neurological consequences by reducing endothelial permeability and immune cell accumulation in the CNS. Our findings suggest Reelin depletion as a therapeutic approach with an inherent good safety margin for the treatment of MS and other diseases where leukocyte extravasation is a major driver of pathogenicity.

Gut Microbiota Promote Angiotensin II-Induced Arterial Hypertension and Vascular Dysfunction.

BackgroundThe gut microbiome is essential for physiological host responses and development of immune functions. The impact of gut microbiota on blood pressure and systemic vascular function, processes that are determined by immune cell function, is unknown.Methods and ResultsUnchallenged germ‐free mice (GF) had a dampened systemic T helper cell type 1 skewing compared to conventionally raised (CONV‐R) mice. Colonization of GF mice with regular gut microbiota induced lymphoid mRNA transcription of T‐box expression in T cells and resulted in mild endothelial dysfunction. Compared to CONV‐R mice, angiotensin II (AngII; 1 mg/kg per day for 7 days) infused GF mice showed reduced reactive oxygen species formation in the vasculature, attenuated vascular mRNA expression of monocyte chemoattractant protein 1 (MCP‐1), inducible nitric oxide synthase (iNOS) and NADPH oxidase subunit Nox2, as well as a reduced upregulation of retinoic‐acid receptor‐related orphan receptor gamma t (Rorγt), the signature transcription factor for interleukin (IL)‐17 synthesis. This resulted in an attenuated vascular leukocyte adhesion, less infiltration of Ly6G+ neutrophils and Ly6C+ monocytes into the aortic vessel wall, protection from kidney inflammation, as well as endothelial dysfunction and attenuation of blood pressure increase in response to AngII. Importantly, cardiac inflammation, fibrosis and systolic dysfunction were attenuated in GF mice, indicating systemic protection from cardiovascular inflammatory stress induced by AngII.ConclusionGut microbiota facilitate AngII‐induced vascular dysfunction and hypertension, at least in part, by supporting an MCP‐1/IL‐17 driven vascular immune cell infiltration and inflammation.

Centella Asiatica and Lipoic Acid, or a combination thereof, inhibit monocyte adhesion to endothelial cells from umbilical cords of gestational diabetic women

Background and aimsDiabetes mellitus is associated with inflammatory endothelial activation and increased vascular leukocyte adhesion molecule expression, both playing a prominent role in the development of vascular complications. Centella asiatica (CA) and Lipoic Acid (LA) have shown anti-inflammatory and anti-oxidant properties in a variety of experimental models; however, their action on human umbilical vein endothelial cells (HUVECs), chronically exposed to hyperglycemia and pro-inflammatory environment during pregnancy, is still unknown. Methods and resultsIn HUVECs from umbilical cords of gestational diabetic (GD) or healthy (C) women, both CA and LA affected tumor necrosis factor-α (TNF-α)-induced inflammation, being associated with a significant decrease in vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression (western blot) and exposure (flow cytometry), as well as monocyte-HUVECs interaction (adhesion assay). Notably, this was associated with a significant reduction of an index of nitro-oxidative stress, such as the intracellular peroxynitrite levels (fluorescence detection by cytometric analysis), Mitogen-Activated Protein kinase (p44/42 MAPK) expression/phosphorylation levels and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) cytoplasm-nucleus translocation (flow cytometry).Overall our results indicate that both CA and LA used separately, and even better when combined, are effective to reduce the inflammatory response in TNF-α-treated HUVECs. Notably, this was more significant in GD than in C-HUVECs and also evident at baseline. ConclusionIn conclusion, our in vitro study demonstrates that both CA and LA, or a combination thereof, are able to mitigate the potentially dangerous effects on the endothelium of chronic exposure to hyperglycemia in vivo.

Abstract W P369: Inhibitory Effect of FTY720 on Neuroinflammation in Cerebral Ischemia-reperfusion

Background: Neuroinflammation is a key contributor to brain injury in cerebral ischemia-reperfusion (CIR). FTY720 has been shown to be neuroprotective in animal stroke models. In most studies, FTY720 treatment was initiated either before or shortly after the cerebral insult. The goal of this study is to investigate the effect of FTY720 on CIR-associated neuroinflammation and outcome using a therapeutic window similar to the one utilized in clinical practice. Methods: We used the rat middle cerebral artery (MCA) occlusion model for CIR. The right MCA was occluded for 1h followed by reperfusion. Animals were treated with vehicle or 0.5 mg/kg FTY720 intraperitoneally at 3h post-reperfusion. Neurobehavioral test battery (scale from 0 to 21 points with lower scores representing increased neurological deficits), grid-walking test, infarct volume, and brain water content were determined 24h post CIR. A cranial window was established at 24h post occlusion and leukocyte trafficking behavior was monitored by direct microscopic observation of surface venules. Pial venular leukocyte adhesion was expressed as the % of vascular area occupied by adherent rhodamine-6G-labeled leukocytes. Statistical analysis was performed by t test. Results: Compared to the vehicle group (n=10), FTY720 animals (n=10) had improved neurological score (8.6±1.9 vs. 13.7±1.9; p<0.001) and better motor performance throughout all subsections of the grid test (p<0.001). FTY720 treatment also decreased infarct volume (vehicle: 342±182; FTY720: 122±138 mm 3 ; p=0.04) and ipsilateral brain edema, measured as water content (vehicle: 84.5±1.05%; FTY720: 79.4±0.87%, p=0.003). Leukocyte trafficking study showed a significant increase in vascular leukocyte adhesion 24 h post reperfusion in the vehicle group which was markedly decreased by FTY720 treatment (sham: 3.0±0.6%; vehicle: 11.4±2.6%; FTY720: 5.2±1.4%; p<0.001). Conclusion: FTY720 given at 3h post reperfusion reduces infarct volume, brain edema, neurological disability, and vascular leukocyte adhesion. These results support the beneficial effect of FTY720 when used in a clinically relevant timeframe and provides direct evidence of the anti-inflammatory effect of FTY720 on CIR.

NADPH Oxidase 1 Plays a Key Role in Diabetes Mellitus–Accelerated Atherosclerosis

In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E-deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E-deficient mice with GKT137831 attenuated atherosclerosis development. These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.

Selectin Mechanokinetics and Two-Dimensional Bond Formation Determine and are Reported by Nano-Motion Dynamic Patterns

Binding between surface-tethered proteins at cellular interfaces has been considered two-dimensional because of the restricted motion of the two binding partners. Two-dimensional protein interactions between cells are critical for many biological processes, such as leukocyte vascular adhesion via selectins. Experimental measurements have yielded data on the kinetics of selectin bond formation and dissociation. Additionally, computational methods have been employed to integrate molecular and cellular properties to elucidate the factors that influence the dynamics of selectin-mediated rolling. Simulation methods focused on biomolecular properties promise to yield additional novel insights into the molecular component of adhesion with the assistance of measurements from improved assays. We performed an in silico investigation on the effects of the kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, two-dimensional bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Statistics describing the motion patterns tied simulated motions to experimentally reported quantities. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve pause times observed in microbead assays. Notably, rebinding events were enhanced by the reduced separation distance following initial sphere capture. The result demonstrates vertical transport can contribute to an enhancement in the apparent bond formation rate. The result also suggests a new mechanism that may be important for the rebinding events characteristic of stable leukocyte rolling. When selectin receptor and ligand are restricted to small, two-dimensional interaction zones during rolling, the resultant wobble was found to be dependent on the confinement model used. Insight into two-dimensional bond formation gained from flow cell assays might also therefore be important to understand processes involving extended cellular interactions, such as immunological synapse formation.

Modifying the anti-inflammatory effects of high-density lipoprotein

The anti-inflammatory effects of high-density lipoproteins (HDL) are well documented and include inhibition of low-density lipoprotein (LDL) oxidation, reduction of inflammatory cytokines and vascular leukocyte adhesion molecules, and participation in innate immunity. However, certain conditions, including coronary disease, diabetes mellitus, systemic inflammation, and a diet high in saturated fat, are associated with modification of HDL such that it paradoxically enhances LDL oxidation and/or vascular inflammation. Treatment with statins and/or apolipoprotein A1 mimetic peptides improves HDL's anti-inflammatory functions, and these as well as other medications may represent a novel pathway through which to target atherosclerosis.

Effect of Topical Nipradilol on Retinal Microvascular Leukocyte Adhesion in Diabetic Rats

Background: Retinal leukostasis plays an important role in the pathogenesis of diabetic retinopathy. Objectives: We studied the effects of nipradilol, a topical antiglaucoma αβ-blocker and nitric oxide donor, on the retinal vascular leukocyte adhesion of rats with diabetes. Methods: Diabetes was induced in seven Brown-Norway rats by one intravenous injection (65 mg/kg) of streptozotocin and confirmed by blood glucose levels >350 mg/dl 48 h after the injection. Nipradilol solution was instilled in the right eye and nipradilol-free base solution in the left eye for 3 weeks, after which the retinal microcirculation was evaluated by acridine orange leukocyte digital fluorography using laser scanning ophthalmoscopy. Leukocytes trapped in the retina were counted around the optic disc in a 5-disc-diameter area and compared between the right and the left eye. Results: The mean retinal leukostasis count in the nipradilol-treated eyes (19 ± 15 cells) was significantly lower than in the untreated eyes (49 ± 19 cells; p < 0.0008). The diameter of the retinal artery in the eyes treated with nipradilol significantly increased (111 ± 13.5%) compared with untreated eyes (p < 0.03). Conclusions: Topical nipradilol significantly reduced retinal leukostasis in the retinal microcirculation in diabetic rats and may be a prophylactic agent for early diabetic retinopathy through its nitric oxide donor effects on the microcirculation.

Role of monocyte l-selectin in the development of post-traumatic organ failure

The vascular leucocyte adhesion molecule, l-selectin, plays an important early role in monocyte trafficking at sites of inflammation, a process which leads to the development of inflammatory organ failure. In this prospective observational study, we investigate whether early numerical and functional changes in circulating monocytes, expression of monocyte l-selectin (CD62L) and monocyte:neutrophil l-selectin ratios are related to the subsequent development of post-traumatic organ failure (OF) and multiple organ dysfunction syndrome (MODS). Monocyte counts and cell surface l-selectin were measured by an automated cell counter and flow cytometry, respectively. Of 164 trauma patients admitted to a university emergency department resuscitation room, 64 had multiple injuries, 51 developed OF, 20 developed MODS and 21 died. Early monocyte counts in patients with multiple injuries were lower in those who developed MODS (0.44×10 9/l) compared with those who did not (0.60×10 9/l; P=0.024). Monocyte l-selectin mean channel fluorescence increased with injury severity and was highest in those who developed MODS ( P=0.033). In the sub-group of patients with multiple injuries, l-selectin mean channel fluorescence was also greater in those patients who developed MODS compared with patients who did not develop MODS ( P=0.042). The monocyte to neutrophil count ratio also decreased with injury severity ( P=0.006). Using optimal cut off values for l-selectin mean channel, fluorescence, the positive and negative predictive values for OF was 43.5 and 91.4%, respectively and for MODS it was 25.4 and 92.9%, respectively. Alterations in early circulating monocyte counts and l-selectin expression after injury are related to the development of post-traumatic organ failure and suggest an area in the inflammatory pathway that may be influenced by l-selectin blockade.

Selective loss of peripheral blood CD45RO + T lymphocytes correlates with increased levels of serum cytokines and endothelial cell-derived soluble cell adhesion molecules in patients with chronic idiopathic neutropenia of adults

The present study was designed to investigate the hypothesis that selective loss of peripheral blood CD45RO+ T lymphocytes in patients with chronic idiopathic neutropenia of adults (CINA), previously reported from our laboratory, may be due to enhanced extravasation into the tissues. Serum levels of endothelial cell-derived soluble cell adhesion molecules (sELAM, sICAM and sVCAM), usually used as indicators of endothelial cell activation, were measured in 73 CINA patients and 32 healthy volunteers using a micro-ELISA method. We found that patients had markedly elevated concentrations of all three soluble cell adhesion molecules studied compared to the controls, and serum levels of sELAM, sICAM and, more importantly, sVCAM correlated inversely with the numbers of both CD4+/CD45RO+ and CD8+/CD45RO+ T cell subsets. Using a micro-ELISA method, we also measured serum levels of two endothelial cell activators, interleukin (IL)-1beta and TNF-alpha, and found that CINA patients had significantly higher cytokine concentrations than control subjects. Serum levels of IL-1beta and TNF-alpha correlated positively with the values of all three soluble cell adhesion molecules and inversely with the numbers of CD4+/CD45RO+ and CD8+/CD45RO+ T cell subsets. Moreover, we measured serum levels of the chemokine RANTES by a micro-ELISA technique and found that CINA patients also had elevated concentrations of the molecule compared to controls. Serum RANTES correlated positively with IL-1beta, TNF-alpha, sICAM, sVCAM and sELAM and inversely with the numbers of both CD4+/CD45RO+ and CD8+/CD45RO+ T cell subsets. These findings strongly suggest that CINA patients have an activated endothelium to which CD45RA+ and CD45RO+ T cells tether and roll, but firm adhesion and transendothelial migration are restricted to CD45RO+ T cell subsets, as endothelial VCAM-1 interacts with the vascular leukocyte adhesion molecule-4 (VLA-4) constitutively expressed on CD45RO+ but not on CD45RA+ T cells. Subsequent subendothelial and tissue migration of CD45RO+ T cells may be facilitated by the chemokine RANTES, which acts mainly on CD45RO+ T cells. We concluded that selective loss of peripheral blood CD45RO+ T lymphocytes in CINA patients is probably due, at least in part, to enhanced extravasation of both CD4+/CD45RO+ and CD8+/CD45RO+ T cell subsets into the tissues.