Reactive oxygen species (ROS) control the integrity of vascular endothelium. Our laboratory has recently shown that transduction human umbilical vein endothelial cells (HUVECs) with an active variant small GTPase Rac promotes production ROS, ROS-dependent activation p38 mitogen-activated protein kinase, and loss vascular/endothelial-cadherin-mediated cell-cell adhesion. Here we show HUVECs express mRNAs for NOX2 as well NOX4 mRNA, but not NOX1 or NOX3. Interestingly, was expressed at...

Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no have been detected at locations other than the cell surface. We studied step intracellular transport by visualizing living cells. For purpose we stably expressed a modified HLA-DR1 beta chain with Green Fluorescent Protein (GFP) coupled its cytoplasmic tail (beta-GFP) II-expressing Mel JuSo This modification of does not affect assembly,...

Rho GTPases are crucial regulators of the actin cytoskeleton and they play a role in control membrane trafficking. In contrast to close family members RhoA RhoC, RhoB localises endosomes delays epidermal growth factor receptor traffic. Here, we show that activated induces peripheral distribution endosomes, which align along subcortical stress fibres surrounded by an coat. The Diaphanous-related formin, Dia1, is recruited RhoB. Dia1 required for formation coat around downstream RhoB,...

Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves integrity. In order to maintain barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation cytoskeletal dynamics. Here, we show loss of results in intercellular gap formation a drop electrical resistance monolayers primary human cells. Detailed analysis revealed adhesion, induced by VE-cadherin-blocking antibodies, preceded dependent on...

Abstract Extravasation of leukocytes is a crucial process in the immunological defense. In response to local concentration chemokines, circulating adhere and migrate across vascular endothelium toward inflamed tissue. The small guanosinetriphosphatase Rap1 plays an important role regulation leukocyte adhesion, polarization, chemotaxis. We investigated guanine nucleotide exchange protein for directly activated by cAMP (Epac1) adhesion chemotaxis promonocytic cell line primary monocytes. found...

Objective— The purpose of this study was to investigate the presence and functionality P-selectin glycoprotein ligand-1 (PSGL-1) on activated endothelial cells (ECs). Methods Results— We show here that PSGL-1 is expressed at mRNA protein levels in umbilical vein microvascular ECs. Furthermore, (ePSGL-1) functional mediates adhesion monocytes or platelet-monocyte complexes (PMCs) endothelium a flow model. ePSGL-1 expression not affected by treating ECs with inflammatory stimuli (tumor...

Wounds in the mouth heal faster and with less scarification inflammation than those skin. Saliva is thought to be essential for superior oral wound healing, but involved mechanism still unclear. We have previously discovered that a human-specific peptide, histatin, might implicated wound-healing properties of saliva. Here we report histatin enhances reepithelialization human full-skin model closely resembling normal The peptide does not stimulate proliferation induces cell spreading...

Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response agonists like thrombin vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition VWF, several other components are known be stored WPBs, osteoprotegerin, monocyte chemoattractant protein-1 angiopoetin-2 (Ang-2). Here, we...

The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading migration mediated by signaling through effector proteins, such as the PAK kinases Scar WAVE proteins. We previously identified series regulatory proteins that associate with its hypervariable C-terminal domain, including activator β-Pix (also known Rho guanine-nucleotide-exchange factor 7) membrane adapter caveolin-1. Here, we show associates, C-terminus, F-BAR domain protein PACSIN2, an inducer...

Directional cell migration is crucially dependent on the spatiotemporal control of intracellular signalling events. These events regulate polarized actin dynamics, resulting in protrusion at front and contraction rear. The cytoskeleton regulated through by Rho-like GTPases, such as RhoA, which stimulates myosin-based contractility, CDC42 Rac1, promote polymerization protrusion. Here, we show that Rac1 binds adapter protein caveolin-1 (Cav1) activity promotes Cav1 accumulation Rac1-positive...

The migration of leukocytes across the vascular endothelium is crucial for immunosurveillance as well inflammatory responses. Uncontrolled leukocyte transendothelial results in pathologies such asthma, rheumatoid arthritis, and atherosclerosis. molecular mechanisms that regulate involve signaling downstream intracellular messengers cAMP, calcium, phosphoinositol lipids, or reactive oxygen species. Among these, cAMP particularly intriguing because it generated both endothelial cells regulates...

RhoGTPases control endothelial cell (EC) migration, adhesion, and barrier formation. Whereas the relevance of RhoA for function is widely accepted, role homologue RhoB poorly defined. are 85% identical, but RhoB’s subcellular localization half-life uniquely different. Here, we studied ubiquitination stability in primary human ECs. We show that K63 polyubiquitination at lysine 162 181 targets protein to lysosomes. Moreover, identified RING E3 ligase complex Cullin-3–Rbx1–KCTD10 as key...

ABSTRACT MHC class II molecules exert their function at the cell surface by presenting to T cells antigenic fragments that are generated in endosomal pathway. The targetted early lysosomal structures, termed MIIC, where they interact with and subsequently transported surface. We previously visualised vesicular transport of II-containing lysosomes from microtubule organising centre (MTOC) region towards living cells. Here we show MIIC move bidirectionally a ‘stop- and-go’ fashion....

Background The shear-stress induced transcription factor KLF2 has been shown to induce an atheroprotective phenotype in endothelial cells (EC) that are exposed prolonged laminar shear. In this study we characterized the effect of shear stress-induced on regulation and composition Weibel-Palade bodies (WPBs) using peripheral blood derived ECs. Methodology Principal Findings Lentiviral expression resulted a 4.5 fold increase number WPBs per cell when compared mock-transduced cells....

Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists raise intracellular Ca2+ or cAMP. We have previously shown cAMP-mediated WPB is dependent on protein kinase A (PKA) and involves activation of small GTPase RalA. Here, we investigated a possible role for another PKA-independent signaling pathway regulation exocytosis, namely guanine nucleotide exchange factor Epac1 its...

Lipoprotein lipase (LPL), a key enzyme in lipoprotein triglyceride metabolism, produces marked increase the retention and uptake of all classes lipoproteins by cultured cells. It was previously shown that two different receptors are involved mediating LPL effects: heparan sulfate proteoglycans (HSPG) low density (LDL) receptor-related protein/alpha 2 macroglobulin receptor (LRP). By immunofluorescence we show here cell surface-bound displays pattern corresponds to described distribution...

In this study, the translocation of NADPH oxidase components p67(phox) and Rac2 was studied during phagocytosis in living cells. For purpose, green fluorescent protein (GFP)-tagged versions these proteins were expressed myeloid cell line PLB-985. First, correct localization p67GFP GFP-Rac2 shown serum-treated zymosan by wild-type PLB-985 cells X-CGD (chronic granulomatous disease) cells, which lack expression flavocytochrome b(558). Subsequently, constructs used for fluorescence recovery...

The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment leukemia and solid tumors. restriction its expression to hematopoietic cells many tumors raised questions regarding cellular functions. Sequence analysis encoding HMHA1 protein revealed presence possible C-terminal RhoGTPase Activating Protein (GAP) domain an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, RhoA are key...

The inflammatory response of endothelial cells triggered by cytokines such as TNFα and IL1β plays a pivotal role in innate immunity. Upon pro-inflammatory cytokine stimulation, produce chemokines that attract activate leukocytes, express high levels leukocyte adhesion molecules. This process is mediated intracellular signaling cascades activation e.g. the receptor (TNFR) lead to NFκB transcription factor MAP kinases, which turn gene transcription. We found small GTPase RhoB was strongly...