Subconfluent Endothelial Cells Research Articles

Endothelial cell-derived nidogen-1 inhibits migration of SK-BR-3 breast cancer cells

BackgroundThe tumour microenvironment is a critical regulator of malignant cancer progression. While endothelial cells have been widely studied in the context of tumour angiogenesis, their role as modulators of cancer cell invasion and migration is poorly understood.MethodsWe have investigated the influence of endothelial cells on the invasive and migratory behaviour of human cancer cells in vitro.ResultsUpon exposure to culture supernatants of endothelial cells, distinct cancer cells, such as SK-BR-3 cells, showed significantly increased invasion and cell migration concomitant with changes in cell morphology and gene expression reminiscent of an epithelial-mesenchymal transition (EMT). Interestingly, the pro-migratory effect on SK-BR-3 cells was significantly enhanced by supernatants obtained from subconfluent, proliferative endothelial cells rather than from confluent, quiescent endothelial cells. Systematically comparing the supernatants of subconfluent and confluent endothelial cells by quantitative MS proteomics revealed eight candidate proteins that were secreted at significantly higher levels by confluent endothelial cells representing potential inhibitors of cancer cell migration. Among these proteins, nidogen-1 was exclusively expressed in confluent endothelial cells and was found to be necessary and sufficient for the inhibition of SK-BR-3 cell migration. Indeed, SK-BR-3 cells exposed to nidogen-1-depleted endothelial supernatants showed increased promigratory STAT3 phosphorylation along with increased cell migration. This reflects the situation of enhanced SK-BR-3 migration upon stimulation with conditioned medium from subconfluent endothelial cells with inherent absence of nidogen-1 expression.ConclusionThe identification of nidogen-1 as an endothelial-derived inhibitor of migration of distinct cancer cell types reveals a novel mechanism of endothelial control over cancer progression.

NIDOGEN1 as a novel regulator of endothelial control over breast cancer invasion and metastasis.

e23005 Background: The microenvironment is a central regulator of cancer biology. While the contribution of fibroblasts has been largely studied, the role of endothelial cells as regulators of cancer cell behavior is still poorly understood. As in a diverse spectrum of physiological processes in normal tissue, endothelial cells may exert a similar regulatory control in cancer progression and metastasis. Methods: To characterize the functional effects of endothelial-cancer interaction we focused on an in vitro co-culture model. Results: Co-culturing human umbilical venous endothelial cells (HUVEC) with SKBR-3 breast cancer cells induced morphological changes with epithelial-mesenchymal transition traits (EMT) and a significantly increased migratory and invasive potential. This activity leading to an elongated phenotype, expression of mesenchymal markers and pro-migratory gene sets in SKBR-3 was contained in HUVEC conditioned medium. The pro-migratory effect on SKBR-3 was significantly more pronounced when the supernatant was obtained from a sub-confluent and highly proliferative endothelial cell culture than from a confluent and resting endothelial cell layer. To identify the secreted regulatory molecules, we analyzed the supernatant of sub-confluent and confluent endothelial cells by quantitative MS proteomics (SILAC analysis). Eight candidate proteins significantly more secreted in conditioned medium from confluent HUVEC represented potential inhibitors of migration. Among them NIDOGEN1 was found to be necessary and sufficient for the inhibition of EMT and migration in SKBR-3. Stimulation of SKBR-3 with supernatant from sub-confluent HUVEC increased p-STAT3 levels in SKBR-3. Silencing nidogen1 in confluent HUVEC re-activated phosphorylation of STAT3 indicating that NIDOGEN1 inhibits the promigratory STAT3 pathway. The STAT3 pathway and migration were also inhibited by overexpression of nidogen1 in MDA-MB-231 LM2 cells.When injected in the mammary fat pad of nude mice these cells formed significantly less lung metastases than controls (p < 0.01). Conclusions: We identified NIDOGEN1 as a novel regulator of endothelial control over cancer cell migration, invasion and metastasis.

TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells.

The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro angiogenesis. To deplete TIMAP, the EC were transfected with TIMAP-specific or nonspecific small interfering (si) RNA. The rate of electrical impedance development across subconfluent EC monolayers, a measure of the time-dependent increase in EC number, was 93 ± 2% lower in TIMAP-depleted than in control EC. This effect on cell proliferation was associated with reduced DNA synthesis and increased apoptosis: TIMAP silencing reduced 5-ethynyl-2'-deoxyuridine incorporation by 38 ± 2% during the exponential phase of EC proliferation, and cleaved caspase 3 as well as caspase 3 activity increased in TIMAP-depleted relative to control cells. Furthermore, TIMAP depletion inhibited the formation of angiogenic sprouts by glomerular EC in three-dimensional culture. TIMAP depletion strongly diminished growth factor-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation, suggesting a specific effect on the PI3K/Akt/PTEN pathway. Endogenous TIMAP and PTEN colocalized in EC and coimmunoprecipitated from EC lysates. The inhibitory PTEN phosphorylation on S370 was significantly reduced in TIMAP-depleted compared with control EC, while phosphorylation of PTEN on the S380/T382/T383 cluster remained unchanged. Finally, the PTEN inhibitor bpV(phen) fully reversed the suppressive effect of TIMAP depletion on Akt phosphorylation. The data indicate that in growing EC, TIMAP is necessary for Akt-dependent EC proliferation, survival, and angiogenic sprout formation and that this effect of TIMAP is mediated by inhibition of the tumor suppressor PTEN.

Roles of cell confluency and fluid shear in 3-dimensional intracellular forces in endothelial cells

We use a novel 3D inter-/intracellular force microscopy technique based on 3D traction force microscopy to measure the cell-cell junctional and intracellular tensions in subconfluent and confluent vascular endothelial cell (EC) monolayers under static and shear flow conditions. We found that z-direction cell-cell junctional tensions are higher in confluent EC monolayers than those in subconfluent ECs, which cannot be revealed in the previous 2D methods. Under static conditions, subconfluent cells are under spatially non-uniform tensions, whereas cells in confluent monolayers are under uniform tensions. The shear modulations of EC cytoskeletal remodeling, extracellular matrix (ECM) adhesions, and cell-cell junctions lead to significant changes in intracellular tensions. When a confluent monolayer is subjected to flow shear stresses with a high forward component comparable to that seen in the straight part of the arterial system, the intracellular and junction tensions preferentially increase along the flow direction over time, which may be related to the relocation of adherens junction proteins. The increases in intracellular tensions are shown to be a result of chemo-mechanical responses of the ECs under flow shear rather than a direct result of mechanical loading. In contrast, the intracellular tensions do not show a preferential orientation under oscillatory flow with a very low mean shear. These differences in the directionality and magnitude of intracellular tensions may modulate translation and transcription of ECs under different flow patterns, thus affecting their susceptibility for atherogenesis.

Glucose‐activated RUNX2 phosphorylation promotes endothelial cell proliferation and an angiogenic phenotype

The runt-related protein-2 (RUNX2) is a DNA-binding transcription factor that regulates bone formation, tumor cell metastasis, endothelial cell (EC) proliferation, and angiogenesis. RUNX2 DNA binding is glucose and cell cycle regulated. We propose that glucose may activate RUNX2 through changes in post-translational phosphorylation that are cell cycle-specific and will regulate EC function. Glucose increased cell cycle progression in EC through both G2/M and G1 phases with entry into S-phase occurring only in subconfluent cells. In the absence of nutrients and growth factors (starvation), subconfluent EC were delayed in G1 when RUNX2 expression was reduced. RUNX2 phosphorylation, activation of DNA binding, and pRb phosphorylation were stimulated by glucose and were necessary to promote cell cycle progression. Glucose increased RUNX2 localization at focal subnuclear sites, which co-incided with RUNX2 occupancy of the cyclin-dependent kinase (cdk) inhibitor p21(Cip1) promoter, a gene normally repressed by RUNX2. Mutation of the RUNX2 cdk phosphorylation site in the C-terminal domain (S451A.RUNX2) reduced RUNX2 phosphorylation and DNA binding. Expression of this cdk site mutant in EC inhibited glucose-stimulated differentiation (in vitro tube formation), monolayer wound healing, and proliferation. These results define a novel relationship between glucose-activated RUNX2 phosphorylation, cell cycle progression, and EC differentiation. These data suggest that inhibition of RUNX2 expression or DNA binding may be a useful strategy to inhibit EC proliferation in tumor angiogenesis.

Abstract 9121: Intra-endoplasmic Reticulum Ca2+ Flow Recharges Preferentially to Subcompartments of ER at Caveolin-rich Cell Edges in Endothelial Cells

Subcellular distribution of caveolin-1, a marker protein of caveolae, is polarized especially in migrating or subconfluent endothelial cells ( Fig. 1 ). In response to ATP, IP 3 production and Ca 2+ release originated at caveolin-1-rich cell edges (CRCE) and then propagated as spatiotemporally organized Ca 2+ waves ( Fig. 2 ). Here in this study, we investigated subcellular Ca 2+ dynamics in the endoplasmic reticulum (ER) Ca 2+ stores by using confocal microscopy and an ER-targeted version of cameleon (D1ER), a GFP-based Ca 2+ -sensing protein. As expected, FRET imaging using an ER-targeted Ca 2+ sensor revealed that the ATP-mediated decrease in local intra-ER Ca 2+ concentration ([Ca 2+ ] er ) at CRCE was more rapid and larger than in other subdomains ( Figs. 2, 3 ). Interestingly, the decreased [Ca 2+ ] er at such CRCE more rapidly returned to the baseline in the presence of normal extracellular Ca 2+ . Even in the absence of extracellular Ca 2+ , when maximal ATP stimulation continued to decrease global [Ca 2+ ] er , [Ca 2+ ] er at CRCE exhibited the opposite incremental recharging. Removal of extracellular Ca 2+ or thapsigargin treatment did not affect such local recharging of Ca 2+ , suggesting no major roles of store-operated Ca 2+ entry (SOCE) or reuptake of released Ca 2+ into the ER by a Ca 2+ -ATPase. Thus, we conclude that the local ER Ca 2+ store comprises functional subcompartments at CRCE and that an ATP-induced [Ca 2+ ] er decrease at the caveolin-1-rich region is preferentially recharged by intra-ER Ca 2+ flow from the other ER subcompartments ( Fig. 4 ).

Primary Monocytes Regulate Endothelial Cell Survival Through Secretion of Angiopoietin-1 and Activation of Endothelial Tie2

Monocyte recruitment and interaction with the endothelium is imperative to vascular recovery. Tie2 plays a key role in endothelial health and vascular remodeling. We studied monocyte-mediated Tie2/angiopoietin signaling following interaction of primary monocytes with endothelial cells and its role in endothelial cell survival. The direct interaction of primary monocytes with subconfluent endothelial cells resulted in transient secretion of angiopoietin-1 from monocytes and the activation of endothelial Tie2. This effect was abolished by preactivation of monocytes with tumor necrosis factor-α. Although primary monocytes contained high levels of both angiopoietin 1 and 2, endothelial cells contained primarily angiopoietin 2. Seeding of monocytes on serum-starved endothelial cells reduced caspase-3 activity by 46 ± 5.1%, and 52 ± 5.8% after tumor necrosis factor-α treatment and decreased detected single-stranded DNA levels by 41 ± 4.2% and 40 ± 3.5%, respectively. This protective effect of monocytes on endothelial cells was reversed by Tie2 silencing with specific short interfering RNA. The antiapoptotic effect of monocytes was further supported by the activation of cell survival signaling pathways involving phosphatidylinositol 3-kinase, STAT3, and AKT. Monocytes and endothelial cells form a unique Tie2/angiopoietin-1 signaling system that affects endothelial cell survival and may play critical a role in vascular remodeling and homeostasis.

A New Role for the Muscle Repair Protein Dysferlin in Endothelial Cell Adhesion and Angiogenesis

Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC. EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing. Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.

Monocyte activation state regulates monocyte-induced endothelial proliferation through Met signaling

AbstractDirect interaction of unactivated primary monocytes with endothelial cells induces a mitogenic effect in subconfluent, injured endothelial monolayers through activation of endothelial Met. We now report that monocytes' contact-dependent mitogenicity is controlled by activation-mediated regulation of hepatocyte growth factor. Direct interaction of unactivated monocytes with subconfluent endothelial cells for 12 hours resulted in 9- and 120-fold increase in monocyte tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) mRNA levels and bitemporal spike in hepatocyte growth factor that closely correlates with endothelial Met and extracellular signal-related kinase (ERK) phosphorylation. Once activated, monocytes cannot induce a second wave of endothelial cell proliferation and endothelial Met phosphorylation and soluble hepatocyte growth factor levels fall off. Monocyte-induced proliferation is dose dependent and limited to the induction of a single cell cycle. Monocytes retain their ability to activate other endothelial cells for up to 8 hours after initial interaction, after which they are committed to the specific cell. There is therefore a profoundly sophisticated mode of vascular repair. Confluent endothelial cells ensure vascular quiescence, whereas subconfluence promotes vessel activation. Simultaneously, circulating monocytes stimulate endothelial cell proliferation, but lose this potential once activated. Such a system provides for the fine balance that can restore vascular and endothelial homeostasis with minimal overcompensation.

The Motor Protein Myosin-X Transports VE-Cadherin along Filopodia To Allow the Formation of Early Endothelial Cell-Cell Contacts

Vascular endothelium (VE), the monolayer of endothelial cells that lines the vascular tree, undergoes damage at the basis of some vascular diseases. Its integrity is maintained by VE-cadherin, an adhesive receptor localized at cell-cell junctions. Here, we show that VE-cadherin is also located at the tip and along filopodia in sparse or subconfluent endothelial cells. We observed that VE-cadherin navigates along intrafilopodial actin filaments. We found that the actin motor protein myosin-X is colocalized and moves synchronously with filopodial VE-cadherin. Immunoprecipitation and pulldown assays confirmed that myosin-X is directly associated with the VE-cadherin complex. Furthermore, expression of a dominant-negative mutant of myosin-X revealed that myosin-X is required for VE-cadherin export to cell edges and filopodia. These features indicate that myosin-X establishes a link between the actin cytoskeleton and VE-cadherin, thereby allowing VE-cadherin transportation along intrafilopodial actin cables. In conclusion, we propose that VE-cadherin trafficking along filopodia using myosin-X motor protein is a prerequisite for cell-cell junction formation. This mechanism may have functional consequences for endothelium repair in pathological settings.

A biohybrid artificial lung prototype with active mixing of endothelialized microporous hollow fibers

Acute respiratory distress syndrome (ARDS) affects nearly 150,000 patients per year in the US, and is associated with high mortality ( approximately 40%) and suboptimal options for patient care. Mechanical ventilation and extracorporeal membrane oxygenation are limited to short-term use due to ventilator-induced lung injury and poor biocompatibility, respectively. In this report, we describe the development of a biohybrid lung prototype, employing a rotating endothelialized microporous hollow fiber (MHF) bundle to improve blood biocompatibility while MHF mixing could contribute to gas transfer efficiency. MHFs were surface modified with radio frequency glow discharge (RFGD) and protein adsorption to promote endothelial cell (EC) attachment and growth. The MHF bundles were placed in the biohybrid lung prototype and rotated up to 1,500 revolutions per minute (rpm) using speed ramping protocols to condition ECs to remain adherent on the fibers. Oxygen transfer, thrombotic deposition, and EC p-selectin expression were evaluated as indicators of biohybrid lung functionality and biocompatibility. A fixed aliquot of blood in contact with MHF bundles rotated at either 250 or 750 rpm reached saturating pO(2) levels more quickly with increased rpm, supporting the concept that fiber rotation would positively contribute to oxygen transfer. The presence of ECs had no effect on the rate of oxygen transfer at lower fiber rpm, but did provide some resistance with increased rpm when the overall rate of mass transfer was higher due to active mixing. RFGD followed by fibronectin adsorption on MHFs facilitated near confluent EC coverage with minimal p-selectin expression under both normoxic and hyperoxic conditions. Indeed, even subconfluent EC coverage on MHFs significantly reduced thrombotic deposition adding further support that endothelialization enhances, blood biocompatibility. Overall these findings demonstrate a proof-of-concept that a rotating endothelialized MHF bundle enhances gas transfer and biocompatibility, potentially producing safer, more efficient artificial lungs.

Bortezomib induces autophagic death in proliferating human endothelial cells

The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

Amyloid beta toxicity dependent upon endothelial cell state

Amyloid beta (Abeta), a peptide family produced and deposited in neurons and endothelial cells (EC), is found at subnanomolar concentrations in the plasma of healthy individuals. Simple conformational changes produce a form of Abeta, Abeta42, which creates toxic plaque in the brains of Alzheimer's patients. Oxidative stress induced blood brain barrier degeneration has been proposed as a key factor for Abeta42 toxicity, but cannot account for lack of injury from the same peptide in healthy tissues. We hypothesized that cell state mediates Abeta effect. Thus, we examined the viability of aortic EC, vascular smooth muscle cells (SMC) and epithelial cells (EPI) in different states in the presence of Abeta secreted from transfected Chinese hamster ovary cells (CHO). Abeta was more toxic to all cell types when they were subconfluent. Subconfluent EC sprouted and SMC and EPI were inhibited by Abeta. Confluent EC were virtually resistant to Abeta and suppressed Abeta production by Abeta+CHO. Products of subconfluent EC overcame this resistant state, stimulating the production and toxicity of Abeta42. Confluent EC overgrew approximately 35% beyond their quiescent state in the presence of Abeta conditioned in media from subconfluent EC. These findings imply that Abeta42 may well be even more cytotoxic to cells in injured or growth states and potentially explain the variable and potent effects of this protein. One may now need to consider tissue and cell state in addition to local concentration of and exposure duration to Abeta. The specific interactions of Abeta and EC in a state-dependent fashion may help understand further the common and divergent forms of vascular and cerebral toxicity of Abeta and the spectrum of AD.

Short-Term Shear Stress Induces Rapid Actin Dynamics in Living Endothelial Cells.

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm(2)). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm(2) followed by 15 dyn/cm(2) 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.

Altered ADAMTS-13 Expression in Human Umbilical Vein Endothelial Cells.

von Willebrand factor (vWF), as secreted by endothelial cells (ECs), is a highly thrombogenic, ultra-large multimeric protein which promotes platelet adhesion and clot formation. This ultra-large vWF would lead to platelet aggregation even without vascular injury if not for the activity of a plasma vWF-cleaving protease, ADAMTS-13, which cleaves the ultra-large vWF multimers upon secretion into the more moderately-sized, less thrombogenic multimers commonly found circulating in normal plasma. The production of ADAMTS-13 was first described in hepatic stellate cells, but has since been found to be produced in a variety of tissues and cell types including ECs. Production of ADAMTS-13 by ECs is of interest as the newly secreted ultra-large vWF is immediately cleaved on the surface of ECs by ADAMTS-13. Quiescent ECs normally exist as a confluent non-thrombogenic monolayer lining the lumen of the vasculature. Damage to the EC monolayer results in cell activation, stimulating not only thrombosis but proliferation as the ECs attempt to prevent blood loss and repair vascular wall damage. To assess the production of ADAMTS-13 by human umbilical vein ECs, as measured by a commercially-available ELISA (American Diagnostica Inc., Stamford, CT), cellular supernatants were collected and cellular proteins extracted from either confluent ECs (EC monolayers), damaged confluent ECs (EC monolayers which had narrow strips of cells removed with a sterile pipette tip), or subconfluent ECs (50% confluence). After four hours, supernatants from subconfluent ECs contained 2.5X as much ADAMTS-13 (161.5±29.1 pg/ml/μg cellular protein; p<0.002) as compared with either confluent or damaged confluent ECs (60.2±22.5 and 56.0±19.2 pg/ml/μg cellular protein, respectively). Similarly, cell lysates from subconfluent ECs contained ∼2.5X as much ADAMTS-13 (645.1±73.1 pg/ml/μg cellular protein; p<0.002) as compared with either confluent or damaged confluent ECs (257.8±32.8 and 311.3±29.3 pg/ml/μg cellular protein, respectively). In time-course experiments, confluent, damaged confluent, and subconfluent ECs were fixed 4, 24, 48, 72, or 96 hours following injury (producing the damaged confluent ECs). ADAMTS-13 expression was subsequently detected by immunohistochemistry using a rabbit polyclonal anti-human ADAMTS-13 antibody directed against the metalloproteinase domain (Abcam Inc., Cambridge, MA) and a fluorescent secondary antibody. Subconfluent ECs expressed more intense staining for ADAMTS-13 throughout the time course when compared with the confluent ECs. By 72 hours, the damaged confluent ECs began expressing comparable intense staining, particularly in regions where the ECs were reestablishing the monolayer. Previous work has suggested that the hepatic stellate cells, as well as the other cell types which express ADAMTS-13, produce this protease constitutively and that this production is not otherwise regulated. The data presented herein demonstrate that ADAMTS-13 production, at least in ECs, is affected by the growth state of the ECs. This enhanced production of ADAMTS-13 may limit the extent of platelet thrombosis at sites of vascular injury.

Effects of D- and L-Glucose and Mannitol on Retinal Capillary Cells: Inhibition by Nanomolar Aminoguanidine

Hyperglycemia may contribute directly to pericyte loss and capillary leakage in early diabetic retinopathy. To elucidate relative contributions of glycation, glycoxidation, sugar autoxidation, osmotic stress and metabolic effects in glucose-mediated capillary damage, we tested the effects of D-glucose, L-glucose, mannitol and the potentially protective effects of aminoguanidine on cultured bovine retinal capillary pericytes and endothelial cells. Media (containing 5 mM D-glucose) were supplemented to increase the concentration of each sugar by 5, 10, or 20 mM. Subconfluent pericytes and endothelial cells were exposed to the supplemented media in the presence or absence of aminoguanidine (1 nM-100 µM) for three days. Cell counts, viability and protein were determined. For both cell types, all three sugars produced concentration-dependent decreases in cell counts and protein content (p

Orientation of apical and basal actin stress fibers in isolated and subconfluent endothelial cells as an early response to cyclic stretching.

We investigated the response of apical and basal actin stress fibers (SFs) and its dependency on cell confluency for endothelial cells subjected to cyclic stretching. Porcine aortic endothelial cells from the 2nd and 5th passages were transferred to a fibronectin-coated silicone chamber with 5000-8000 cells/cm2 (isolated condition), positioning the cells apart, or with 25,000-27,000 cells/cm2 (subconfluent condition), allowing cell-to-cell contact. The substrate was stretched cyclically by 0.5 Hz for 2 h with a peak strain on the substrate that was 15% in the stretch direction and -4% in the transverse direction. The actin filaments (AFs) were stained with rhodamine phalloidin and their orientations were examined under a confocal laser scanning microscope. In the basal region, SFs formed in all of the cells under both the isolated and subconfluent conditions. We observed an average of 5 and 9 SFs per cell under the isolated and subconfluent conditions, respectively, in the fluorescent images of the apical region. We also observed cells that were bush-like without apical AFs or apical SFs. On average, the SFs in the subconfluent cells oriented in the direction of minimal strain, while the SFs in the isolated cells oriented in the direction of a 2% compressive strain. These results suggest that such differential response may be due to differences in the transmission of mechanical stretching to the central and apical regions of the cell through the SFs. We also speculate that cell-to-cell contact might change the strength, orientation, and anchorage of apical AFs and play a critical role in mechanical signal transduction.

Early Response of Endothelial Cells to Flow Is Mediated by VE-Cadherin

Endothelial cells are known to respond to flow onset by increasing actin turnover rate. Current models assume that an increase in the actin turnover rate should result in a rise in cell crawling speed. Here we report that confluent endothelial monolayer shows an unexpected behavior: cell crawling speed decreases by approximately 40% within the first 30 min of flow onset. A drop in crawling speed has not been observed in either subconfluent endothelial cells or in VE-cadherin-deficient cells. We found that flow onset caused an increase in the number of VE-cadherin-GFP molecules in the junctions and elicited changes in the cytoskeleton-associated fractions of alpha, beta -catenins and VE-cadherin. Flow application also increased the strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin. These observations suggest that endothelial cell junctional proteins respond to flow transiently by increasing the strength of intercellular attachments early after flow onset and support the view on the active role of intercellular adhesions in mechanotransduction.

Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling

A growing body of evidence implicates Wnt signaling in the control of angiogenesis. To better understand the role of the Wnt/beta-catenin pathway in endothelial cells (EC), we examined endogenous signaling activity and signaling component expression in vascular cells. We observed stabilization of cytosolic beta-catenin and activation of a T-cell factor (TCF) -luciferase promoter, hallmarks of canonical Wnt signaling activity, in cultured EC. This activity was increased in subconfluent EC, which are known to display characteristics of angiogenic EC, compared with confluent EC, which have a more differentiated phenotype. Endogenous TCF activity was inhibited by transfection with a secreted inhibitor of canonical Wnt signaling. A systematic analysis of Wnt, Fzd, SFRP, and Dkk gene expression in human EC (cultured and freshly isolated), smooth muscle cells (cultured), and aorta demonstrated that numerous Wnt signaling components are expressed by vascular cells. We conclude that Wnt signaling components are expressed and active in cultured EC.