Intact Endothelial Cells Research Articles

Stiffness of Intact Endothelial Cells From Fresh Aortic Bifurcations of Atherosclerotic Rabbits-Atomic Force Microscopic Study.

Stiffness of intact endothelial cells (ECs) in the abdominal aorta (AA) and in the medial and lateral wall of the common iliac artery (CIA(Medial) and CIA(Lateral), respectively), which were freshly obtained from cholesterol-fed rabbits, were measured with an atomic force microscopic indentation method. In the areas away from atherosclerotic plaques (Off-plaque), ECs were significantly stiffer in CIA(Medial) than in the other two locations; this result was similar to that from normal diet-fed animals. On the other hand, there were no significant differences in the stiffness of ECs located on atherosclerotic plaques (On-plaque) among the three sites; the stiffness was equal to those in "Off-plaque" wall of CIA(Lateral) and AA. Moreover, the stiffness of ECs covering plaques decreased with the progression of atherosclerosis. The precise quantification of the stiffness of vascular ECs would provide a better understanding of cellular remodeling and adaptation in atherosclerosis. J. Cell. Physiol. 232: 7-13, 2017. © 2016 Wiley Periodicals, Inc.

Intracellular sources of ornithine for polyamine synthesis in endothelial cells.

Polyamines are essential for proliferation of endothelial cells (EC)and angiogenesis. This study was conducted to identify the metabolic source(s) of ornithine for polyamine synthesis in EC, using N(ω)-hydroxy-nor-L-arginine (Nor-NOHA, an inhibitor of arginase) and gabaculine (an inhibitor of ornithine aminotransferase; OAT). Nor-NOHA inhibited arginase with an IC50 value of 10µM for intact EC. Nor-NOHA (0.5mM) alone inhibited arginase activity in EC by 98%, increased total cellular concentrations of arginine by 14%, and decreased total cellular concentrations of ornithine, putrescine and spermidine by 17, 65 and 74%, respectively. Arginine and glutamine contributed to 73 and 26% of the ornithine produced by EC, respectively. Gabaculine (1mM) alone decreased the total cellular concentrations of arginine, ornithine, putrescine, and spermidine by 14, 96, 32, and 42%, respectively. A combination of both Nor-NOHA and gabaculine completely blocked ornithine production in EC, resulting in no detectable cellular ornithine and almost complete depletion of cellular putrescine and spermidine. Addition of 0.5mM ornithine restored the intracellular concentrations of polyamines in EC treated with Nor-NOHA plus gabaculine, indicating that Nor-NOHA and gabaculine did not inhibit ornithine decarboxylase activity. Our results suggest that the arginase and OAT pathways are the exclusive sources of ornithine in EC when there is little extracellular ornithine and that there is intracellular compartmentalization of arginine and ornithine for endothelial synthesis of polyamines. These novel findings may have important implications for improving placental vascular growth, wound healing, and cancer therapy.

Peptide-stimulated angiogenesis: Role of lung endothelial caveolar signaling and nitric oxide

Endothelial nitric oxide (NO) synthase (eNOS)-derived NO plays a critical role in the modulation of angiogenesis in the pulmonary vasculature. We recently reported that an eleven amino acid (SSWRRKRKESS) cell penetrating synthetic peptide (P1) activates caveolar signaling, caveloae/eNOS dissociation, and enhance NO production in lung endothelial cells (EC). This study examines whether P1 promote angiogenesis via modulation of caveolar signaling and the level of NO generation in EC and pulmonary artery (PA) segments. P1-enhanced tube formation and cell sprouting were abolished by caveolae disruptor Filipin (FIL) in EC and PA, respectively. P1 enhanced eNOS activity and angiogenesis were attenuated by inhibition of eNOS as well as PLCγ-1, PKC-α but not PI3K-mediated caveolar signaling in intact EC and/or PA. P1 failed to enhance the catalytic activity of eNOS and angiogenesis in caveolae disrupted EC by FIL. Lower (0.01 mM) concentration of NOC-18 enhanced angiogenesis without inhibition of eNOS activity whereas higher concentration of NOC-18 (1.0 mM) inhibited eNOS activity and angiogenesis in EC. Inhibition of eNOS by l-NAME in the presence of P1 resulted in near total loss of tube formation in EC. Although P1 enhanced angiogenesis mimicked only by lower concentrations of NO generated by NOC-18, this response is independent of caveolar signaling/integrity. These results suggest that P1-enhanced angiogenesis is regulated by dynamic process involving caveolar signaling-mediated increased eNOS/NO activity or by the direct exposure to NOC-18 generating only physiologic range of NO independent of caveolae in lung EC and PA segments.

Engineering of a Biomimetic Pericyte-Covered 3D Microvascular Network.

Pericytes enveloping the endothelium play an important role in the physiology and pathology of microvessels, especially in vessel maturation and stabilization. However, our understanding of fundamental pericyte biology is limited by the lack of a robust in vitro model system that allows researchers to evaluate the interactions among multiple cell types in perfusable blood vessels. The present work describes a microfluidic platform that can be used to investigate interactions between pericytes and endothelial cells (ECs) during the sprouting, growth, and maturation steps of neovessel formation. A mixture of ECs and pericytes was attached to the side of a pre-patterned three dimensional fibrin matrix and allowed to sprout across the matrix. The effects of intact coverage and EC maturation by the pericytes on the perfused EC network were confirmed using a confocal microscope. Compared with EC monoculture conditions, EC-pericyte co-cultured vessels showed a significant reduction in diameter, increased numbers of junctions and branches and decreased permeability. In response to biochemical factors, ECs and pericytes in the platform showed the similar features with previous reports from in vivo experiments, thus reflect various pathophysiological conditions of in vivo microvessels. Taken together, these results support the physiological relevancy of our three-dimensional microfluidic culture system but also that the system can be used to screen drug effect on EC-pericyte biology.

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10–100μM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.

Human Endothelial Progenitor Cell Application in Vascular Diseases Seen in Metabolic Syndrome

Intact endothelial cells and their progenitors receive, integrate and response to physiological and pathological environment and play an important role in cardiovascular disease development and angiogenesis. When Furchgott et al. demonstrated that a healthy intact endothelium is necessary to continuation of vascular function, it truly revealed a different point of view [1]. The new era, vascular adaptation requires intact healthy endothelial cells to response physiological and pathological environment, displayed different therapeutic approaches in cardiovascular diseases. Generation of more information such as endothelial cells release a short-lived vasodilator agent which called nitric oxide via eNOS also was shook down the previous approaches in vascular disease management [1]. Furthermore, Asahara et al. were isolated endothelial progenitors from human peripheral blood in 1997 and they clearly demonstrated that those progenitors are actually differentiated into endothelial cells in adults [2]. It was completely started to predominant paradigm in previous treatment modalities regarding a new vessel formation probable via their progenitors in adults after embryonic development [2]. Because, until that time it was believed that endothelial differentiation could only occur during embryonic development [2]. Thus, endothelial progenitors are important as intact endothelial cells and the information “during different physiological and pathological stress a larger amount of endothelial progenitors released and mobilized into the peripheral blood furthermore display vasodilatation and generate a new vessel formation to re-establish blood flow” is impressive as intact endothelium necessary to vessel function knowledge.

Role of platelet CD40 ligand for endothelial cell-monocyte interaction in the presence of flow

CD40 ligand (CD154)-induced ultra-large von Willebrand factor (vWF) multimer-mediated endothelial cell-platelet-monocyte interaction may play an important role in adaptive and maladaptive vascular remodeling processes. Here we analyzed the impact of and conditions favouring the deposition of these multimers on the endothelial cell (EC) surface by way of CD40-CD154 co-stimulation in settings mimicking different forms of blood flow. Upon exposure to low oscillatory shear stress and sCD154, a release of vWF multimers comparable to histamine stimulation was monitored on the EC surface in a string-like fashion. Moreover, ex vivo perfused carotid arteries of wild type mice at low laminar shear stress rates showed a luminal release of vWF as ultra-large vWF multimers (ULVWF) upon stimulation with sCD154 which was absent in blood vessels of CD40 knockout mice. The observed CD40- and flow-dependent vWF release from intact endothelial cells and subsequent vWF multimer formation may facilitate adhesion and subsequent activation of circulating platelets at atherosclerotic predilection sites, which are characterized by disturbed flow patterns. This in turn may amplify endothelial cell-monocyte interaction, thus possibly initiating or promoting early atherosclerotic lesion formation.

Interacting mechanisms in the pathogenesis of cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is the major cause of late graft loss in heart transplant recipients. Histological studies of characteristic end-stage lesions reveal arterial changes consisting of a diffuse, confluent, and concentric intimal expansion containing graft-derived cells expressing smooth muscle markers, extracellular matrix, penetrating microvessels, and a host mononuclear cell infiltrate concentrated subjacent to an intact graft-derived luminal endothelial cell lining with little evidence of acute injury. This intimal expansion combined with inadequate compensatory outward remodeling produces severe generalized stenosis extending throughout the epicardial and intramyocardial arterial tree that causes ischemic graft failure. Cardiac allograft vasculopathy lesions affect ≥50% of transplant recipients and are both progressive and refractory to treatment, resulting in ≈5% graft loss per year through the first 10 years after transplant. Lesions typically stop at the suture line, implicating alloimmunity as the primary driver, but pathogenesis may be multifactorial. Here, we will discuss 6 potential contributors to lesion formation (1) conventional risk factors of atherosclerosis; (2) pre- or peritransplant injuries; (3) infection; (4) innate immunity; (5) T-cell-mediated immunity; and (6) B-cell-mediated immunity through production of donor-specific antibody. Finally, we will consider how these various mechanisms may interact with each other.

Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria.

We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested that glycocalyx disruption preceded cerebral manifestations. The contribution of this loss to pathogenesis should be studied further.

The effects of low-frequency ultrasound and microbubbles on rabbit hepatic tumors

High-intensity focused ultrasound in combination with microbubbles (MBs) is able to inhibit the growth of VX2 rabbit liver tumors in vivo and prolong the survival time of the animals. In this study, we attempt to investigate the feasibility of VX2 tumor growth inhibition using low-frequency ultrasound (US)-mediated MB disruption. Forty-eight New Zealand rabbits with hepatic VX2 tumors were divided into four groups: control, MBs group, low-frequency US group, and US + MB group. The parameters of the US were 20 kHz, 2 W/cm², 40% duty cycle, 5 min, and once every other day for 2 weeks. At the end of the therapy experiment, 24 rabbits were euthanized, and the cancers were collected and cut into five sections for histological examination, immunohistochemistry, laser confocal microscopy, western blotting assays, and transmission electron microscopy (TEM). Another 24 rabbits were saved, and overall survival time was recorded. The tumor volumes in control, MB, US, and US + MB groups were 6.36 ± 0.58, 5.68 ± 0.42, 5.29 ± 0.26, and 2.04 ± 0.14 cm³, respectively (US + MB versus the other three groups, P < 0.01). Tumor cells manifested coagulation necrosis with internal calcification. Hematoxylin and eosin (H–E) staining revealed interstitial hemorrhage and intravascular thrombosis. The intensity of cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF) in the US + MB group in the immunohistochemical staining, laser confocal microscopy, and western blotting assays was lower than that of the other three groups (P < 0.05). TEM of the US + MB group revealed vascular endothelial cell wall rupture, widened endothelial cell gaps, interstitial erythrocyte leakage, and microvascular thrombosis, while intact vascular endothelial cells and normal erythrocytes in the tumor vessels were observed in control, MB, and US groups. Rabbits treated with US + MB had a significantly longer overall survival than those in the other three groups (χ2 = 9.328, P = 0.0242). VX2 tumor growth could be inhibited by cavitation induced using low-frequency US and MB.

Intima/medulla reconstruction and vascular contraction–relaxation recovery for acellular small diameter vessels prepared by hyperosmotic electrolyte solution treatment

This study aims at the evaluation of blood vessel reconstruction process of decellularized small diameter vessels prepared by a hyperosmotic electrolyte solution treatment not only histologically but also physiologically in rat transplantation model. Complete cell removal by a hyperosmotic electrolyte solution treatment was confirmed by hematoxylin/eosin staining and scanning electron microscopic observation. All acellular vessels transplanted into the rat abdominal aorta were patent up to 14months. One week post-transplantation, the vWF-positive cells were observed on the luminal surface but the layer formation did not complete. Five weeks following transplantation, the vWF-positive endothelial cells were located on the intima consistent with intact endothelial cells. Beneath the endothelial cells, α-SMA-positive smooth muscle cells were distributed. The harvested vessels displayed formation of tunica intima (endothelial cells) and tunica medulla (smooth muscle cell) layers. We also examined the physiological properties of the vessels 12months post-transplantation using a wire myograph system. The transplanted vessels contracted upon addition of norepinephrine and relaxed upon addition of sodium nitroprusside as well as the native vessels. In conclusion, the acellular vessels prepared with hyperosmotic electrolytic solution showed excellent and long-term patency, which may be related to the successful preservation of vascular ECM. In addition, the acellular vessels revealed the intima/medulla regeneration with the physiological contraction-relaxation functions in response to the each substance.

N-isopropylacrylamide-co-glycidylmethacrylate as a thermoresponsive substrate for corneal endothelial cell sheet engineering.

Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

ABSTRACTCandida albicans invades endothelial cells by binding to N-cadherin and other cell surface receptors. This binding induces rearrangement of endothelial cell actin microfilaments, which results in the formation of pseudopods that surround the organism and pull it into the endothelial cell. Here, we investigated the role of endothelial cell septin 7 (SEPT7) in the endocytosis of C. albicans hyphae. Using confocal microscopy, we determined that SEPT7 accumulated with N-cadherin and actin microfilaments around C. albicans as it was endocytosed by endothelial cells. Affinity purification studies indicated that a complex containing N-cadherin and SEPT7 was recruited by C. albicans and that formation of this complex around C. albicans was mediated by the fungal Als3 and Ssa1 invasins. Knockdown of N-cadherin by small interfering RNA (siRNA) reduced recruitment of SEPT7 to C. albicans, suggesting that N-cadherin functions as a link between SEPT7 and the fungus. Also, depolymerization of actin microfilaments with cytochalasin D decreased the association between SEPT7 and N-cadherin and inhibited recruitment of both SEPT7 and N-cadherin to C. albicans, indicating the necessity of an intact cytoskeleton in the functional interaction between SEPT7 and N-cadherin. Importantly, knockdown of SEPT7 decreased accumulation of N-cadherin around C. albicans in intact endothelial cells and reduced binding of N-cadherin to this organism, as revealed by the affinity purification assay. Furthermore, SEPT7 knockdown significantly inhibited the endocytosis of C. albicans. Therefore, in response to C. albicans infection, SEPT7 forms a complex with endothelial cell N-cadherin, is required for normal accumulation of N-cadherin around C. albicans hyphae, and is necessary for maximal endocytosis of the organism.

Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles.

Transepithelial corneal collagen cross‐linking by iontophoresis of riboflavin

To evaluate the effectiveness of transepithelial cornea impregnation with riboflavin 0.1% by iontophoresis for collagen cross-linking. Transepithelial collagen cross-linking by iontophoresis of riboflavin was performed in a series of 22 eyes of 19 patients with progressive keratoconus I-II of Amsler classification. The riboflavin solution was administered by iontophoresis for 10 min in total, after which standard surface UVA irradiation (370 nm, 3 mW/cm(2) ) was performed at a 5-cm distance for 30 min. The riboflavin/UVA treatment resulted in a decrease in the average keratometry level from 46.47 ± 1.03 to 44.12 ± 1.12 D 1 year after the procedure. Corneal astigmatism decreased from 3.44 ± 0.48 to 2.95 ± 0.23 D. Uncorrected distance visual acuity improved from 0.61 ± 0.44 up to 0.48 ± 0.41 (LogMAR). Preoperative and postoperative endothelial cell density remained unchanged at 2765 ± 21.15 cells/mm(2) . Transepithelial collagen cross-linking by iontophoresis might become an effective method for riboflavin impregnation of the corneal stroma reducing the duration of the procedure and being more comfortable for the patients. Further long-term studies are necessary to complete the evaluation of the efficacy and risk spectrum of the modified cross-linking technique.

Histological and ultrastructural findings of corneal tissue after failed descemet membrane endothelial keratoplasty

To investigate in a retrospective review the histological and ultrastructural findings after failed primary and early Descemet membrane endothelial keratoplasty (DMEK), propose possible pathomechanisms of graft failure and give clinical implications. The explanted grafts underwent light- and electromicroscopical investigations in eight failed DMEK cases. Haematoxylin - Eosin, periodic acid Schiff and Alcian blue stainings were performed. Special note was given to any residual stromal remnants, absence of endothelial cells, lamellar structure and 'activation' of keratocytes. Of the eight cases, six were re-DMEKs and two penetrating keratoplasties. Partial graft separation was seen in six and no graft separation in two of the cases. The average time-interval to the re-DMEK or penetrating keratoplasty was 4.6 months. Light and electron microscopy of the two explanted stromal specimens showed varying degrees of keratocyte activation. Endothelial cell loss was observed in essentially all explants with varying degrees and positive correlation with intraoperative difficulty. Assumed upside-down situations showed large areas of intact endothelial cells. In addition, a new layer, situated between the endothelial cell layer and the posterior nonbanded layer, was observed with loose intercellular structure. A loss of the endothelial cell layer of varying degrees and positive correlation with intraoperative difficulty are the prominent feature of primary and early DMEK graft failure. Of note is the upside-down situation, in which in some cases, the endothelial cell layer not only remains intact but also demonstrates metabolical activity in forming a novel cellular layer.

Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways

Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs), the first stage in leukocyte trafficking, plays a pivotal role in inflammation and injury. Acute mechanical stretch has been closely associated with vascular inflammation, although the precise mechanism is unknown. Here, we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial cells (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways. Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs, promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane. We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo. Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCγ1/calcium pathway. Interestingly, stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway. Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments, as well as in acute hypertensive mouse models. These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis, which is modulated by VEGFR2 signaling. Thus, VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.

Depolarization of collecting lymphatic endothelium with acetylcholine or TRPV4 activation

Smooth muscle cells of collecting lymphatic vessels undergo coordinated contractions to propel lymph centrally. Whether endothelial‐derived hyperpolarization [via activation of small‐ and intermediate‐conductance Ca2+ activated K+ channels (SKCa/IKCa)] shown in blood vessels is manifest in governing lymphatic function has not been established. To test this possibility, lymphangions dissected from mouse popliteal fossae were treated to yield intact lymphatic endothelial cell tubes (LECTs; width: ~50μm; length: ~500 μm) for intracellular recording (bath pH 7.4; 30°C). Resting membrane potential (Vm) of LECTs was −60±5 mV and depolarized (P&lt;0.05) to −49±5 mV with BaCl2 (100 μM; n=6). Activation (NS309, 1 μM) or blockade [apamin (300 nM) + charybdotoxin (100 nM)] of SKCa/IKCa did not alter Vm (n=4). Acetylcholine (ACh, 1 μM) or GSK (TRPV4 channel agonist; 100 nM) depolarized LECTs by 29±7 and 49±4 mV, respectively (P&lt;0.05; n=4 each). Thus blockade of inward‐rectifying K+ channels and agents promoting Ca2+influx (via TRPV4) as well as release from the endoplasmic reticulum (ACh) evoke LECT depolarization that is facilitated by lack of functional SKCa/IKCa expression. With myoendothelial coupling, endothelium‐derived depolarization may coordinate contraction of lymphatic smooth muscle cells and thereby determine the effectiveness of lymph transport. (NIH R37HL041026, R01HL086483, F32HL110701, R01HL089784)

Impaired Ca2+ signaling following acutely elevated glucose in mouse endothelial cell tubes

Calcium signaling is integral to endothelial function and may be altered by hyperglycemia, a hallmark of diabetes. We tested the hypothesis that acute exposure to elevated glucose impairs Ca2+ signaling of native endothelium from resistance arteries of skeletal muscle. Intact endothelial cell tubes were isolated from superior epigastric arteries of male C57BL/6 mice (3–4 mos). Monitoring Ca2+ levels during exposure to control (10 mM) or elevated (20 mM) glucose for 30 min had no effect on resting Ca2+ (Δ fura‐2 ratio: 0.13±0.03 vs. 0.14±0.01, respectively; n=3). After loading with fluo‐4 dye, Ca2+ responses were evoked with 1 μM acetylcholine (ACh) in physiological solutions with normal (10 mM) and elevated (20 or 30 mM) glucose (with mannitol as an osmotic control) and imaged using spinning‐disc confocal microscopy. Exposure to 20 mM glucose for 30 min diminished peak Ca2+ responses (F/F0) to ACh (from 3.14±0.27 to 2.11±0.12; n=3, P&lt;0.05) that were otherwise maintained in 10 mM glucose. These reductions in F/F0 with 20 mM glucose persisted for 1 hr following restoration of 10 mM glucose. Elevating glucose to 30 mM effectively abolished Ca2+ responses to ACh, which did not recover within 1 hr. Acute exposure to elevated glucose can impair endothelial Ca2+ signaling in the endothelium of resistance arteries and thereby contribute to endothelial dysfunction in diabetes. (NIH R37HL041026, R01HL086483, F32HL107050)

Spatial and temporal characteristics of Ca2+ signaling in endothelial cells of intact rat tail artery

Very few studies have described briefly the spatial and temporal characteristics of Ca2+ signaling using ATP, bradykinin, histamine and thrombin in cultured endothelium. However so far, the spatial and temporal characteristics in intact endothelium have not been elucidated.The aim of this work was to increase the understanding of characteristics of Ca2+ signaling in endothelial cells using real time confocal imaging of Fluo-4 loaded intact rat tail arteries. Of all the three agonist used i.e. histamine, bradykinin and carbachol; only CCh produced strong dose dependent stimulant action on the endothelial cells of intact rat tail artery. Local endothelial heterogeneity was observed in fresh intact cells in response to the three agonist used which was absent upon culture.In agreement with the previous studies, each of the three agonists produced complex Ca2+ signaling consisting of initial fast followed by sustained component with Ca2+ oscillations superimposing it. The data supports the following conclusions: (i) Ca2+ puffs were evoked in 18% and 14% of cells by low concentrations of CCh (0.1 μM and 1 μM), respectively. The average diameter and width of Ca2+ puffs initiation sites was 1.9 ± 0.4 μm, 1.6 ± 0.1 μm, respectively. Length varied between 2 and 6 μm (n = 25 vessels). (ii) ECs responding with Ca2+ waves and oscillations were dependent on concentration of CCh: 0.1 μM (20%), 1 μM (65%) and 10 μM (99%). Speed of wave propagation and frequency of Ca2+ oscillations varied among different cells even at the same agonist concentration. (iii) Ca2+ waves were regenerative; initiated by one or both ends of the cell and propagated across the cell at a speed of 14–44 μm/s (iv) With increasing concentrations of CCh (0.1–10 μM), the frequency of Ca2+ oscillations ranged from 0.02 to 0.3 Hz with optimal frequency being about 0.2 ± 0.02 Hz (n = 15 vessels). In conclusions, this data shows the morphology, heterogeneity and detailed characteristics of Ca2+ signal in intact endothelial cells.