Endothelial Ca2 Research Articles

Calcium/calmodulin‐dependent protein kinase II modulates inositol‐3‐phosphate receptors in vascular endothelium from mouse mesenteric arteries (1075.1)

Endothelial Ca2+ pulsar consists in a spontaneous Ca2+ release from IP3R localized within myoendothelial projections (MEP). We recently showed CaMKII activation by Ca2+ pulsars, thus modulating endothelial function through NO production. Moreover, CaMKII is a known regulator of intracellular Ca2+ homeostasis in several cell types. Our hypothesis was that Ca2+ pulsar‐activated CaMKII modulates endothelial intracellular Ca2+ dynamics. Our investigation showed that preincubation with KN‐93 (10 µM), CaMKII inhibitor, significantly altered endoplasmic reticulum (ER) Ca2+ content (≍ ‐60%). A similar KN‐93‐induced alteration of ER Ca2+ levels (≍ ‐55%) was observed while SERCA pumps were inhibited (Thapsigargin; 1 µM). However, inhibition of IP3R (2APB; 100 µM) precluded KN‐93 effect on ER Ca2+ content. Colocalization (<40nm) of CaMKII and IP3R was observed by PLA in situ hybridization. In addition, an isoform‐specific distribution within MEP (IP3R2>IP3R1>IP3R3) has been found by in situ immunofluorescence. Finally, KN‐93 induced recruitment of new Ca2+ pulsars sites (1.8‐Fold). These results suggest that CaMKII modulates intracellular Ca2+ dynamics through modulation of IP3R activity. Thereby, CaMKII is involved in a negative feedback loop as an endothelial calcium‐sensor switch regulating Ca2+ homeostasis.Grant Funding Source: Supported by FRQS, FICM, CFI, SQHA and HSFC

Ca2+ entry via transient receptor potential vanilloid channel 4 mediates ventilation‐induced lung vascular barrier failure (1176.3)

Study objective: High tidal volume ventilation causes lung vascular barrier failure, yet underlying mechanisms are unclear. Here, we tested whether endothelial Ca2+ influx via mechanosensitive TRPV4 may contribute to stretch‐induced pulmonary hyperpermeability in vivo and ex vivo. Next, we probed for the role of putative upstream (serum/glucocorticoid‐regulated kinase (SGK) 1) and downstream (Ca2+‐activated K+ channels) regulators of TRPV4 in this scenario.Methods: Mice were ventilated for 2 h with low (7 ml/kg) and high (20 ml/kg) tidal volumes (LVT/HVT) in vivo. Isolated‐perfused lungs were inflated with 5 or 15 cmH2O (LPIP/HPIP) and [Ca2+]i was quantified by real‐time imaging with or without pharmacological inhibition of SGK1, TRPV4 or Ca2+‐activated K+ channels.Results: TRPV4 deficiency or inhibition by HC‐067047 protected from HVT‐induced lung injury in vivo, and attenuated the endothelial Ca2+ response to HPIP ex vivo. Involvement of SGK1 and Ca2+‐activated K+ channels was evident as reduced lung hyperpermeability in HVT mice following inhibition by GSK650394 (SGK1), apamin or charybdotoxin.Conclusions: Activation of TRPV4 by HVT mediates endothelial Ca2+ influx and promotes lung injury and barrier failure via a signaling pathway that involves both SGK1 and Ca2+‐activated K+ channels, which may present new targets for the prevention or treatment of stretch‐induced vascular pathologies.Grant Funding Source: Supported by an operational grant of the Deutsche Forschungsgemeinschaft to W.M.K.

Heterogeneous endothelial Ca2+ signal modalities in wild type and TRPC4 knockout rats in pulmonary arterial hypertension (1176.4)

Pulmonary arterial hypertension (PAH) is a deadly disease characterized by high pulmonary arterial pressure, and there are currently no effective treatments. Recently, our lab found that rats genetically lacking the canonical transient receptor potential channel 4 (TRPC4), a Ca2+‐permeable channel, exhibit a survival benefit in a model of PAH. Here we tested the hypothesis that heterogeneous endothelial Ca2+ signaling modalities exist in wild type and TRPC4 knockout rats, and that these modalities are altered in PAH. We compared the cytosolic endothelial Ca2+ activity of wild type and knockout rats in both normal and PAH conditions using opened artery preparations and confocal microscopy. We found that the amplitude of basal endothelial Ca2+ activity was significantly greater for knockout (mean F/F0: 1.46) than for wild type (mean F/F0: 1.11) rats. Additionally, both genotypes exhibited considerable Ca2+ signal heterogeneity within opened artery segments, which was altered in response to PAH. Furthermore, we found that Ca2+ responses to Acetylcholine were significantly truncated in knockout (mean duration: 9.69 s) compared to wild type (mean duration: 26.7 s) rats. We conclude that heterogeneous Ca2+ signaling modalities exist in the endothelium of wild type and TRPC4 knockout rats, and that these modalities are altered differentially in PAH. This phenomenon may be a key determinant of PAH mortality.Grant Funding Source: HL66299, HL60024

New insights on endothelial CaMKII in angiotensin II‐induced hypertension (851.6)

Angiotensin II‐induced arterial hypertension (AngII‐AH) is associated with an endothelial dysfunction characterized by a Ca2+ dyshomeostasis. Alteration of the delicate balance between reactive oxygen species (ROS) and nitric oxide (NO) then arise. Recent evidences suggest that hypertension stimulates Ca2+ pulsars and CaMKII but the contribution of AngII and the concomitant ROS remains elusive. The aim of this study was to establish a relationship between endothelial Ca2+ pulsars, ROS and CaMKII in AngII‐AH. Acute exposure to high (> 10‐8 M) concentrations of AngII stimulated Ca2+ pulsars and ROS production in high‐speed confocal microscopy. However, lower AngII level (10‐9 M) only increased Ca2+ pulsars frequency, suggesting a role for ROS in the Ca2+ pulsars stimulation. All AngII concentrations induced endothelial CaMKII recruitment within myoendothelial projections. AngII also stimulated NO production, an effect blunted by CaMKII inhibition (KN‐93). Candesartan, an AT1 blocker, partially reduced Ca2+ pulsars stimulation, suggesting roles for both AT receptors. Moreover, an increased in Ca2+ pulsars frequency and ROS production, associated with CaMKII clustering, is also observed in AngII‐AH mice. Our findings shed a new light on hypertension and Ca2+ dyshomeostasis along with their relationships with CaMKII, a key regulator of endothelial function.Grant Funding Source: Supported by CIHR, FRQS, CFI, HSFC and SQHA.

Cholesterol regulates both store‐ and agonist‐induced Ca2+ entry in pulmonary arterial endothelium (1089.9)

Endothelial Ca2+ entry is decreased in pulmonary arteries from rats exposed to chronic hypoxia (CH) (4 wk, PB = 380 torr). In addition, membrane cholesterol (Chol) is diminished in these cells and Chol supplementation restores ATP‐induced Ca2+ entry. Since both agonist‐induced and store‐operated Ca2+ entry (SOCE) are decreased after CH, we hypothesized that both require functional membrane Chol. To test this hypothesis, we administered epicholesterol (EpiCh; epimeric form of cholesterol) or vehicle to pulmonary arterial endothelial cells (PAEC) from control and CH rats to replace native Chol. The efficacy of EpiCh to diminish membrane Chol was confirmed by filipin staining in cultured ECs. EpiCh treatment greatly reduced both cyclopiazonic acid (CPA) ‐induced SOCE and ATP‐induced Ca2+ entry in PAECs from control and CH rats. We next assessed the effect of EpiCh on ATP and CPA‐induced Ca2+ currents in PAEC from control rats using patch clamp. Both EpiCh and La3+ inhibited ATP‐induced current similarly. Although T‐type voltage gated Ca2+ channels contribute to ATP responses, mibefradil (T‐type inhibitor) had no further effect in EpiCh treated cells. Likewise, CPA‐induced current was reduced in cells treated with EpiCh. We conclude that membrane Chol is required for both agonist‐induced and store‐operated Ca2+ entry in PAEC and that reduced Ca2+ seen after CH is possibly due to loss of this key regulator.Grant Funding Source: Supported by NIH grant HL95640

Phospholipase C isoforms expression in mouse endothelium (1075.2)

Phospholipase C (PLC) is a major signal transduction component through the regulation of various intracellular pathways, including PKC and intracellular Ca2+ channels. Up to 13 mammalian PLC isoforms have been identified and are classified in 6 families: PLC‐β, γ, δ, ε, ζ and η. Although expression of PLCs is tissue‐specific, combined expression of different PLCs has been reported. However, endothelial PLC expression remains to be established. Therefore, we sought to determine the expression pattern of PLC in mouse resistance arteries. We first studied the expression of PLC isoforms in mesenteric arteries. We found mRNA encoding for most PLCs with the exception of ζ1, η1 and η2 in whole mesenteric arteries. Similar results were obtained in middle cerebral, coronary and pulmonary arteries. We then elucidated the intracellular distribution of PLCs β3, γ1 and δ1, the main mammalian isoforms, in endothelial cells with in situ immunocytochemistry. We observed a diffuse staining in endothelium with no clearly defined heterogeneous distribution of all three PLCs. Further investigation is necessary to determine the specific PLC isoforms involved in endothelial localized Ca2+ dynamics. Taken together, these studies might strengthen our understanding of endothelial Ca2+ homeostasis mechanisms and therefore endothelial function.Grant Funding Source: Supported by FRQS, HSFC, SQHA and FICM.

Mitochondrial modulation of calcium pulsars in native endothelial cells (1075.3)

Calcium homeostasis is the crux of endothelial function and the regulation of vascular tone. Endothelial Ca2+ is thus highly dynamic and finely tuned. Amongst Ca2+ dynamics, Ca2+ pulsars are spontaneous Ca2+ release by IP3 receptors within myoendothelial projections (MEP). However, regulatory mechanisms of endothelial Ca2+ pulsars remain to be determined. Mitochondria play a major role in intracellular Ca2+ regulation in several cell types and may represent a modulator of endothelial Ca2+ dynamics like Ca2+ pulsars. We then hypothesized that mitochondria are active regulators of Ca2+ pulsars. Given the specific localization of Ca2+ pulsars, we first established the mitochondrial distribution in native mouse endothelium by confocal and electron microscopy. Mitochondria appeared heterogeneously distributed in endothelial cells but were found to be three times more concentrated near MEP. The functional interaction between mitochondria and Ca2+ pulsars was then investigated with real‐time confocal microscopy. Interestingly, mitochondrial uncouplers FCCP and CCCP significantly decreased Ca2+ pulsars frequency. Our data suggest that mitochondria are clustered near MEP to modulate Ca2+ pulsars and might therefore be an important regulator of endothelial function.Grant Funding Source: Supported by FRQS, HSFC, CFI, SQHA and CIHR.

TRPV4‐associated alterations of Ca2+ pulsars in a murine model of pulmonary hypertension secondary to heart failure (1090.11)

Heart failure is the most frequent cause of pulmonary hypertension (PH) (group 2). Pathophysiological mechanisms modulating pulmonary vascular tone and leading to group 2‐PH are poorly defined. Since Ca2+ homeostasis is crucial to endothelial control of vascular tone, mechanisms involved, including TRPV4 channels, may be altered. This study aimed to characterize intracellular Ca2+ dynamics in pulmonary endothelium and their alterations in group 2 PH. A mouse model of group 2 PH was developed and 3rd order resistance arteries were isolated to investigate endothelial intracellular Ca2+. TRPV4 involvement was studied using a high‐speed confocal microscope. Characterization of endothelial localized calcium transients, similar to Ca2+ pulsars, was carried out in pulmonary arteries from control mice. Exposure of Sham arteries to GSK1016790A (100 nM), a TRPV4‐agonist, increased the frequency (1.5 fold) of Ca2+ pulsars. However, exposure to GSK1016790A had little if any effect on Ca2+ dynamics in PH arteries. TRPV4 channel expression at both mRNA and protein levels within pulmonary endothelium was confirmed by qPCR, IHC and WB approaches. Our data suggest a potential role for endothelial TRPV4 in vasoregulatory alterations involving local Ca2+ dynamics. This study strengthens our understanding of endothelial Ca2+ dyshomeostasis occurring in a clinically relevant model of PH.Grant Funding Source: FRQS, HSFC, FICM, CIHR

Characterization of vascular endothelial and smooth muscle cell pathways and their contributions to myogenic and agonist‐mediated control (677.16)

Vascular smooth muscle cell contraction and relaxation is central to the local regulation of blood flow to tissue. At the arteriolar level, interactions between vascular endothelial cells and smooth muscel cells play critical role in determining the level of vascular tone. During mechanical and/or agonist stimulations, vascular Ca2+ dynamics are finely adjusted via a number of signaling pathways and the Ca2+ levels in the smooth muscle cells determine the level of contraction. To quantify how cellular Ca2+ dynamics are transformed into changes in vascular tone, we have constructed a cellular‐based model of isolated resistance vessel by integrating models of vessel wall mechanics, smooth muscle force generation, smooth muscle Ca2+ handling and electrophysiology with an endothelial electrophysiology and Ca2+ handling model. The dose‐response vasodilation and vasoconstriction data of different sizes of vessels in response to pharmacological vasoconstrictors and vasodilators, measured using a novel isovolumic myograph developed by Lu and Kassab are analyzed. The model simulates how changes in intraluminal pressure are used to maintain a constant vessel diameter during application of phenylephrine and acetylcholine. In order to simulate these sets of experimental data the model captures integrated vessel behavior including ionic (Ca2+, K+, Na+, etc.), IP3 and nitric oxide myoendothelial transport. Model analysis shows that α‐adrenergic pathways in smooth muscle and NO pathways in endothelium are sufficient to capture the experimentally observed phenylephrine and acetylcholine mediated responses.Grant Funding Source: NIH U01 HL118738, NSF CBET 1133260 and NIH P50‐GM094503

A pharmacologic activator of endothelial KCa channels improves coronary function in the hearts of type 2 diabetic rats (1078.10)

Endothelial dysfunction is a common early pathogenic event in patients with type 2 diabetes (T2D) who exhibit cardiovascular disease. In the present study, we have examined the effect of SKA‐31, a positive modulator of endothelial Ca2+‐activated K+ (KCa) channels, on total coronary flow in isolated hearts from Goto‐Kakizaki (GK) rats, a non‐obese model of T2D and age‐matched controls. Total coronary flow and left ventricular developed pressure were monitored simultaneously in spontaneously beating, Langendorff‐perfused hearts. Acute, bolus administrations of bradykinin (BK, 1 μg) or adenosine (ADO, 10 μg) increased coronary flow, and these responses were blunted in diabetic hearts at 10‐12 and 18‐20 weeks of age. In contrast, SKA‐31 dose‐dependently (0.01‐5 μg bolus exposures) increased coronary flow to comparable levels in both control and diabetic rat hearts at both ages. Flow responses to sodium nitroprusside were not different between control and diabetic hearts, suggesting normal coronary smooth muscle function. Importantly, continuous exposure to a sub‐threshold concentration of SKA‐31 (i.e. 0.3 μM) did not alter basal coronary flow or heart rate, but did ameliorate the blunted BK and ADO‐evoked flow responses in diabetic hearts. In summary, these data demonstrate that SKA‐31 is an effective coronary vasodilator in a rat model of T2D exhibiting endothelial dysfunction and metabolic syndrome, and further rescues impaired vasodilatory responses to BK and ADO in the coronary circulation.Grant Funding Source: Supported by CIHR

Impact of mitochondrial complex I inhibition on Ca2+ transients in pulmonary microvascular endothelium (1176.2)

In intact rat lung, we have shown that the decrease in lung ATP and endothelial barrier integrity resulting from rotenone‐induced mitochondrial complex I (CI) inhibition are prevented by the quinone CoQ1 (Free Radic Biol Med. doi: 10.1016/j.freeradbiomed.2013.07.040). To test whether impact of CI inhibition on Ca2+ handling might contribute, we evaluated Ca2+ dynamics using confocal microscopy and automated region of interest (ROI) detection and event tracking software in confluent monolayers of Fluo 4‐AM loaded rat pulmonary microvascular endothelial cell (PMVEC), in low glucose (0.2mM) HEPES buffer (25 °C) to limit glycolysis. Ca2+ transients were assessed at 1) baseline, 2) after vehicle, rotenone (0.5 µM) or rotenone + CoQ1 (10 µM), and 3) during activation of transient receptor potential vanilloid 4 channels with 4α‐phorbol 12,13‐didecanoate (4αPDD, 5 µM). Rotenone depressed the number of 4αPDD‐induced Ca2+ events/100 cells/sec, compared to vehicle or rotenone + CoQ1 (p<0.05, 2‐way ANOVA with Bonferroni post hoc tests), but did not significantly impact Ca2+ signal amplitude or spread. The addition of CoQ1 concomitant with rotenone limited both the duration and decay (sec) of Ca2+ signals compared to vehicle or rotenone alone (p<0.05). These data suggest that while CI inhibition does not break down structured endothelial Ca2+ domains in PMVEC, it blunts recovery of Ca2+ transients after TRPV4 activation.

Visualizing the endothelium from inside intact arteries at physiological pressures reveals pulsatile linear, circular and spiral Ca2+ waves (668.1)

Ca2+ signalling in the endothelium is central to the control of vascular function and to the changes that occur in vascular disease. However, visualizing Ca2+ signals in endothelial cells in intact, pressurised arteries is difficult (particularly in the larger arteries where atherosclerosis and re‐stenosis occurs), due to medial and adventitial thickness. A novel wide‐field, epi‐fluorescent microscope with subcellular resolution, that enables endothelium imaging from the inside of large pressurised arteries, has been developed. A side‐viewing Gradient Index (GRIN) relay lens, inserted into the lumen of pressurised arteries, permits the endothelium to be imaged with a 350 µm diameter field of view ‐ an area encompassing approximately 200 cells. Acetylcholine‐activation of the endothelium evoked pulsatile, repetitive Ca2+ waves that initiated at several sites (measured by fluorescent indicators). Waves progressed through the endothelium linearly, in expanding circles and in spiral forms. The Ca2+ waves show properties of excitable systems i.e. regenerative propagation and annihilation of colliding wavefronts. The GRIN lens approach offers a new level of understanding in the study of endothelial Ca2+ signalling and we propose the endothelium, via Ca2+ signals, behaves as an excitable medium in physiological conditions.Grant Funding Source: Wellcome Trust; British Heart Foundation; EPSRC

Activation of canonical transient receptor potential channels preserves Ca2+ entry and endothelium-derived hyperpolarizing factor–mediated function in vitro in porcine coronary endothelial cells and coronary arteries under conditions of hyperkalemia

Although membrane depolarization by hyperkalemia is known to reduce Ca2+ influx in endothelial cells, the mechanism by which endothelial Ca2+ channel is affected by hyperkalemia remains poorly studied. We studied the effect of hyperkalemia on canonical transient receptor potential channels, in particular canonical transient receptor potential channel 3, in modulation of endothelial intracellular Ca2+ concentration. Endothelium-derived hyperpolarizing factor-mediated function is Ca2+ dependent, and hyperkalemic cardioplegia/organ preservation solutions impair endothelium-derived hyperpolarizing factor-mediated function. We explored the role of canonical transient receptor potential channel 3 in endothelium-derived hyperpolarizing factor-mediated function and investigated whether modulation of these channels preserves endothelial Ca2+ influx and endothelium-derived hyperpolarizing factor-mediated function under the condition of hyperkalemic/cardioplegic exposure. Intracellular Ca2+ concentration was measured with fluorescent dye in primary cultured porcine coronary endothelial cells exposed to hyperkalemic/cardioplegic solutions containing mild to extreme high K+ concentration. Endothelium-derived hyperpolarizing factor-mediated relaxation under hyperkalemic/cardioplegic exposure was studied in small porcine coronary arteries in a myograph in the presence of cyclooxygenase and nitric oxide synthase inhibitors and nitric oxide scavenger. Canonical transient receptor potential channel 3 blocker inhibited bradykinin-induced Ca2+ influx and attenuated endothelium-derived hyperpolarizing factor-mediated response. Hyperkalemic exposure inhibited canonical transient receptor potential channel 3-mediated Ca2+ influx in a K+ concentration-dependent manner (120>20>10 mmol/L). Ca2+ influx decreased in porcine coronary endothelial cells exposed to histidine-tryptophan-ketoglutarate, St Thomas' Hospital, and University of Wisconsin solutions that contained mild (10 mmol/L), moderate (20 mmol/L), and extreme high (125 mmol/L) K+ concentration, respectively. Canonical transient receptor potential channel activator prevented the reduction of Ca2+ influx in porcine coronary endothelial cells exposed to solutions containing mild to moderate high [K+]o and restored endothelium-derived hyperpolarizing factor-mediated response that was impaired by hyperkalemic exposure. Canonical transient receptor potential channel 3 is involved in endothelium-derived hyperpolarizing factor-mediated function in coronary arteries. Hyperkalemia inhibited canonical transient receptor potential channel 3-mediated Ca2+ influx in endothelial cells. Canonical transient receptor potential channel activation restores Ca2+ influx suppressed by hyperkalemia and prevents dysfunction of endothelium-derived hyperpolarizing factor.

Characterization of blood pressure and endothelial function in TRPV4-deficient mice with l -NAME- and angiotensin II-induced hypertension

Transient receptor potential vanilloid type 4 (TRPV4) is an endothelial Ca2+ entry channel contributing to endothelium‐mediated dilation in conduit and resistance arteries. We investigated the role of TRPV4 in the regulation of blood pressure and endothelial function under hypertensive conditions. TRPV4‐deficient (TRPV4−/−) and wild‐type (WT) control mice were given l‐NAME (0.5 g/L) in drinking water for 7 days or subcutaneously infused with angiotensin (Ang) II (600 ng/kg per minute) for 14 days, and blood pressure measured by radiotelemetry. TRPV4−/− mice had a lower baseline mean arterial pressure (MAP) (12‐h daytime MAP, 94 ± 2 vs. 99 ± 2 mmHg in WT controls). l‐NAME treatment induced a slightly greater increase in MAP in TRPV4−/− mice (day 7, 13 ± 4%) compared to WT controls (6 ± 2%), but Ang II‐induced increases in MAP were similar in TRPV4−/− and WT mice (day 14, 53 ± 6% and 37 ± 11%, respectively, P < 0.05). Chronic infusion of WT mice with Ang II reduced both acetylcholine (ACh)‐induced dilation (dilation to 10−5 mol/L ACh, 71 ± 5% vs. 92 ± 2% of controls) and the TRPV4 agonist GSK1016790A‐induced dilation of small mesenteric arteries (10−8 mol/L GSK1016790A, 14 ± 5% vs. 77 ± 7% of controls). However, Ang II treatment did not affect ACh dilation in TRPV4−/− mice. Mechanistically, Ang II did not significantly alter either TRPV4 total protein expression in mesenteric arteries or TRPV4 agonist‐induced Ca2+ response in mesenteric endothelial cells in situ. These results suggest that TRPV4 channels play a minor role in blood pressure regulation in l‐NAME‐ but not Ang II‐induced hypertension, but may be importantly involved in Ang II‐induced endothelial dysfunction.

Pregnancy programming and preeclampsia: identifying a human endothelial model to study pregnancy-adapted endothelial function and endothelial adaptive failure in preeclamptic subjects.

We have previously reported that the increase in vasodilator production in an ovine model pregnancy is underpinned by an increase in connexin 43 (Cx43) gap junction function, so allowing more uterine artery endothelial cells to produce a more sustained Ca(2+) burst response to agonist stimulation. Since activation of endothelial nitric oxide synthase (eNOS) requires elevated [Ca(2+)]i, it follows that the direct result of enhanced bursting in turn is an increase in nitric oxide (NO) production per cell from more cells, and for a longer period of time. Preeclampsia (PE) is associated with endothelial vasodilatory dysfunction, and the endocrine profile of women with PE includes an increase in a number of factors found in wound sites. The common action of these growth factors and cytokines in wound sites is to mediate Cx43 dysfunction through kinase phosphorylation and closure. Translational studies are now needed to establish if inhibitory phosphorylation of Cx43 in human endothelium is the cause of endothelial dysfunction in PE subjects and if so, to identify the kinase pathways best targeted for therapy in PE subjects. Consistent with this we have already shown endothelial Ca(2+) and NO responses of human umbilical vein from normal subjects are similar to that of ovine pregnant uterine artery, and that those same responses in cords from PE subjects are blunted to levels more typical of nonpregnant uterine artery. In this review we summarize the further evidence that growth factors and cytokines may indeed mediate endothelial dysfunction in PE subjects through closure of Cx43 gap junctions. We also consider how we may clinically translate our studies to humans by using intact umbilical vein and isolated HUVEC in primary culture for an initial screen of drugs to prevent deleterious Cx43 phosphorylation, with the ultimate goal of reversing PE-related endothelial dysfunction.

Local Effects of Pregnancy on Connexin Proteins That Mediate Ca 2+ -Associated Uterine Endothelial NO Synthesis

Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth. local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and NO production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43, and P(635)eNOS protein levels in uterine artery and systemic artery (omental and renal) endothelium and connexins in vascular smooth muscle. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy, uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P(635)eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P(635)eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37-specific Gap peptides, abrogated uterine artery endothelial ATP-induced Ca(2+)-mediated NO production. Thus, uterine artery endothelial connexin 43, but not connexin 37, regulates Ca(2+)-mediated NO production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.

Klebsiella pneumoniae Induces an Inflammatory Response in an In Vitro Model of Blood-Retinal Barrier

Klebsiella pneumoniae has become an important pathogen in recent years. Although most cases of K. pneumoniae endogenous endophthalmitis occur via hematogenous spread, it is not yet clear which microbial and host factors are responsible for the ability of K. pneumoniae to cross the blood-retinal barrier (BRB). In the present study, we show that in an in vitro model of BRB based on coculturing primary bovine retinal endothelial cells (BREC) and primary bovine retinal pericytes (BRPC), K. pneumoniae infection determines changes of transendothelial electrical resistance (TEER) and permeability to sodium fluorescein. In the coculture model, bacteria are able to stimulate the enzyme activities of endothelial cytosolic and Ca(2+)-independent phospholipase A2s (cPLA2 and iPLA2). These results were confirmed by the incremental expression of cPLA2, iPLA2, cyclo-oxygenase-1 (COX1), and COX2 in BREC, as well as by cPLA2 phosphorylation. In supernatants of K. pneumoniae-stimulated cocultures, increases in prostaglandin E2 (PGE2), interleukin-6 (IL-6), IL-8, and vascular endothelial growth factor (VEGF) production were found. Incubation with K. pneumoniae in the presence of arachidonoyl trifluoromethyl ketone (AACOCF3) or bromoenol lactone (BEL) caused decreased PGE2 and VEGF release. Scanning electron microscopy and transmission electron microscopy images of BREC and BRPC showed adhesion of K. pneumoniae to the cells, but no invasion occurred. K. pneumoniae infection also produced reductions in pericyte numbers; transfection of BREC cocultured with BRPC and of human retinal endothelial cells (HREC) cocultured with human retinal pericytes (HRPC) with small interfering RNAs (siRNAs) targeted to cPLA2 and iPLA2 restored the pericyte numbers and the TEER and permeability values. Our results show the proinflammatory effect of K. pneumoniae on BREC, suggest a possible mechanism by which BREC and BRPC react to the K. pneumoniae infection, and may provide physicians and patients with new ways of fighting blinding diseases.

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g. endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g. nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel. To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g. endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g. nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

Positive Feedback Regulation of Agonist-Stimulated Endothelial Ca 2+ Dynamics by K Ca 3.1 Channels in Mouse Mesenteric Arteries

Intermediate and small conductance KCa channels IK1 (KCa3.1) and SK3 (KCa2.3) are primary targets of endothelial Ca(2+) signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca(2+) transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1(-/-)) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca(2+) dynamics. Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca(2+) dynamics was not different between IK1(-/-) and wild-type mice (P>0.05), the frequency of acetylcholine-stimulated (2 μmol/L) Ca(2+) dynamics was greatly decreased in IK1(-/-) endothelium (515±153 versus 1860±319 events; P<0.01). In IK1(-/-)/SK3(T/T) mice, ancillary suppression (+Dox) or overexpression (-Dox) of SK3 channels had little additional effect on the occurrence of events under basal or acetylcholine-stimulated conditions. However, SK3 overexpression did restore the decreased event amplitudes. Removal of extracellular Ca(2+) reduced acetylcholine-induced Ca(2+) dynamics to the same level in wild-type and IK1(-/-) arteries. Blockade of IK1 and SK3 with the combination of charybdotoxin (0.1 μmol/L) and apamin (0.5 μmol/L) or transient receptor potential vanilloid 4 channels with HC-067047 (1 μmol/L) reduced acetylcholine Ca(2+) dynamics in wild-type arteries to the level of IK1(-/-)/SK3(T/T)+Dox arteries. These drug effects were not additive. IK1, and to some extent SK3, channels exert a substantial positive feedback influence on endothelial Ca(2+) dynamics.