Release In Endothelial Cells Research Articles

Leeches alleviates diabetic macroangiopathy by increasing nitric oxide production in rat aorta and restoring endothelium-dependent vasodilatation

IntroductionDiabetes mellitus (DM) is a chronic metabolic disorder characterized by elevated blood glucose level over a prolonged period, leading to severe damage in tissues including the heart, blood vessels, eyes and the kidneys. Danzhi Jiangtang Capsule (DJC) is an effective drug for diabetes, but the mechanism responsible for its efficacy remains unknown. This study aimed to explore the effective ingredient of DJC that ameliorated diabetes and the possible mechanisms.Material and methodsWe orally treated streptozotocin (STZ)-induced diabetic rats with 540 mg/kg DIC or the same dose of its four active components, namely Leeches, Pseudostellaria Polysaccharides (PP), Paeonia Suffruticosa Andr (PSA) and Rehmannia Glutinosa Libosch (RGL), respectively for 8 weeks.ResultsAlthough all of these components could reduce blood glucose levels in diabetic rats, the extent of alleviation of DJC was more pronounced than all of its four ingredients. Unlike the other three components, Leeches is the only effective ingredient of DJC that decreased tetrahydrobiopterin (BH4) oxidation to activate endothelial nitric oxide synthase (eNOS), and increased nitric oxide (NO) production, leading to improved endothelium-dependent relaxation both in diabetic rats and in immortalized human mesangial cells under the stimulation of high glucose.ConclusionsLeeches could alleviate diabetic macroangiopathy by inhibiting NO release in endothelial cells under high-glucose condition.

Control of TRPML1 Channel Activity and Lysosome Trafficking by Acid Ceramidase in Mouse Coronary Arterial Endothelial Cells

Previous studies have demonstrated that lysosomal acid ceramidase (AC) regulates lysosome-multivesicular body (MVB) interaction and exosome release in mouse coronary arterial endothelial cells (CAECs). However, it remains unknown how exosome secretion is controlled by AC. In this study, we tested a hypothesis that enhancement of lysosome trafficking by AC activity increases lysosome-MVB interaction determining MVB fate to promote exosome release, which may be attributed to its action on lysosomal TRPML1 channel-mediated Ca2+ release in CAECs. By GCaMP3 Ca2+ imaging, we found that ML-SA1 as a TRPML1 channel agonist remarkably increased lysosomal Ca2+ release through TRPML1 channel in WT/WT CAECs. In Asah1fl/fl/ECcre CAECs, however, ML-SA1 failed to influence TRPML1 channel-mediated Ca2+ release. As an AC product, sphingosine remarkably increased lysosomal Ca2+ release through TRPML1 channel in both WT/WT and Asah1fl/fl/ECcre CAECs. With whole-lysosome patch clamp recording, we detected ML-SA1-induced Ca2+ currents in a dose-dependent manner through the membrane of lysosomes isolated from WT/WT CAECs, while intact lysosomes isolated from Asah1fl/fl/ECcre CAECs had evidently blunted opening of TRPML1 channels in response to ML-SA1. Different effects of AC associated sphingolipids on TRPML1 channel activity were also examined in lysosomes isolated from WT/WT CAECs. It was found that sphingomyelin inhibited, but sphingosine enhanced lysosomal TRPML1 channel activity. Ceramide did not alter this lysosomal channel activity. In lysosomes isolated from Asah1fl/fl/ECcre CAECs, only sphingosine increased ML-SA1-induced TRPML1 channel-mediated Ca2+ release. Using confocal microscopy for measurement of lysosomes movement, we demonstrated that lysosomal TRPML1 channel activation by ML-SA1 in CAECs markedly enhanced lysosome trafficking toward nucleus, which was blocked by genetic deletion of AC. These results suggest that AC-mediated sphingolipid metabolism controls lysosomal TRPML1 channel-mediated Ca2+ release and lysosome trafficking in CAECs, which may contribute to the regulation of lysosome-MVB interaction and exosome release by AC (supported by NIH grant DK120491).

Aprocitentan (a Dual Endothelin-Receptor Antagonist) for Treatment-Resistant Hypertension.

Treatment-resistant hypertension (TRH) is associated with increased cardiovascular risks and progression of chronic kidney disease. The pathophysiology of TRH is multifactorial, including overactivity of the renin-angiotensin-aldosterone system and sympathetic nervous system, endothelial dysfunction, and volume overload. Endothelin-1 is a vasoconstrictive peptide that causes neurohormonal and sympathetic activation, increased aldosterone synthesis and secretion, endothelial dysfunction, vascular hypertrophy and remodeling, and fibrosis. Endothelin-1 acts through 2 receptors, ETA and ETB. Activation of ETA receptors in vascular smooth muscle cells results in vasoconstriction, whereas ETB receptor activation results in vasoconstriction in the vascular smooth muscle cells and vasodilation through nitric oxide release in endothelial cells. Aprocitentan is novel, oral, dual endothelin-receptor antagonist that has demonstrated a more favorable tolerability and safety profile in early clinical trials compared with other endothelin-receptor antagonists studied. Phase 2 trial data support a significant reduction in blood pressure compared to placebo and similar blood pressure reduction compared to a moderately dosed angiotensin-converting enzyme inhibitor in patients with essential hypertension. An ongoing phase 3 randomized clinical trial is evaluating aprocitentan's efficacy and safety in patients with TRH receiving multiple antihypertensives. Additional research is needed to determine aprocitentan's role in therapy, but this agent may be a suitable treatment option for TRH.

MESENCHYMAL STROMAL CELLS PROTECT AGAINST VASCULAR DAMAGE AND DEPRESSION-LIKE BEHAVIOR IN MICE SURVIVING CEREBRAL MALARIA

<h3>Introduction</h3> Cerebral malaria (CM) is a severe systemic inflammatory disease, that leads to behavioral impairments, such as depression. Currently, there are no therapies to treat the neurological sequelae of CM, and mesenchymal stromal cells (MSC) may be an alternative. <h3>Objectives</h3> To investigate the effects of MSC therapy on the blood-brain barrier (BBB), leukocyte rolling and adherence in the brain, and depression like-behavior in experimental CM. <h3>Methods</h3> Male C57BL/6 mice were infected with Plasmodium berghei ANKA (PbA, 1 × 106, intraperitoneally). At day 6, PbA-infected animals received chloroquine (25 mg/kg orally for seven days) and were then randomized to receive MSC (105 cells) or saline intravenously. All animal experiments were approved by the ethics committee of the Oswaldo Cruz Institute (L-012/2015), and had a number of 3 to 12 animals per group. In vitro, endothelial cells were used to evaluate the effects of conditioned media derived from MSC (CMMSC) on cell viability by lactate dehydrogenase (LDH) release. We used a one-way analysis of variance with post-hoc Tukey test to assess the differences between more than two groups and Student‘s t test when data from only two groups were analyzed. <h3>Results</h3> MSC therapy improved the vascular damage at 7 days after infection (dpi). Evans blue assay and intravital microscopy showed that infected mice, who received only saline, had an increase in BBB permeability (n=7; 0.0084±0.0016) and adherent leukocytes (n=3; 3.76±0.23). PbA+MSC mice had reduced BBB permeability (n=4; 0.0063±0.0012) and leukocyte adhesion in the brain microvasculature (n=4; 2.18±0.37). PbA-infected mice presented reduced levels of BDNF 7 dpi (n=4; 0.025±0.0015), and depression-like behavior 15 dpi evaluated by the tail suspension test (TST, n=5; 44.38±4) and the forced swim test (FST, n=5; 45.5±4.8). MSC therapy restored BDNF levels (n=11; 0.029±0.0008) and mitigated behavioral alterations (n=5; TST:18±2.7; FST:18.2±2.8). CMMSC reduced LDH release in endothelial cells stimulated with heme (group heme: 1,89±0,11; group CMMSC: 0,96±0,06), suggesting a paracrine mechanism of action. <h3>Conclusion</h3> A single dose of MSC as an adjuvant therapy protected against vascular damage and improved depression-like behavior in mice that survived experimental CM.

The antihypertensive effect of MK on spontaneously hypertensive rats through the AMPK/Akt/eNOS/NO and ERK1/2/Cx43 signaling pathways

We investigated the antihypertensive effects of maximakinin (MK) on spontaneously hypertensive rats (SHRs). The effects of MK on arterial blood pressure in SHRs were observed, and flow cytometry and 4,5-diaminofluorescein-2 staining were used to examine MK-induced nitric oxide (NO) release in human umbilical vein endothelial cells (HUVECs). Western blotting was used to analyze the effects of MK on the expression of AMP-activated protein kinase (AMPK), Akt, Connexin 43, ERK1/2, p38, and p-eNOS in HUVECs. The results showed that MK induced a more significant antihypertensive effect on SHRs than bradykinin (BK). MK induced significant increases in endothelial nitric oxide synthase (eNOS) phosphorylation and NO release in HUVECs. MK also significantly increased the phosphorylation of Akt and AMPK in HUVECs. The AMPK inhibitor compound C blocked the effect of MK on the generation of NO. MK induced the phosphorylation of ERK1/2, p38, and Connexin 43. The expression of p-Connexin 43 was significantly decreased in the presence of the ERK1/2 inhibitor U0126 but not the p38 inhibitor SB203580. The effects of MK on the phosphorylation of AMPK and ERK1/2 were significantly decreased by the BK B2 receptor inhibitor HOE-140. In summary, MK can significantly reduce blood pressure in SHRs. The antihypertensive effect might be mediated through the activation of the BK B2 receptor, while the downstream AMPK/PI3K/Akt/eNOS/NO and ERK1/2/Connexin 43 signaling pathways play additional roles.

Barrier protective functions of hederacolchiside-E against HMGB1-mediated septic responses

The role of high mobility group box 1 (HMGB1) has been recognized as important, and suppression of HMGB1 release and restoration of vascular barrier integrity are regarded as potentially promising therapeutic strategies against sepsis. Hederacolchiside-E (HCE), namely 3-O-{α-L-rhamnopyranosyl (1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl}-28-O-[α-L-rhamnopyranosyl (1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester, is a bidesmosidic oleanane saponin first isolated in 1970 from the leaves of Hedera colchica. We tested our hypothesis that HCE inhibits HMGB1-induced vascular hyperpermeability and thereby increases the survival of septic mouse model from suppression of HMGB1 release upon lipopolysaccharide (LPS)-stimulation. In LPS-activated human endothelial cells and a sepsis mouse model by cecal ligation and puncture (CLP), antiseptic activity of HCE was investigated from suppression of vascular permeability, pro-inflammatory proteins, and tissue injury markers. Post-treatment of HCE significantly suppressed HMGB1 release both in LPS-activated human endothelial cells and the CLP-induced sepsis mouse model. HCE inhibited hyperpermeability and alleviated HMGB1-mediated vascular disruptions, and reduced sepsis-related mortality and tissue injury in mice. Our results suggest that reduction of HMGB1 release and septic mortality by HCE may be useful for the drug candidate of sepsis, indicating a possibility of successful repositioning of HCE.

Penicillin causes non-allergic anaphylaxis by activating the contact system

Immediate hypersensitivity reaction (IHR) can be divided into allergic- and non-allergic-mediated, while “anaphylaxis” is reserved for severe IHR. Clinically, true penicillin allergy is rare and most reported penicillin allergy is “spurious”. Penicillin-initiated anaphylaxis is possible to occur in skin test- and specific IgE-negative patients. The contact system is a plasma protease cascade initiated by activation of factor XII (FXII). Many agents with negative ion surface can activate FXII to drive contact system. Our data showed that penicillin significantly induced hypothermia in propranolol- or pertussis toxin-pretreated mice. It also caused a rapid and reversible drop in rat blood pressure, which did not overlap with IgE-mediated hypotension. These effects could be countered by a bradykinin-B2 receptor antagonist icatibant, and consistently, penicillin indeed increased rat plasma bradykinin. Moreover, penicillin not only directly activated contact system FXII-dependently, but also promoted bradykinin release in plasma incubated-human umbilical vein endothelial cells. In fact, besides penicillin, other beta-lactams also activated the contact system in vitro. Since the autoactivation of FXII can be affected by multiple-factors, plasma from different healthy individuals showed vastly different amidolytic activity in response to penicillin, suggesting the necessity of determining the potency of penicillin to induce individual plasma FXII activation. These results clarify that penicillin-initiated non-allergic anaphylaxis is attributed to contact system activation, which might bring more effective diagnosis options for predicting penicillin-induced fatal risk and avoiding costly and inappropriate treatment clinically.

Phosphorylation of STAT3 and ERBB2 mediates hypoxia‑induced VEGF release in ARPE‑19 cells

Neovascularization in the retina can cause loss of vision. Vascular endothelial growth factor (VEGF) serves an important role in the pathogenesis of retinal vascular diseases. Hypoxia is a notable cause of VEGF release and both STAT3 and ERBB2 are known to be associated with VEGF. In addition, STAT3 and ERBB2 interact with each other. In the present study, it was hypothesized that signal transducer and activator of transcription 3 (STAT3) and erbB-2 receptor tyrosine kinase 2 (ERBB2) may be involved in the regulation of hypoxia-induced VEGF in the retina. Cells of the retinal pigment epithelium (RPE) are an important source of VEGF. Therefore, the RPE-derived human cell line ARPE-19 was exposed to hypoxia. Hypoxia-induced phosphorylation of STAT3 and ERBB2 in ARPE-19 cells was decreased by AG490, an inhibitor of Janus kinase 2, as were hypoxia-induced VEGF release and tube formation in human umbilical vein endothelial cells. Thus, phosphorylation of ERBB2 and STAT3 regulates hypoxia-induced VEGF release in ARPE-19 cells. The results of the present study suggested that inhibition of ERBB2 and STAT3-mediated pathways under hypoxia may represent a new strategy for treating retinal vascular disease.

Molecular mechanisms in lipopolysaccharide-induced interleukin 6 release in lymphatic endothelial cells

The lymphatic vasculature is involved in the transportation of tissue fluids, extravasated plasma proteins and cells back into blood circulation. Formation of lymphatic vessels by lymphatic endothelial cells (LECs) occurs both in normal tissues as well as in pathological processes including inflammation, lymphedema and tumor metastasis. Recent reports demonstrated that lymphatic vasculature is not just a major conduit for immune cell transport. It seems to be directly involved in both the induction and the resolution of inflammation. However, little is known about how lymphatic vessels themselves respond to inflammation. The purpose of this study was to investigate the molecular mechanism by which interleukin-6 release in LECs exposure to lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria. LPS was shown to cause an increase in IL-6 release in LECs. Pharmacological inhibitors of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38MAPK or c-jun N-terminal kinase (JNK), significantly abrogated the LPS-induced IL-6 release. In addition, LPS was shown to activate ERK, p38MAPK and JNK in LECs, suggesting functional cross-talk. The results of reporter assay further indicated that LPS increased the transcriptional activity of NF-κB, a critical transcription factor in inducing IL-6 expression. Our data suggest that MAPKs and NF-κB activation may contribute to LPS-induced IL-6 release in LECs. Interventions of MAPKs and NF-κB signaling may be beneficial in the treatment of lymphatic-associated inflammation.

Nicotinic acid adenine dinucleotide phosphate activates two-pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony-forming cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+ -releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP3 )-sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca2+ release also required ER-embedded InsP3 receptors. Finally, NAADP-induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.

Sex-specific stress response and HMGB1 release in pulmonary endothelial cells.

Women are known to be associated with a higher susceptibility to pulmonary arterial hypertension (PAH). In contrast, male PAH patients have a worse survival prognosis. In this study, we investigated whether the contribution of sex goes beyond the effects of sex hormones by comparing the ability of isolated male and female pulmonary endothelial cells to respire, proliferate and tolerate the stress. Mouse lung endothelial cells (MLEC) were isolated from the lungs of male and female 3-week old mice. Male MLEC showed an increased basal mitochondrial respiration rate, elevated maximal respiration, a significantly greater level of mitochondrial polarization, and a higher rate of proliferation. Exposure of cells to hypoxia (2% of O2 for 24 hours) induced a strong apoptotic response in female but not male MLEC. In contrast, treatment with mitochondrial respiratory Complex III inhibitor Antimycin A (AA, 50μM) mediated severe necrosis specifically in male MLEC, while female cells again responded primarily by apoptosis. The same effect with female cells responding to the stress by apoptosis and male cells responding by necrosis was confirmed in starved pulmonary endothelial cells isolated from human donors. Elevated necrosis seen in male cells was associated with a significant release of damage-associated alarmin, HMGB1. No stimuli induced a significant elevation of HMGB1 secretion in females. We conclude that male cells appear to be protected against mild stress conditions, such as hypoxia, possibly due to increased mitochondrial respiration. In contrast, they are more sensitive to impaired mitochondrial function, to which they respond by necrotic death. Necrosis in male vascular cells releases a significant amount of HMGB1 that could contribute to the pro-inflammatory phenotype known to be associated with the male gender.

The Effects of Novel Myristoylated PKC Epsilon Peptide Modulators on Real‐Time Nitric Oxide Release in Human Umbilical Vein Endothelial Cells

Endothelial dysfunction is a major consequence of ischemia‐reperfusion (I/R) injury, such as during the re‐establishment of blood flow after endovascular thrombus removal. It is characterized by limited nitric oxide (NO) bioavailability and production of superoxide (SO) instead of NO, in part from uncoupled endothelial NO synthase (eNOS) activity when the dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4) ratio is elevated during reperfusion following prolonged ischemia. The protein kinase C epsilon (PKCɛ) isoform is known to participate in pre‐conditioning (PKCɛ activation treatment prior to ischemia) and post‐conditioning (PKCɛ inhibition treatment during reperfusion) to attenuate rat myocardial I/R injury. Normally following diacylglycerol‐dependent activation, PKCɛ binds to isoform‐specific receptors for activated C‐kinase (RACK) and translocates from the cytosol to the cell membrane to phosphorylate its targets, such as eNOS at Ser‐1177 to augment NO release when the BH2 to BH4 ratio is reduced to promote coupled eNOS activity that is normally seen prior to prolonged ischemia. Cell permeable myristic acid conjugated PKCɛ peptide activator (myr‐HDAPIGYD; myr‐PKCɛ+) and inhibitor (N‐myr‐EAVSLKPT; myr‐PKCɛ‐) has been shown to increase and decrease NO release, respectively, in rat aortic tissue by regulating PKCɛ translocation to eNOS. However, modulation of PKCɛ‐mediated NO release with these peptides in human cells remains undetermined. We hypothesize that myr‐PKCɛ+ will augment and myr‐PKCɛ‐ will attenuate NO release in cultured human umbilical vein endothelial cells (HUVECs). Real‐time HUVEC NO release (pmol) was measured using a calibrated NO electrode following administration of myr‐PKCɛ+ or myr‐PKCɛ‐ ± acetylcholine (Ach; positive control). Data were analyzed by ANOVA using Fishers post‐hoc analysis. Basal NO levels (66±6; n=35) were determined as the pmol difference between cell‐populated (106 cells/well) and reagent only wells. Control Ach (10 μM) enhanced NO release (102±7; n=37), myr‐PKCɛ+ (10 μM) enhanced NO release with Ach (107±16; n=8) and without Ach (107±13; n=7), and myr‐PKCɛ‐ (10 μM) attenuated NO release with Ach (37±9; n=13) and without Ach (7±29; n=9) compared to basal levels (all p&lt;0.05). Results suggest that myr‐PKCɛ +/− effects on NO release is translational across mammalian species, presumably through activation and inhibition of PKCɛ‐mediated phosphorylation of eNOS. Thus, therapeutic intervention that targets inhibition of PKCɛ to reduce uncoupled eNOS activity during reperfusion may yield protective effects via attenuating the extent of I/R injury seen in clinical myocardial infarction, coronary bypass, coronary angioplasty, and organ transplantation.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current research license is supported by Young Therapeutics, LLC.

Effects of substrate mechanics on angiogenic capacity and nitric oxide release in human endothelial cells.

Loss of vascular elasticity results from progressive degeneration of the extracellular matrix of elastic arteries under the effect of aging and certain diseases, including atherosclerosis. To investigate the influence of vessel wall stiffening on endothelial cell (EC) function, we seeded human umbilical vein ECs onto variably compliant polydimethylsiloxane substrates. When plated on the more compliant substrate, ECs assembled into capillary-like structures. By contrast, they failed to form a network on stiff substrates, even in the presence of vascular endothelial growth factor (VEGF). Cell proliferation and migration increased with stiffness, while ECs released more nitric oxide (NO) on the soft substrate. Treatment with VEGF increased migration and NO release in a stiffness-dependent manner. Atomic force microscopy measurement of cell elasticity along with actin fiber analysis revealed that ECs plated on the more compliant surface were mechanically softer, with mostly diffuse actin arrangement. Our results demonstrate that matrix stiffening induces actin reorganizations, reflected by cortical stiffening in ECs, which may lead to a decrease in their angiogenic capacity and NO release. Hence, the mechanical properties of ECs display a prognostic and therapeutic potential and might serve as a reliable biomarker of vascular function.

RIPK3 collaborates with GSDMD to drive tissue injury in lethal polymicrobial sepsis.

Sepsis is a systemic inflammatory disease causing life-threatening multi-organ dysfunction. Accumulating evidences suggest that two forms of programmed necrosis, necroptosis and pyroptosis triggered by the pathogen component lipopolysaccharide (LPS) and inflammatory cytokines, play important roles in the development of bacterial sepsis-induced shock and tissue injury. Sepsis-induced shock and tissue injury required receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) phosphorylation, caspase11 activation and gasdermin D (GSDMD) cleavage. However, the synergistic effect of necroptosis and pyroptosis in the pathological progress of sepsis remains elusive. In this study, we found that blockage of both necroptosis and pyroptosis (double deletion of Ripk3/Gsdmd or Mlkl/Gsdmd) resulted in accumulative protection against septic shock, systemic blood clotting and multi-organ injury in mice. Bone marrow transplantation confirmed that necroptosis and pyroptosis in both myeloid and nonmyeloid cells are indispensable in the progression of sepsis-induced multi-organ injury. Both RIPK3 and GSDMD signaling collaborated to amplify necroinflammation and tissue factor release in macrophages and endothelial cells, which led to tissue injury. Furthermore, cell death induced by inflammatory cytokines and high-mobility group box 1 could be prevented by double ablation of Ripk3/Gsdmd or Mlkl/Gsdmd, suggesting that a positive feedback loop interconnecting RIPK3/MLKL and GSDMD machinery and inflammation facilitated sepsis progression. Collectively, our findings demonstrated that RIPK3-mediated necroptosis and GSDMD-mediated pyroptosis collaborated to amply inflammatory signaling and enhance tissue injury in the process of sepsis, which may shed new light on two potential targets of combined therapeutic interventions for this highly lethal disorder.

Curcumane C and (±)-curcumane D, an unusual seco-cadinane sesquiterpenoid and a pair of unusual nor-bisabolane enantiomers with significant vasorelaxant activity from Curcuma longa

A new seco-cadinane sesquiterpenoid (curcumane C, 1) and a pair of new nor-bisabolene enantiomers [(+)- and (−)-curcumane D, 2a and 2b] were isolated from C. longa. Compound 1 possesses an unusual 4,5-seco-cadinane skeleton with a tetrahydrophthalide moiety, while 2a and 2b contain an unusual 15-nor-bisabolene skeleton with a chromone core. All compounds exhibited significant vasorelaxant effects against KCl-induced contraction of rat aortic rings. Compound 1 also exhibited a vasorelaxant effect against phenylephrine-induced contraction of rat aortic rings. Meanwhile, compound 1 showed a stronger vasorelaxant effect in endothelium-intact rat aortic rings compared with endothelium-denuded rat aortic rings, indicating that vasodilation by 1 involved both endothelium-dependent and endothelium-independent pathways. Furthermore, compound 1 increased the NO content in human umbilical vein endothelial cells and its vasorelaxant effect could be attenuated by treatment with L-NAME, an endothelium NO synthase inhibitor. Thus, the underlying vasodilatory mechanisms of 1 may be mediated via abrogation of extracellular Ca2+ influx and regulation of NO release in vascular endothelial cells.

Group 1 metabotropic glutamate receptors trigger glutamate-induced intracellular Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity causes an increase in local cerebral blood flow (CBF) to ensure local supply of oxygen and nutrients to the activated areas. The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-D-aspartate receptors to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO, thereby triggering NVC. Recent work suggested that endothelial Ca2+ signals could underpin NVC by recruiting the endothelial NO synthase. For instance, acetylcholine induced intracellular Ca2+ signals followed by NO release by activating muscarinic 5 receptors in hCMEC/D3 cells, a widely employed model of human brain microvascular endothelial cells. Herein, we sought to assess whether also glutamate elicits metabotropic Ca2+ signals and NO release in hCMEC/D3 cells. Glutamate induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) that was blocked by α-methyl-4-carboxyphenylglycine and phenocopied by trans-1-amino-1,3-cyclopentanedicarboxylic acid, which, respectively, block and activate group 1 metabotropic glutamate receptors (mGluRs). Accordingly, hCMEC/D3 expressed both mGluR1 and mGluR5 and the Ca2+ response to glutamate was inhibited by their pharmacological blockade with, respectively, CPCCOEt and MTEP hydrochloride. The Ca2+ response to glutamate was initiated by endogenous Ca2+ release from the endoplasmic reticulum and endolysosomal Ca2+ store through inositol-1,4,5-trisphosphate receptors and two-pore channels, respectively, and sustained by store-operated Ca2+ entry. In addition, glutamate induced robust NO release that was suppressed by pharmacological blockade of the accompanying increase in [Ca2+]i. These data demonstrate for the first time that glutamate may induce metabotropic Ca2+ signals in human brain microvascular endothelial cells. The Ca2+ response to glutamate is likely to support NVC during neuronal activity, thereby reinforcing the emerging role of brain microvascular endothelial cells in the regulation of CBF.

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.

IP3‐evoked Ca2+ release in Endothelial Cells is Modulated by the Immunosuppressant FK506‐binding Protein (FKBP12)

The endoplasmic reticulum (ER) is integral to the regulation of cytoplasmic Ca2+ concentration in endothelial cells. Ca2+ release from the ER is controlled by two intracellular receptor/channel complexes, the inositol 1,4,5‐triphosphate receptor (IP3R) and the ryanodine receptor (RyR). These receptors have been shown to be regulated by the accessory FK506‐binding protein (FKBP). FK506 is a potent immunosuppressant, which is routinely used after organ transplant to lower the risk of rejection, and has also been used in drug‐eluting stents. FK506 has been shown to act on smooth muscle cells either directly by binding to the channel, or indirectly via FKBP modulation of two targets, calcineurin or the mammalian target of rapamycin (mTOR). However very little is known on the role FKBPs play in endothelial cell Ca2+ signaling.To investigate this, Ca2+ signals were measured in the endothelium of second order mesenteric arteries using Cal‐520 (5 μM). IP3Rs were activated by localised photo‐uncaging of IP3 (5 μM), and RyR by the application of Caffeine (10 mM). FK506 (10 μM), which displaces FKBP from each receptor to inhibit calceinurin, increased the IP3‐evoked Ca2+ rise. The phosphatase inhibitors cypermethrin and okadaic acid also increased the IP3‐mediated Ca2+ release. Cypermethrin and okadaic acid did not prevent FK506‐induced increase in Ca2+ release. These results suggest that while calceinurin may modulate IP3‐evoked Ca2+ release, the phosphatase is not necessary for the FK506‐mediated potentiation of IP3‐evoked Ca2+ increase.Rapamycin (10 μM), which disrupts the binding of FKBP12 to the receptor and inhibits mTOR, did not significantly alter IP3‐induced Ca2+ release in endothelial cells, indicating that mTOR does not exert a significant role in endothelial IP3‐induced Ca2+ release.Interestingly Caffeine, the activator of RyR‐mediated Ca2+ release, did not activate Ca2+ signals in the endothelium either in intact tissue or in freshly isolated endothelial patches, suggesting that Caffeine‐activated RyRs do not play a role in endothelial Ca2+ release.These results indicate that FKBPs play an integral role in regulating endothelial IP3‐mediated Ca2+ release, and may underlie the severe endothelial dysfunction associated with FK506 immunosuppressants.Support or Funding InformationWellcome TrustBritish Heart FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium and the modulatory mechanism of D39

Non‐muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor‐α (TNF‐α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose‐dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF‐κB p65 nuclear translocation and I B degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3 and suppressed NF‐κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF‐α‐stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model. Moreover, D39 inhibited tissue factor expression and procoagulant activities in HUVECs and decreased thrombus weight in inferior venacava‐ligated mice dose‐dependently. Serial affinity chromatography and molecular docking analysis suggested that D39 bound to NMMHC IIA. In HEK293T cells, D39 inhibited tissue factor expression evoked by NMMHC IIA overexpression. This effect was blocked by NMMHC IIA knockdown in HUVECs. D39 inhibited dissociation of NMMHC IIA from TNFR2, which subsequently modulated the Akt/GSK3β–NF‐κB signalling pathways. Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders. In addition, we identified a new natural product that targeted NMMHC IIA, as a potential treatment for thrombotic disorders and other vasculopathies.Support or Funding InformationThe present research was supported by the National Natural Science Foundation of China (Grant No. 81274131 and 81601034), 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inositol 1,4,5-Trisphosphate Receptors in Endothelial Cells Play an Essential Role in Vasodilation and Blood Pressure Regulation.

BackgroundEndothelial NO synthase plays a central role in regulating vasodilation and blood pressure. Intracellular Ca2+ mobilization is a critical modulator of endothelial NO synthase function, and increased cytosolic Ca2+ concentration in endothelial cells is able to induce endothelial NO synthase phosphorylation. Ca2+ release mediated by 3 subtypes of inositol 1,4,5‐trisphosphate receptors (IP 3Rs) from the endoplasmic reticulum and subsequent Ca2+ entry after endoplasmic reticulum Ca2+ store depletion has been proposed to be the major pathway to mobilize Ca2+ in endothelial cells. However, the physiological role of IP 3Rs in regulating blood pressure remains largely unclear.Methods and ResultsTo investigate the role of endothelial IP 3Rs in blood pressure regulation, we first generated an inducible endothelial cell–specific IP 3R1 knockout mouse model and found that deletion of IP 3R1 in adult endothelial cells did not affect vasodilation and blood pressure. Considering all 3 subtypes of IP 3Rs are expressed in mouse endothelial cells, we further generated inducible endothelial cell–specific IP 3R triple knockout mice and found that deletion of all 3 IP 3R subtypes decreased plasma NO concentration and increased basal blood pressure. Furthermore, IP 3R deficiency reduced acetylcholine‐induced vasodilation and endothelial NO synthase phosphorylation at Ser1177.ConclusionsOur results reveal that IP 3R‐mediated Ca2+ release in vascular endothelial cells plays an important role in regulating vasodilation and physiological blood pressure.