Stimulation Of Vascular Endothelial Cells Research Articles

PEGylated graphene oxide-mediated stimulation of vascular endothelial cells and responsive release of PD-1/PD-L1 inhibitor for efficient chemo-immunotherapy against cancer

Immune checkpoint blockade (ICB) has emerged as a promising immunotherapeutic modality against cancer in the clinic. However, only 10–30% of patients respond to ICB, primarily due to poor immunogenicity and insufficient T cell infiltration in solid tumors. Herein, we presented an approach for high-performance cancer treatment using the programmed cell death protein-1 and programmed cell death ligand-1 (PD-1/PD-L1) inhibitor (BMS-202)-loaded PEGylated graphene oxide (GPi). On the one hand, GPi dissociated tight junctions of vascular endothelial cells (VECs) in tumor, thus promoting the extravasation and intratumoral accumulation of liposomal doxorubicin (LipDox), which then effectively induced immunogenic cell death of tumor cells. On the other hand, GPi also stimulated VECs to upregulate the expression of cell-cell interaction molecules, such as intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1, which facilitated the infiltration of T cells in tumor. Beyond acting as a stimulator of VECs, GPi could exert responsive release of BMS-202 under the acidic tumor microenvironment and blockade PD-1/PD-L1 axis in tumors. Finally, the alternating administration of GPi and LipDox effectively inhibited tumor growth in a 4T1 tumor model, providing a novel treatment mode for chemo-immunotherapy.

Nanomaterial's interfacial stimulation of vascular endothelial cells and divergent guidances for nanomedicine treating vasculature-associated diseases

Blood vasculature plays an essential role in transporting nanomaterials in vivo. However, the interaction between vascular endothelial cells (VECs) and nanomaterials lacked deep understanding, impeding the development of safe and efficient nanomedicine. Here, we discover that VECs can be significantly stimulated by two-dimensional PEGylated lamellar materials (PLMs) without being internalized. Additional experiments and computational simulations suggest that the unique VEC stimulation can be ascribed to the interaction between the PLMs and the membrane lipids, which induces ion-transport-related conformational change of L-type calcium ion channel Cav1.3. Additionally, the in vivo studies demonstrate that PLMs-stimulated VECs have divergent impacts on two types of vasculature-associated diseases: exacerbating the development of atherosclerosis while magnifying the accumulation of nanomedicine within tumors. Thus, the current study not only advances our understanding of how specific proteins on VEC membranes could be stimulated by PLMs but also guides the rational use of promising PLMs in nanomedicine.

Arbitrary Ca2+ regulation for endothelial nitric oxide, NFAT and NF-κB activities by an optogenetic approach.

Modern western dietary habits and low physical activity cause metabolic abnormalities and abnormally elevated levels of metabolites such as low-density lipoprotein, which can lead to immune cell activation, and inflammatory reactions, and atherosclerosis. Appropriate stimulation of vascular endothelial cells can confer protective responses against inflammatory reactions and atherosclerotic conditions. This study aims to determine whether a designed optogenetic approach is capable of affecting functional changes in vascular endothelial cells and to evaluate its potential for therapeutic regulation of vascular inflammatory responses in vitro. We employed a genetically engineered, blue light-activated Ca2+ channel switch molecule that utilizes an endogenous store-operated calcium entry system and induces intracellular Ca2+ influx through blue light irradiation and observed an increase in intracellular Ca2+ in vascular endothelial cells. Ca2+-dependent activation of the nuclear factor of activated T cells and nitric oxide production were also detected. Microarray analysis of Ca2+-induced changes in vascular endothelial cells explored several genes involved in cellular contractility and inflammatory responses. Indeed, there was an increase in the gene expression of molecules related to anti-inflammatory and vasorelaxant effects. Thus, a combination of human blue light-activated Ca2+ channel switch 2 (hBACCS2) and blue light possibly attenuates TNFα-induced inflammatory NF-κB activity. We propose that extrinsic cellular Ca2+ regulation could be a novel approach against vascular inflammation.

Gastric Cancer Cell-Derived Exosomal GRP78 Enhances Angiogenesis upon Stimulation of Vascular Endothelial Cells.

Exosomes containing glucose-regulated protein 78 (GRP78) are involved in cancer malignancy. GRP78 is thought to promote the tumor microenvironment, leading to angiogenesis. No direct evidence for this role has been reported, however, mainly because of difficulties in accurately measuring the GRP78 concentration in the exosomes. Recently, exosomal GRP78 concentrations were successfully measured using an ultrasensitive ELISA. In the present study, GRP78 concentrations in exosomes collected from gastric cancer AGS cells with overexpression of GRP78 (OE), knockdown of GRP78 (KD), or mock GRP78 (mock) were quantified. These three types of exosomes were then incubated with vascular endothelial cells to examine their effects on endothelial cell angiogenesis. Based on the results of a tube formation assay, GRP78-OE exosomes accelerated angiogenesis compared with GRP78-KD or GRP78-mock exosomes. To investigate the mechanisms underlying this effect, we examined the Ser473 phosphorylation state ratio of AKT, which is involved in the angiogenesis process, and found that AKT phosphorylation was increased by GRP78-OE exosome application to the endothelial cells. An MTT assay showed that GRP78-OE exosome treatment increased the proliferation rate of endothelial cells, and a wound healing assay showed that this treatment increased the migration capacity of the endothelial cells. These findings demonstrated that GRP78-containing exosomes promote the tumor microenvironment and induce angiogenesis.

Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma.

Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within the tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cell themselves, thus making it a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and holds tremendous promise for cancer treatment. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of invivo tumor-targeted delivery systems. Here, we screened an OS cell-specific aptamer (LC09) and developed a LC09-functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. Our results demonstrated that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity. The delivery system simultaneously restrained autocrine and paracrine VEGFA signaling in tumor cells and could facilitate translating CRISPR-Cas9 into clinical cancer treatment.

Salt-induced Na+/K+-ATPase-α/β expression involves soluble adenylyl cyclase in endothelial cells.

High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca2+- and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na+/K+-ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150mM Na+)-induced Na+/K+-ATPase-α and Na+/K+-ATPase-β protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na+/K+-ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na+/K+-ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na+/K+-ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na+/K+-ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na+/K+-ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.

Tissue Specificity: SOCE: Implications for Ca2+ Handling in Endothelial Cells.

Many cellular functions of the vascular endothelium are regulated by fine-tuned global and local, microdomain-confined changes of cytosolic free Ca2+ ([Ca2+]i). Vasoactive agonist-induced stimulation of vascular endothelial cells (VECs) typically induces Ca2+ release through IP3 receptor Ca2+ release channels embedded in the membrane of the endoplasmic reticulum (ER) Ca2+ store, followed by Ca2+ entry from the extracellular space elicited by Ca2+ store depletion and referred to as capacitative or store-operated Ca2+ entry (SOCE). In vascular endothelial cells, SOCE is graded with the degree of store depletion and controlled locally in the subcellular microdomain where depletion occurs. SOCE provides distinct Ca2+ signals that selectively control specific endothelial functions: in calf pulmonary artery endothelial cells, the SOCE Ca2+ signal drives nitric oxide (an endothelium-derived relaxing factor of the vascular smooth muscle) production and controls activation and nuclear translocation of the transcription factor NFAT. Both cellular events are not affected by Ca2+ signals of comparable magnitude arising directly from Ca2+ release from intracellular stores, clearly indicating that SOCE regulates specific Ca2+-dependent cellular tasks by a unique and exclusive mechanism. This review discusses the mechanisms of intracellular Ca2+ regulation in vascular endothelial cells and the role of store-operated Ca2+ entry for endothelium-dependent smooth muscle relaxation and nitric oxide signaling, endothelial oxidative stress response, and excitation-transcription coupling in the vascular endothelium.

Pregnancy-Induced Shifts in Von Willebrand Factor (VWF) Parameters Support a Prolongation in VWF Survival

Abstract 33Levels of the hemostatic protein von Willebrand Factor (VWF) are known to increase markedly during pregnancy. The mechanism by which VWF levels are increased has long been thought to be due to estrogen stimulation of vascular endothelial cells to synthesize and release more VWF. We hypothesized an alternate scenario in which prolongation of VWF survival contributes to the overall increase in VWF levels during pregnancy.To test this hypothesis, we measured VWF parameters in a repository of plasma samples acquired longitudinally over healthy pregnancies. Third trimester and postpartum (≥ 6 weeks) samples were available for 45 pregnancies, while prepregnancy, first, and second trimester samples were also available on a subset of pregnancies. VWF propeptide (VWFpp) and VWF antigen (VWF:Ag) levels were determined by ELISA (GTI Diagnostics), Factor VIII:C (FVIII:C) was performed by a one-stage assay, and ADAMTS13 activity was measured via cleavage of an enzyme-linked peptide substrate. ABO blood group, a known modifier of VWF:Ag levels and VWF survival, was determined by ABO allele-specific PCR (GTI Diagnostics).We found VWFpp, VWF:Ag, and FVIII:C levels were all significantly increased in the third trimester, as expected, while ADAMTS13 activity remained unchanged (see Table 1). Furthermore, the VWFpp:Ag ratio dropped by nearly half, consistent with a prolongation in VWF survival, while the VWFpp also rose, supporting a coexistent increase in VWF production. Unexpectedly, the decrease in the VWFpp:Ag ratio could be detected early during pregnancy, before the second trimester. Also surprisingly, we observed the VWF:Ag/FVIII:C ratio increase significantly during pregnancy. In a subset analysis of the data categorized by ABO blood group (group O, A, or B), the observed differences in VWF parameters between the third trimester and baseline remained statistically significant for all blood types.Table 1VWF parameters (+/− 1SD) in 45 healthy pregnancies.3rd TrimesterPostpartump-valueVWF:Ag (U/dL)192 +/− 7282 +/− 308.3 × 10−15VWFpp (U/dL)160 +/− 42132 +/− 310.00015FVIII:C (% Activity)200% +/− 63%114% +/− 38%6.9 × 10−11ADAMTS13 (% Activity)119 +/− 30122 +/− 330.599VWFpp/Ag Ratio0.91 +/− 0.271.75 +/− 0.631.4 × 10−13VWF/FVIII Ratio1.03 +/− 0.450.77 +/− 0.270.00054p-values determined using Student's two-tailed, paired t-testCollectively, these data point to previously unsuspected and biologically relevant qualitative changes in VWF during pregnancy. In a model accounting for the volume expansion of pregnancy and evidence of increased VWF production (and assuming one compartment, single-order kinetics, and a stable VWFpp half-life), we calculated that an increase in the half-life of VWF (1.9 fold) would account for the observed excess in VWF:Ag in the third trimester. Furthermore, the change in the VWF/FVIII ratio suggests a pregnancy-induced alteration in the VWF:FVIII stoichiometry such that pregnancy-associated VWF carries less FVIII. This new model of pregnancy-induced VWF change has direct relevance to the prothrombotic state of pregnancy, and could relate to the increased risk of venous thromboembolism (VTE), the major cause of maternal mortality in the developed world. This fresh perspective on the shifts in VWF and FVIII levels which occur in healthy pregnancy may also lend new insight into the mechanisms underlying the derangements of VWF which accompany other life-threatening pregnancy-associated complications, such as preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet syndrome). Disclosures:No relevant conflicts of interest to declare.

Regulation of arterial blood pressure by Akt1-dependent vascular relaxation

Endothelial cell-dependent vascular relaxation plays an important role in the regulation of blood pressure. Here, we show that stimulation of vascular endothelial cells with platelet-derived growth factor (PDGF) results in vascular relaxation through Akt1-dependent activation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Stimulation of both human umbilical artery endothelial cells and abdominal aortic vessels with PDGF induced NO production. PDGF-dependent production of NO was completely abolished by inhibition of phosphatidylinositol 3-kinase with wortmannin (100nM). Stimulation of aortic vessels with PDGF resulted in the activation of Akt phosphorylation and eNOS phosphorylation: however, eNOS phosphorylation and production of NO were abolished in aortic vessels of mice lacking Akt1. PDGF strongly induced vascular relaxation in the presence of endothelium, and inhibition of NO production by N-nitro-L: -arginine-methyl ester completely blocked PDGF-dependent vascular relaxation. In addition, PDGF-dependent relaxation was completely abolished by inhibition of PI3K with wortmannin (100nM). Furthermore, vessels from Akt1 heterozygotes showed normal relaxation after PDGF stimulation, whereas vessels from Akt1 knockout littermates did not respond to PDGF stimulation. Finally, administration of PDGF (5ng/ml) significantly lowered blood pressure in Akt1 heterozygotes, whereas a blood pressure-lowering effect was not observed in Akt1 knockout littermates. These results suggest that Akt1 regulates blood pressure through regulation of vascular relaxation by eNOS phosphorylation and subsequent production of NO.

Metalloproteinase‐dependent cleavage of neuregulin and autocrine stimulation of vascular endothelial cells

Inflammation is often accompanied by robust angiogenesis. Vascular endothelial cells (ECs) express erbB receptors and their ligand, neuregulin-1, and can respond to neuregulin by proliferation and angiogenesis. We hypothesized that some growth factor-like responses of ECs to inflammatory cytokines can be explained by cleavage of transmembrane neuregulin with subsequent release of its extracellular epidermal growth factor-like-containing domain and autocrine activation. Using a model of cultured human ECs, we found that interleukin-6 or interferon-gamma causes rapid cleavage and release of transmembrane neuregulin. Inhibitors of metalloproteinases abolish this effect. The addition of an inhibitor of tumor necrosis factor-alpha converting enzyme (TACE) blocks cytokine-induced neuregulin release. Silencing of TACE expression increases the amount of basal proneuregulin present in ECs but does not block neuregulin release in response to phorbol myristate acetate (PMA), suggesting that other proteinases are responsible for mediating protein kinase C-dependent cleavage. Cytokines capable of inducing neuregulin cleavage stimulated ERK activation and in vitro angiogenesis (Matrigel cord formation). This effect is blocked by inhibitors that block neuregulin cleavage, erbB protein tyrosine kinase inhibitors, or antineuregulin-neutralizing antibodies. Cytokine-activated metalloproteinase cleavage of neuregulin may play an important role in autocrine activation of EC signaling pathways, contributing to key biological effects, perhaps including inflammation-associated angiogenesis.

Role of arachidonic acid lipoxygenase metabolites in the regulation of vascular tone

Stimulation of vascular endothelial cells with agonists such as acetylcholine (ACh) or bradykinin or with shear stress activates phospholipases and releases arachidonic acid (AA). AA is metabolized by cyclooxygenases, cytochrome P-450s, and lipoxygenases (LOs) to vasoactive products. In some arteries, a substantial component of the vasodilator response is dependent on LO metabolites of AA. Nitric oxide (NO)- and prostaglandin (PG)-independent vasodilatory responses to ACh and AA are reduced by inhibitors of LO and by antisense oligonucleotides specifically against 15-LO-1. Vasoactive 15-LO metabolites derived from the vascular endothelium include 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-HEETA) that is hydrolyzed by soluble epoxide hydrolase to 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). HEETA and THETA are endothelium-derived hyperpolarizing factors that induce vascular relaxations by activation of smooth muscle apamin-sensitive, calcium-activated, small-conductance K(+) channels causing hyperpolarization. In other arteries, the 12-LO metabolite 12-hydroxyeicosatetraenoic acid is synthesized by the vascular endothelium and relaxes smooth muscle by large-conductance, calcium-activated K(+) channel activation. Thus formation of vasodilator eicosanoids derived from LO pathways contributes to the regulation of vascular tone, local blood flow, and blood pressure.

Expression of hyaluronan synthase in intraocular proliferative diseases: regulation of expression in human vascular endothelial cells by transforming growth factor-β

Purpose To investigate the role of hyaluronan (HA) and elucidate the mechanisms that regulate the expression of hyaluronan-synthesizing enzymes in vascular endothelial cells (VECs) in intraocular proliferative diseases. Methods Cultured VECs were used. Hyaluronan synthase (HAS) expression was determined on the mRNA products obtained by reverse transcription polymerase chain reaction (RT-PCR). The effect of transforming growth factor-β 1(TGF-β 1) and/or platelet-derived growth factor-BB (PDGF-BB) on HAS expression was examined by quantitative RT-PCR and Western blot analysis. HAS expression in intraocular proliferative membranes was observed by immunohistochemistry. Results Cultured VECs expressed the three HAS isoforms. Stimulation of VECs with TGF-β 1 induced a marked increase in the expression level of HAS2 mRNA and protein. The stimulatory effect of PDGF-BB was less potent. A synergistic or additive effect between TGF-β 1 and PDGF-BB-induced HA synthesis was not observed. Furthermore, HAS1 and HAS2 exhibited differential expression in VECs and non-VECs populating intraocular proliferative membranes. Conclusions The expression of each HAS isoform is regulated differently by growth factors and cytokines in VECs. Importantly, HA-synthesizing enzymes were expressed in cells populating proliferative membranes obtained from eyes of patients with proliferative vitreoretinal diseases, and thus may be key molecules in the events that control progression of the proliferative diseases.

Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells

BackgroundThe cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) – dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors.ResultsIn this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs), and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1.ConclusionsThus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.

Substrate specificity of the agonist-stimulated release of polyunsaturated fatty acids from vascular endothelial cells

Stimulation of vascular endothelial cells with agonists such as histamine and thrombin results in release of arachidonic acid from membrane lipids and subsequent eicosanoid synthesis. As shown previously, the agonist-stimulated deacylation is specific for arachidonate, eicosapentaenoate, and 5,8,11-eicosatrienoate. This study has utilized radiolabeled fatty acids differing in chain length and position of double bonds to further elucidate the fatty acyl specificity of agonist-stimulated deacylation. Replicate wells of confluent human umbilical vein endothelial cells were incubated with 14C-labeled fatty acids and then challenged with histamine, thrombin, or the calcium ionophore A23187. Comparison of the results obtained with isomeric eicosatetraenoic fatty acids with initial double bonds at carbons 4, 5, or 6 indicated that the deacylation induced by all three agonists exhibited marked specificity for the cis-5 double bond. Lack of stringent chain length specificity was indicated by agonist-stimulated release of 5,8,11,14-tetraenoic fatty acids with 18, 19, 20, and 21 carbons. Release of 5,8,14-[ 14C]eicosatrienoate was two-to threefold that of 5,11,14-[ 14C]eicosatrienoate, thus indicating that the cis-8 double bond may also contribute to the stringent recognition by the agonist-sensitive phospholipase. The present study has also demonstrated that histamine, thrombin, and A23187 do not stimulate release of docosahexaenoate from endothelial cells.

Effect of fibrinogen-clotting enzymes on secretion of plasminogen activators from cultured human endothelial cells

Fibrinogen-clotting enzymes purified from snake venoms, ancrod, batroxobin and crotalase induce a fibrinolytic state in the circulation of humans and animals. In vivo, but not in vitro, the enzymes indirectly convert fibrinogen into plasmin-derived degradation products. To explain this effect, we investigated the secretion of plasminogen activators by cultured human umbilical vein endothelial cells in response to purified snake venom enzymes. The activity of plasminogen activators was measured by a solid-phase 125I-fibrin lysis assay and the type demonstrated by fibrin zymography. Unstimulated primary or passaged cultures secreted activators at a mean rate of 0.183 or 46.6 milliunits/10 6 cells, respectively. Ancrod, batroxobin and crotalase did not affect biosynthesis and secretion of tissue plasminogen activator and did not induce pro-urokinase from primary cell cultures after 4 h. However, in passaged cells treated for 4 h, ancrod and batroxobin stimulated the secretion of plasminogen activators in a dose-dependent manner. Maximum stimulation by the two enzymes at 3 and 2 u/ml, respectively, was twofold higher than control values. In contrast, crotalase (1 u/ml), and thrombin (1 u/ml) decreased activator secretion to 32 and 11% of control values, respectively. The use of specific antibodies showed that the secreted plasminogen activators were inhibited by 90% using anti-urokinase antibodies and by 10% with anti-tissue plasminogen activator antibodies. Fibrin zymography supported the data and demonstrated that fibrinogen-clotting enzymes affected both extracellular and intracellular pro-urokinase levels. Intracellular plasminogen activators increased in cells treated with crotalase to 130% but decreased with ancrod to 40%, batroxobin to 80% and thrombin to 23%. The data indicated that the effects of the four enzymes on the secretion of plasminogen activators from passaged endothelial cells were independent of their action on fibrinogen. It is concluded that ancrod or batroxobin may activate the fibrinolytic system in vivo by stimulation of vascular endothelial cells to selectively increase pro-urokinase secretion without alteration of tissue plasminogen activator output.