The Effect of Race and Shear Stress on CRP-Induced Responses in Endothelial Cells

Background. C-reactive protein (CRP) is an independent biomarker of systemic inflammation and a predictor of future cardiovascular disease (CVD). More than just a pure bystander, CRP directly interacts with endothelial cells to decrease endothelial nitric oxide synthase (eNOS) expression and bioactivity, decrease nitric oxide (NO) production, and increase the release of vasoconstrictors and adhesion molecules. Race is significantly associated with CRP levels and CVD risks. With aerobic exercise, the vessel wall is exposed to chronic high laminar shear stress (HiLSS) that shifts the endothelium phenotype towards an anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative environment. Thus, the purpose of this study was to assess the racial differences concerning the CRP-induced effects in endothelial cells and the potential role of HiLSS in mitigating these differences. Methods. Human umbilical vein endothelial cells (HUVECs) from four African American (AA) and four Caucasian (CA) donors were cultured and incubated under the following conditions: (1) static control, (2) CRP (10 μg/mL, 24 hours), (3) CRP receptor (FcγRIIB) inhibitor followed by CRP stimulation, (4) HiLSS (20 dyne/cm2, 24 hours), and (5) HiLSS followed by CRP stimulation. Results. AA HUVECs had significantly higher FcγRIIB receptor expression under both basal and CRP incubation conditions. Blocking FcγRIIB receptor significantly attenuated the CRP-induced decrements in eNOS expression only in AA HUVECs. Finally, HiLSS significantly counteracted CRP-induced effects. Conclusion. Understanding potential racial differences in endothelial function is important to improve CVD prevention. Our results shed light on FcγRIIB receptor as a potential contributor to racial differences in endothelial function in AA.

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Modulation of pulmonary endothelial endothelin B receptor expression and signaling: implications for experimental hepatopulmonary syndrome

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00446.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. L1467-L1472 ◽

Cited By ~ 31

Author(s):

Liping Tang ◽

Bao Luo ◽

Rakesh P. Patel ◽

Yiqun Ling ◽

Junlan Zhang ◽

...

Keyword(s):

Nitric Oxide ◽

Shear Stress ◽

Portal Hypertension ◽

Hepatopulmonary Syndrome ◽

Receptor Expression ◽

No Production ◽

Laminar Shear Stress ◽

Akt Activation ◽

Microvascular Endothelial ◽

Endothelin B

The hepatopulmonary syndrome (HPS) results from intrapulmonary vasodilation in the setting of cirrhosis and portal hypertension. In experimental HPS, pulmonary endothelial endothelin B (ETB) receptor overexpression and increased circulating endothelin-1 (ET-1) contribute to vasodilation through enhanced endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. In both experimental cirrhosis and prehepatic portal hypertension, ETB receptor overexpression correlates with increased vascular shear stress, a known modulator of ETB receptor expression. We investigated the mechanisms of pulmonary endothelial ETB receptor-mediated eNOS activation by ET-1 in vitro and in vivo. The effect of shear stress on ETB receptor expression was assessed in rat pulmonary microvascular endothelial cells (RPMVECs). The consequences of ETB receptor overexpression on ET-1-dependent ETB receptor-mediated eNOS activation were evaluated in RPMVECs and in prehepatic portal hypertensive animals exposed to exogenous ET-1. Laminar shear stress increased ETB receptor expression in RPMVECs without altering mRNA stability. Both shear-mediated and targeted overexpression of the ETB receptor enhanced ET-1-mediated ETB receptor-dependent eNOS activation in RPMVECs through Ca2+-mediated signaling pathways and independent of Akt activation. In prehepatic portal hypertensive animals relative to control, ET-1 administration also activated eNOS independent of Akt activation and triggered HPS. These findings support that increased pulmonary microvascular endothelial ETB receptor expression modulates ET-1-mediated eNOS activation, independent of Akt, and contributes to the development of HPS.

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Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells

Pharmaceuticals ◽

10.3390/ph14060577 ◽

2021 ◽

Vol 14 (6) ◽

pp. 577

Author(s):

Chin-Feng Hsuan ◽

Thung-Lip Lee ◽

Wei-Kung Tseng ◽

Chau-Chung Wu ◽

Chi-Chang Chang ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Western Blot ◽

Umbilical Vein ◽

No Production ◽

Enos Expression ◽

Human Umbilical Vein ◽

Whole Plant ◽

Umbilical Vein Endothelial Cells ◽

Glossogyne Tenuifolia

The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

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eNOS activation and NO function: Pregnancy adaptive programming of capacitative entry responses alters nitric oxide (NO) output in vascular endothelium–new insights into eNOS regulation through adaptive cell signaling

Journal of Endocrinology ◽

10.1530/joe-11-0053 ◽

2011 ◽

Vol 210 (3) ◽

pp. 243-258 ◽

Cited By ~ 27

Author(s):

D S Boeldt ◽

F X Yi ◽

I M Bird

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Gap Junction ◽

Uterine Artery ◽

Umbilical Vein ◽

Normal Pregnancy ◽

Human Hand ◽

No Production ◽

Trpc Channels ◽

Enos Expression

In pregnancy, vascular nitric oxide (NO) production is increased in the systemic and more so in the uterine vasculature, thereby supporting maximal perfusion of the uterus. This high level of functionality is matched in the umbilical vein, and in corresponding disease states such as pre-eclampsia, reduced vascular responses are seen in both uterine artery and umbilical vein. In any endothelial cell, NO actually produced by endothelial NO synthase (eNOS) is determined by the maximum capacity of the cell (eNOS expression levels), eNOS phosphorylation state, and the intracellular [Ca2+]iconcentration in response to circulating hormones or physical forces. Herein, we discuss how pregnancy-specific reprogramming of NO output is determined as much by pregnancy adaptation of [Ca2+]isignaling responses as it is by eNOS expression and phosphorylation. By examining the changes in [Ca2+]isignaling responses from human hand vein endothelial cells, uterine artery endothelial cells, and human umbilical vein endothelial cells in (where appropriate) nonpregnant, normal pregnant, and pathological pregnant (pre-eclamptic) state, it is clear that pregnancy adaptation of NO output occurs at the level of sustained phase ‘capacitative entry’ [Ca2+]iresponse, and the adapted response is lacking in pre-eclamptic pregnancies. Moreover, gap junction function is an essential permissive regulator of the capacitative response and impairment of NO output results from any inhibitor of gap junction function, or capacitative entry using TRPC channels. Identifying these [Ca2+]isignaling mechanisms underlying normal pregnancy adaptation of NO output not only provides novel targets for future treatment of diseases of pregnancy but may also apply to other common forms of hypertension.

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Cilostazol Induces eNOS and TM Expression via Activation with Sirtuin 1/Krüppel-like Factor 2 Pathway in Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms221910287 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10287

Author(s):

Chih-Hsien Wu ◽

Yi-Lin Chiu ◽

Chung-Yueh Hsieh ◽

Guo-Shiang Tsung ◽

Lian-Shan Wu ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Nitric Oxide Synthase ◽

Umbilical Vein ◽

Sirtuin 1 ◽

No Production ◽

Beneficial Effects ◽

Umbilical Vein Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

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Primary cilia of human endothelial cells disassemble under laminar shear stress

The Journal of Cell Biology ◽

10.1083/jcb.200312133 ◽

2004 ◽

Vol 164 (6) ◽

pp. 811-817 ◽

Cited By ~ 145

Author(s):

Carlo Iomini ◽

Karla Tejada ◽

Wenjun Mo ◽

Heikki Vaananen ◽

Gianni Piperno

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Primary Cilia ◽

Umbilical Vein ◽

Intraflagellar Transport ◽

Mechanical Stimuli ◽

Laminar Shear Stress ◽

Human Endothelial Cells ◽

Umbilical Vein Endothelial Cells ◽

Laminar Shear

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-α-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.

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Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

Circulation Research ◽

10.1161/circresaha.121.318982 ◽

2021 ◽

Author(s):

Sarah Basehore ◽

Samantha Bohlman ◽

Callie Weber ◽

Swathi Swaminathan ◽

Yuji Zhang ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Shear Stress ◽

Laminar Flow ◽

No Production ◽

Disturbed Flow ◽

Enos Activity ◽

Enos Phosphorylation ◽

Steady Laminar ◽

In Cells

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

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Abstract 1432: Nitric Oxide Bioavailability is Lower in Endothelium of Healthy Mexican American Donors as Compared to Non-Hispanic White Donors: Effect of Nebivolol

Circulation ◽

10.1161/circ.116.suppl_16.ii_295 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

R. P Mason ◽

Ruslan Kubant ◽

Christopher Malinski ◽

Adam Jacoby ◽

Robert F Jacob ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Function ◽

Mexican Americans ◽

Mexican American ◽

Umbilical Vein ◽

Coupling Efficiency ◽

Calcium Ionophore ◽

Stress Factors ◽

No Bioavailability

Background: Epidemiologic studies indicate that Mexican Americans (MA) have a higher prevalence of CV risk factors and disease as compared with non-Hispanic whites (NHW). This increase in CV risk may be due, in part, to differences in endothelial function. In this study, we measured endothelial function in cells from normotensive, age-matched MA and NHW donors, as well as the effects of treatment with nebivolol, a new β 1 -selective blocker with vasodilating properties. Methods: Endothelial nitric oxide (NO) and peroxynitrite (ONOO − ) release in human umbilical vein endothelial cells (HUVECs) from age-matched MA and NHW donors were measured simultaneously using a nanosensor array. The effects of nebivolol on NO and ONOO − release were evaluated following pretreatment (24 h) with a calcium ionophore (CaI) as a receptor-independent stimulus. Endothelial NO synthase (eNOS) levels were measured by Western blot analysis, and drug-membrane interactions were determined by small angle x-ray diffraction approaches. Results: NO bioavailability in endothelial cells of MA donors was 30% lower than that of cells from NHW donors (383 ± 10 nM versus 543 ± 8 nM, n=6) following stimulation with CaI (1.0 μM). Pretreatment with nebivolol (1.0 μM) eliminated these interracial differences and enhanced NO release disproportionately in MA cells (57%) versus NHW cells (20%). Nebivolol also reduced ONOO − levels in MA endothelium by 75% (746 ± 12 nM to 195 ± 10 nM) and by 50% in NHW cells (416 ± 7 nM to 191 ± 13 nM). The ratio of NO to ONOO − , an indicator of eNOS coupling, increased more than 5-fold in MA cells following nebivolol treatment. In addition, eNOS levels were 40% lower in MA endothelium compared to NHW, but increased 2-fold with nebivolol treatment. These effects were not observed with atenolol, a hydrophilic β 1 -selective antagonist. Conclusion: We observed differences between Mexican Americans and non-Hispanic whites in endothelial NO bioavailability and nitroxidative stress–factors that may contribute to increased CV risk. Treatment with nebivolol, but not atenolol, enhanced both the expression and coupling efficiency of eNOS in Mexican American endothelium.

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Shear stress enhances human endothelial cell wound closure in vitro

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.1.h293 ◽

2000 ◽

Vol 279 (1) ◽

pp. H293-H302 ◽

Cited By ~ 77

Author(s):

Maria Luiza C. Albuquerque ◽

Christopher M. Waters ◽

Ushma Savla ◽

H. William Schnaper ◽

Annette S. Flozak

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Endothelial Cell ◽

Wound Closure ◽

Type I Collagen ◽

Umbilical Vein ◽

Type I ◽

Laminar Shear Stress ◽

Human Coronary Artery ◽

Closure Rate

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm2 to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm2 under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm2 closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.

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Salidroside Stimulates Mitochondrial Biogenesis and Protects against H2O2-Induced Endothelial Dysfunction

Oxidative Medicine and Cellular Longevity ◽

10.1155/2014/904834 ◽

2014 ◽

Vol 2014 ◽

pp. 1-13 ◽

Cited By ~ 38

Author(s):

Shasha Xing ◽

Xiaoyan Yang ◽

Wenjing Li ◽

Fang Bian ◽

Dan Wu ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Transcription Factor ◽

Endothelial Dysfunction ◽

Mitochondrial Biogenesis ◽

Umbilical Vein ◽

Adenosine Monophosphate ◽

No Production ◽

Peroxisome Proliferator ◽

Atp Production

Salidroside (SAL) is an active component ofRhodiola roseawith documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙-) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways.

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Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase

Biochemistry and Cell Biology ◽

10.1139/o07-146 ◽

2008 ◽

Vol 86 (1) ◽

pp. 1-10 ◽

Cited By ~ 34

Author(s):

Syamantak Majumder ◽

Ajit Muley ◽

Gopi Krishna Kolluru ◽

Samir Saurabh ◽

K. P. Tamilarasan ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Function ◽

Egg Yolk ◽

Cellular Migration ◽

No Production ◽

Enos Phosphorylation ◽

Low Levels ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.

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