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This study aimed to examine the influence of the Neuregulin-1 (NRG1)/ERBB4 signaling pathway on the function of human pulmonary artery endothelial cells (HPAECs) and investigate the underlying mechanisms. Enzyme-linked immunosorbent assay indicated that ERBB4 levels in the serum of patients with pulmonary embolism (PE) were significantly higher than those of healthy controls ( p  < 0.05). In cellular studies, thrombin stimulation for 6 h led to a significant decrease in cell viability and overexpression of ERBB4 compared to control ( p  < 0.05). In the NRG1 group, apoptosis of HPAECs was reduced ( p  < 0.05), accompanied by a decrease in ERBB4 expression and an increase in p-ERBB4, phosphorylated serine/threonine kinase proteins (Akt) (p-Akt), and p-phosphoinositide 3-kinase (PI3K) expression ( p  < 0.05). In the AG1478 group, there was a significant increase in HPAEC apoptosis and a significant decrease in p-ERBB4 and ERBB4 expression compared to the Con group ( p  < 0.05). In the AG1478 + NRG1 group, there was an increase in the apoptosis rate and a significant decrease in the expression of p-ERBB4, ERBB4, p-Akt, and phosphorylated PI3K compared to the NRG1 group ( p  < 0.05). In animal studies, the PE group showed an increase in the expression of ERBB4 and p-ERBB4 compared to the Con group ( p  < 0.05). NRG1 treatment led to a significant reduction in embolism severity with decreased ERBB4 expression and increased p-ERBB4 expression ( p  < 0.05). Gene set enrichment analysis identified five pathways that were significantly associated with high ERBB4 expression, including CHOLESTEROL HOMEOSTASIS, OXIDATIVE PHOSPHORYLATION, and FATTY ACID METABOLISM ( p  < 0.05). Therefore, NRG1 inhibits apoptosis of HPAECs, accompanied by a decrease in ERBB4 and an increase in p-ERBB4. NRG1 inhibition in HPAECs apoptosis can be partially reversed by inhibiting ERBB4 expression with AG1478. ERBB4 has the potential to be a novel biological marker of PE., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Pulmonary Circulation published by John Wiley & Sons Ltd on behalf of Pulmonary Vascular Research Institute.)

Hong Xue,1,2 Baosong Xie,1,2 Nengluan Xu,1,2 Hongru Li,1,2 Qianshun Chen,2,3 Weiping Xie,4 Hong Wang4 1Department of Respiratory and Critical Care Medicine, Fujian Provincial Hospital, Fuzhou, Fujian, People’s Republic of China; 2Provincial School of Clinical Medicine, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China; 3Department of Thoracic Surgery, Fujian Provincial Hospital, Fuzhou, Fujian, People’s Republic of China; 4Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital with Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of ChinaCorrespondence: Baosong Xie; Hong XueDepartment of Respiratory and Critical Care Medicine, Fujian Provincial Hospital, No. 134, East Street, Fuzhou, Fujian, 350000, People’s Republic of ChinaTel +86 591-88217531Email xiebaos_baosong@163.com; xuehong_xhx@163.comPurpose: Etanercept (ETN), a tumor necrosis factor-α (TNF-α) inhibitor, has been applied in the treatment of many diseases. However, whether it has effects on chronic obstructive pulmonary disease (COPD) and its interaction with tumor necrosis factor receptor 1 (TNFR1) remained unknown.Methods: Histopathological analysis of lung tissues from non-smokers and smokers with or without COPD was conducted using hematoxylin–eosin (H&E) staining, Van Gieson (VG) staining, and terminal transferase-mediated biotin dUTP nick end labeling (TUNEL). TNF-α content was measured using Immunohistochemistry. Correlation analysis among apoptosis rate, smoke index, the FEV1/FVC ratio, and TNF-α-positive cells was performed. After ETN treatment and transfection of overexpressed or silenced TNFR1, levels of inflammatory cytokines, apoptosis and related genes expressions in cigarette smoke extract (CSE)-treated human pulmonary artery endothelial cells (HPAECs) were detected using enzyme-linked immunosorbent assay (ELISA), Hoechst 33342 staining, flow cytometry, quantitative real-time PCR (qRT-PCR) and Western blot.Results: Pulmonary arterial remodeling and increased apoptotic and TNF-α+ HPAECs were found in lung tissue of smokers with or without COPD, with higher degrees in smokers with COPD. The numbers of apoptotic and TNF-α+ HPAECs were positively correlated with smoke index, while the FEV1/FVC ratio was negatively correlated with apoptotic HPAECs. In HPAECs, ETN downregulated the expressions of proteins related to CSE-induced apoptosis and the TNF receptor family, decreased CSE-induced cell apoptosis and inflammatory cytokine levels, and inhibited TNFR1 expression and p65 phosphorylation. Overexpressed TNFR1 reversed the effects of ETN on CSE-treated HPAECs, whereas silencing TNFR1 did the opposite.Conclusion: ETN protected HPAECs against CSE-induced inflammation and apoptosis via downregulating TNFR1, thus providing a potential therapy for smoking-induced COPD.Keywords: chronic obstructive pulmonary disease, etanercept, apoptosis, cigarette smoke extract, human pulmonary artery endothelial cells, tumor necrosis factor receptor 1

The development and use of nanomaterials, especially of nickel oxide nanoparticles (NiONPs), is expected to provide many benefits but also has raised concerns about the potential human health risks. Inhaled NPs are known to exert deleterious cardiovascular side effects, including pulmonary hypertension. Consequently, patients with pulmonary hypertension (PH) could be at increased risk for morbidity. The objective of this study was to compare the toxic effects of NiONPs on human pulmonary artery endothelial cells (HPAEC) under physiological and pathological conditions. The study was conducted with an in vitro model mimicking the endothelial dysfunction observed in PH. HPAEC were cultured under physiological (static and normoxic) or pathological (20% cycle stretch and hypoxia) conditions and exposed to NiONPs (0.5–5 μg/cm2) for 4 or 24 h. The following endpoints were studied: (i) ROS production using CM-H2DCF-DA and MitoSOX probes, (ii) nitrite production by the Griess reaction, (iii) IL-6 secretion by ELISA, (iv) calcium signaling with a Fluo-4 AM probe, and (v) mitochondrial dysfunction with TMRM and MitoTracker probes. Our results evidenced that under pathological conditions, ROS and nitrite production, IL-6 secretions, calcium signaling, and mitochondria alterations increased compared to physiological conditions. Human exposure to NiONPs may be associated with adverse effects in vulnerable populations with cardiovascular risks. [ABSTRACT FROM AUTHOR]

The development and use of nanomaterials, especially of nickel oxide nanoparticles (NiONPs), is expected to provide many benefits but also has raised concerns about the potential human health risks. Inhaled NPs are known to exert deleterious cardiovascular side effects, including pulmonary hypertension. Consequently, patients with pulmonary hypertension (PH) could be at increased risk for morbidity. The objective of this study was to compare the toxic effects of NiONPs on human pulmonary artery endothelial cells (HPAEC) under physiological and pathological conditions. The study was conducted with an in vitro model mimicking the endothelial dysfunction observed in PH. HPAEC were cultured under physiological (static and normoxic) or pathological (20% cycle stretch and hypoxia) conditions and exposed to NiONPs (0.5–5 μg/cm2) for 4 or 24 h. The following endpoints were studied: (i) ROS production using CM-H2DCF-DA and MitoSOX probes, (ii) nitrite production by the Griess reaction, (iii) IL-6 secretion by ELISA, (iv) calcium signaling with a Fluo-4 AM probe, and (v) mitochondrial dysfunction with TMRM and MitoTracker probes. Our results evidenced that under pathological conditions, ROS and nitrite production, IL-6 secretions, calcium signaling, and mitochondria alterations increased compared to physiological conditions. Human exposure to NiONPs may be associated with adverse effects in vulnerable populations with cardiovascular risks.

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity. This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs). Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-β2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH's pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH. Unlabelled Image • Administration of EPA ameliorated cardiovascular remodeling in PAH rats. • EPA and RvE1 suppressed vasocontraction in rats and human PA. • Fyn activity is a key regulator of STAT3 phosphorylation in HPAECs and HPASMCs. • EPA and RvE1 downregulate SRC family activity in HPAECs and HPASMCs. • EPA and RvE1 suppressed proliferation of IPAH-PASMCs. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension is a progressive heart and lung disease that is caused by irreversible pulmonary vascular remodeling. Sinomenine hydrochloride is an alkaloid that is extracted from sinomenium acutum, which has strong anti-inflammatory effects. In this study, male rats were injected with monocrotaline, and endothelial cells were exposed to hypoxia for 24 hours to induce pulmonary arterial hypertension. Apoptosis, inflammation, and oxidative stress pathways were observed the in lungs and cells. Sinomenine hydrochloride repressed the increased right ventricular systolic pressure and attenuated the right ventricular hypertrophy and pulmonary artery remodeling in model rats. It reversed the expression of BCL2 and BAX and prevented the apoptosis of endothelial cells. Additionally, it increased the contents of IKBα and NRF2. P65, P-P65, TNFα, IL1β, and IL6 levels in the lungs decreased by it. Malondialdehyde contents decreased, and the superoxide dismutase and glutathione peroxidase activity and HO-1 level increased in the treatment group. In vivo, it promoted apoptosis of pulmonary artery endothelial cells. Moreover, by activating PPAR-γ, sinomenine hydrochloride attains the above effects. These data suggested that sinomenine hydrochloride could protect endothelial cells, restrain inflammation and oxidative stress, and enhance pulmonary vascular remodeling.

Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades, PAH remains an incurable disease with high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. The pathological mechanisms leading to this fatal disorder remain mostly undetermined, although structural remodeling of the pulmonary vessels is known to be an early feature of PAH. Pulmonary vascular remodeling includes proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). The use of in vitro approaches is useful to delineate the mechanisms involved in the pathogenesis of PAH and to identify new therapeutic strategies for PAH. In this chapter, we describe protocols for culturing and assessing proliferation and migration of human PASMCs and PAECs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Endothelial dysfunction plays an important role in structural remodeling occurring in the pulmonary vasculature during pulmonary hypertension (PH). Endothelial injury causes apoptosis and activation of endothelial cells. However, some endothelial cells show apoptosis-resistance and later proliferate extensively leading to vascular oculopathy and formation of plexiform lesions in PH. Studies have shown that rapidly proliferating cells exhibit increased expression of Fatty acid synthase (FAS), a regulatory enzyme responsible for the production of fatty acids. Our previous study has shown that FAS inhibition prevented smooth muscle cell proliferation, reversed pulmonary vascular remodeling and improved pulmonary vasoreactivity in monocrotaline induced PH model. However, the role of FAS in pulmonary artery endothelial cell proliferation and angiogenesis has not been explored. The present study was designed to explore the role of FAS in proliferation, metabolic dysfunctions, and angiogenesis in endothelial dysfunction associated with PH. The human pulmonary artery endothelial cells (HPAECs) were exposed to hypoxia and FAS siRNA (60 nM) was used for the FAS inhibition. Increased expression and activity of FAS were observed in hypoxic HPAECs. Inhibition of FAS increased apoptosis and glucose oxidation, but decreased cellular proliferation, markers of autophagy and glycolysis in hypoxic HPAECs. FAS inhibition decreased the angiogenesis as evident by decreased tubule length and VEGF expression in hypoxic HPAECs. Inhibition of FAS also increased expression of endothelial NOS in hypoxic HPAECs, a marker of endothelial function. Our results proved, and further supported previous findings, that inhibition of FAS is beneficial for endothelial function in pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptide ( CPP) intracellular delivery of receptor signaling motifs provides an opportunity to regulate specific receptor tyrosine kinase signal transductions. We targeted tyrosine residues Y740 and Y751 of the PDGF receptor β ( PDGFR β) and Y1175 of the VEGF receptor 2 ( VEGFR2). The Y740 and Y751 motifs activated ERK and Akt, while the Y1175 motif activated ERK. Targeting either Y740 or Y751 of the PDGFR β in human pulmonary artery smooth muscle cells ( HPASMC) effectively inhibited PDGF activation of ERK or Akt. Interfering with the Y751 region of the PDGFR β proved more effective than targeting the Y740 region. The phosphorylation of Y751 of the CPP and the length and exact sequence of the mimicking peptide proved crucial. On the other hand, in human pulmonary artery endothelial cell phosphorylation of the VEGFR2 Y1175 CPP was not a determinant in blockage of ERK activation. Likewise, the length of the peptide mimic was not crucial with a very small sequence containing the Y1175 remaining effective. Physiologic proof of concept for the effectiveness of the CPP was confirmed by blockage of HPASMC migration in response to PDGF following culture injury. Thus targeted blockage of tyrosine kinase receptor signaling can be very effective. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) has a prognostic role in cardiovascular and pulmonary diseases. Recent data suggest its pro-inflammatory effects in atherosclerotic lesion formation. This raises the hypothesis of whether or not CRP has pro-inflammatory effects on pulmonary vasculature by inducing the production of endothelin-1 (ET)-1, a potent vasoconstrictor and proliferative cytokine, and expression of adhesion molecules which could culminate in inflammatory cell recruitment and vascular injury. Human pulmonary artery endothelial cells (HPAECs) were cultured and incubated with 25μg/ml of human recombinant CRP and with interleukin (IL)-1β 10ng/ml, a well-known activator of endothelial cells, which served as a positive control for 24 hours. Expression of vascular cell adhesion molecule (VCAM)-l and intercellular adhesion molecule (ICAM)-l was assessed by flow cytometry. Secretion of ET-1 from HPAECs was also evaluated. In this study we show that incubation of HPAECs with human recombinant CRP for 24 hours induced a significant increase in ICAM-1 expression (from 610 to 6553 mean fluorescence intensity, p < 0.005) and VCAM-1 expression (from 212 to 303 mean fluorescence intensity, p < 0.05), as compared to control. Adhesion molecule induction was similar to that observed in endothelial cells activated with IL-lß. Likewise, CRP potentiated the ET-1 production by HPAECs. The levels of ET-1 were significantly higher at 24 hours (control 19.94±3 vs CRP 46.54±18 pg/ml, p < 0.05). In conclusion, this study makes a novel observation that CRP induces expression of adhesion molecules and secretion of ET-1 in HPAECs. Our study provides the first evidence that CRP exerts direct proinflammatory effects on pulmonary artery endothelial cells. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular adenosine is a physiologically relevant agonist released by various sources, including endothelial cells (EC) and activated platelets, with complex effects mediated via activation of P1 purinergic receptors. Adenosine-induced EC production of glutathione peroxidase1 and nitric oxide is recognized, and an anti-inflammatory mechanism has been described. Effects of extracellular adenosine on the pulmonary EC barrier function and vascular permeability, however, remain poorly characterized. In this study, we demonstrated the adenosine-induced rapid dose-dependent barrier enhancement in human pulmonary artery EC (HPAEC) as measured by an increase in transendothelial electrical resistance (TER). We have shown that HPAEC express only A2A and A2B adenosine receptors. Pharmacological and siRNA depletion studies indicate that A2A, but not A2B receptor activation is required for the adenosine-induced TER increase. Depletion of Gαs with a specific siRNA significantly attenuated the adenosine-induced TER response in HPAEC. In contrast, depletion of either Gαq or Gαi2 did not affect the adenosine-induced TER increase. This suggests that the adenosine-induced TER increase is cAMP-dependent. The adenosine-induced barrier enhancement effects were associated with a rearrangement of the EC F-actin component of the cytoskeleton, enhanced cell-surface presentation of cell–cell junctional protein VE-cadherin and an involvement of Myosin-light-chain phosphatase (MLCP). Our results suggest, for the first time, that adenosine regulates the EC barrier function via A2A receptors followed by Gαs engagement and is associated with cytoskeletal activation. [Copyright &y& Elsevier]

Abstract: Cigarette smoke mediated oxidative stress and endothelial dysfunction are important processes in the pathogenesis of several lung disorders. In this study we evaluated the effect of PDE5 inhibition on pulmonary artery endothelial dysfunction induced by cigarette smoke in vitro. Human pulmonary artery endothelial cells (HPAEC) were incubated in the absence or presence of PDE5 inhibitor sildenafil (10nM–1μM), PKG agonist 8-Br-cGMP (1mM), or the antioxidants dyphenyleneiodonium (DPI 1μM) and N-acetylcysteine (NAC 1mM) for 30min. Then, cigarette smoke extract (CSE) was added for 24h. CSE (2.5–10%)-induced ROS generation was suppressed by DPI, and partially reversed by sildenafil and 8-Br-cGMP. Decreases in intracellular levels of cGMP and extracellular NO induced by CSE were reversed by sildenafil and DPI. Furthermore, CSE-induced pg91 phox and PDE5 mRNA overexpression were suppressed by both sildenafil and DPI. CSE (2.5–10%) induced upregulation of IL-6, IL-8 and Ang-2, and decreased Ang-1 expression in parallel to apoptosis which were partially suppressed by sildenafil, 8-Br-cGMP, DPI and NAC. This study demonstrates that PDE5 inhibition attenuates the oxidant burden and the inflammatory and remodeling effects of CSE in human HPAEC which may contribute to the therapeutic value of PDE5 inhibitors for pulmonary disorders coursing with endothelial dysfunction. [Copyright &y& Elsevier]

T-cadherin (H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in tumor-associated neovascularization and is deregulated in many cancers. Unlike canonical cadherins, it lacks transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. T-cadherin is thought to function in signaling rather than as an adhesion molecule. Some interactive partners of T-cadherin at the plasma membrane have recently been identified. We examined T-cadherin location in human endothelial cells using confocal microscopy and subcellular fractionation. We found that a considerable proportion of T-cadhenn is located in the nucleus and in the centrosomes. T-cadherin colocalized with a centrosomal marker γ-tubulin uniformly throughout the cell cycle at least in human umbilical vein endothelial cells. In the telophase, T-cadherin transiently concentrated in the midbody and was apparently degraded. Its overexpression resulted in an increase in the number of multinuclear cells, whereas its downregulation by small interfering RNA led to an increase in the number of cells with multiple centrosomes. These findings indicate that deregulation of T-cadherin in endothelial cells may lead to disturbances in cytokinesis or centrosomal replication. [ABSTRACT FROM AUTHOR]

The effect of alkaline stress, or an increase in extracellular pH (pHext), on cell viability is poorly defined. Human pulmonary artery endothelial cells (HPAEC) were subjected to alkaline stress using different methods of increasing pHext. Viability and mode of cell death following alkaline stress were determined by assessing nuclear morphology, ultrastructural features, and caspase-3 activity. Incubation of monolayers in media set to different pHext values (7.4–8.4) for 24-h induced morphological changes suggesting apoptosis (35–45% apoptotic cells) following severe alkaline stress. The magnitude of apoptosis was related to the severity of alkaline stress. These findings were confirmed with an assessment of ultrastructural changes and caspase-3 activation. While there was no difference in the intracellular calcium level ([Ca2+] i ) in monolayers set to pHext 7.4 versus 8.4 following the first hour of alkaline stress, blockade of calcium uptake with the chelator, EGTA, potentiated the magnitude of apoptosis under these conditions. Potentiation of apoptosis was reduced by calcium supplementation of the media. Finally, alkaline stress was associated with an increase in intracellular pH. This is the first report of apoptosis following alkaline stress in endothelial cells in the absence of other cell death stimuli. [ABSTRACT FROM AUTHOR]

Objective: To observe the senescent effect of human pulmonary arterial endothelial cells (HPAEC) stimulated by cigarette smoke extract (CSE) and the effect of secretion of senescent cells on human pulmonary arterial smooth muscles cell (HPASMC) proliferation and migration. Methods: HPAEC was treated with different concentrations of CSE in vitro and cell proliferation was determined by CCK8, senescence cells analyzed by detecting the β-gal activity, and the senescent proteins of cells measured by Western blot. The concentration of senescence-associated secretory phenotype (SASP) was detected by ELISA and the expression of MCP-1 and TGF-β1 was measured by Real-time PCR. The number of the proliferated cells was measured by Transwell assay and immunoflurescence. Results: The HPAEC was aging with the stimulation concentration of CSE increasing and the stimulation time prolonging ( P <0.05). Western blot indicated that the senescent associated protein p53 or p21 increased markedly after 48 h and 72 h CSE-exposure ( n =3, P <0.05). The SA-β-Gal staining showed that the number of senescent cells increased as the exposure time prolonged. Compared with the control group, cell viability of 48 h group(1.8±0.1) and 72 h group (1.8±0.1) decreased significantly. The flow cytometry showed a significant difference between the CSE group(14.1±1.2) and the control group(28.5±1.8) in S phase( P <0.01), indicating cell cycle arrest. The SASP was increasing as the CSE-exposure prolonged. Compared with the control group(177±39), the 48 h group(460±43) and the 72 h group(609±64) showed a marked increase in MCP-1( P <0.05). For TGF-β1, it had a same tendency and a significant difference between the control group(121±18) and the 48 h group(413±32) or 72 h group(606±67, both P <0.05). In the meantime, the bFGF increased after 48 h stimulation(291±13, P <0.05). Besides MCP-1, TGF-β1 showed a significant difference between the control group and the 72 h CSE-exposure group ( P <0.01). Premature cells could secrete SASP which induced HPASMC proliferation. After different times of conditioned medium stimulation, HPASMC proliferated especially at 72 h( P <0.05) . The immnoflorescence and Transwell assay confirmed this finding. Conclusion: CSE could induce senescence of HPAEC and SASP production which improved HPASMC proliferation and migration.

Introduction: PrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity. Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts., Methods: The anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1-200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol--a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB)., Results: At higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC50 values compared to other modes of measurement, however, the statistically significant differences in EC50 values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract>resveratrol>walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay., Discussion: In conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided., (© 2013.)