Exposure Of Human Endothelial Cells Research Articles

Transcriptome-centric approach to the derivation of adverse outcome pathway networks of vascular dysfunction after long-term low-level exposure of human endothelial cells to dibutyl phthalate

Dibutyl phthalate (DBP) is an endocrine disruptor that adversely affects reproduction; however, evidence suggests it can also impact other systems, including vascular function. The mechanisms underlying DBP-induced vascular dysfunction, particularly after long-term low-level exposure of endothelial cells to this phthalate, remain largely unknown. To address this gap, we used experimentally derived data on differentially expressed genes (DEGs) obtained after 12 weeks of exposure of human vascular endothelial cells EA.hy926 to the concentrations of DBP to which humans are routinely exposed (10−9 M, 10−8 M, and 10−7 M) and various computational tools and manual data curation to build the first adverse outcome pathway (AOP) network relevant to DBP-induced vascular toxicity. DEGs were used to infer transcription factors (molecular initiating events) and molecular functions and biological processes (key events, KEs) using the Enrichr database. The AOP-helpFinder 2.0, an artificial intelligence-based web tool, was used to link genes and KEs and assign confidence scores to co-occurred terms. We constructed the AOP networks using Cytoscape and then manually arranged KEs to depict the flow of mechanistic information across different levels of network organization. An AOP network was created for each DBP concentration, revealing several distinct high-confidence subnetworks that could be involved in DBP-induced vascular toxicity: the insulin-like growth factor subnetwork for 10−7 M DBP, the CXCL8-dependent chemokine subnetwork for 10−8 M DBP, and the fatty acid subnetwork for 10−9 M DBP. We also developed an AOP network providing a mechanistic insight into the dose-dependent effects of DBP in endothelial cells leading to vascular dysfunction. In summary, we present novel putative AOP networks describing the mechanistic flow of information involved in DBP-induced vascular dysfunction in a long-term low-level exposure scenario.

Targeting the High-Density Lipoprotein Proteome for the Treatment of Post-Acute Sequelae of SARS-CoV-2.

Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.

Role of adropin in reducing arterial stiffness in type 2 diabetes

Adropin is a peptide primarily expressed and secreted by the liver and is known to regulate energy homeostasis. Increasing evidence also indicates that adropin can exert vascular effects and its low circulating levels are associated with increased arterial stiffness, obesity, and type 2 diabetes. However, whether reduced adropin contributes to arterial stiffening in type 2 diabetes remains unknown. Herein, we tested the hypothesis that loss of adropin in non‐diabetic mice causes arterial stiffening and that the converse is also true. That is, adropin exposure reduces arterial stiffness in diabetic mice. In alignment with this hypothesis, we found that 1) mesenteric arteries from adropin knockout male mice are stiffer than those from wild‐type littermates; 2) exposure of human endothelial cells and mesenteric arteries from diabetic (i.e., db/db) male mice to adropin reduces actin polymerization and stiffness; 3) adropin‐induced reduction of actin polymerization and softening of endothelial cells and diabetic arteries is abrogated by inhibition of nitric oxide synthase; and 4) treatment of diabetic male mice with adropin for four weeks reduces arterial stiffness in mesenteric arteries. Collectively, these findings support the notion that reduced adropin may be implicated in arterial stiffening and thus represent a novel therapeutic target to lessen arterial stiffness in type 2 diabetes.

Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension.

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222-OR: High Glucose Level Increases Expression of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptors

Introduction: Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by a novel coronavirus, SARS-CoV-2. The SARS-CoV-2 virus infects the host human cells through its surface receptors human angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2). This study aims to investigate the effects of high glucose on the expression of ACE2 and TMPRSS2 receptors using macrophage (CD14+ve cells) as a biomarker in vivo and human endothelial cells in vitro. Methods: Mononuclear cells were isolated by Ficoll density centrifuge method from whole blood. CD14 cells were isolated from MNCs by MACS kit. The migratory capacity of CD14 cells was evaluated using the CytoSelect Migration Assay kit. CFU-Hill colony formation assay was also done on MNCs. In vitro studies using HUVEC were done by culturing the cells in normal glucose (NG, 5.5mmol/L) or high glucose (HG, 20 mmol/L), followed by mRNA assays at day 3 and 7. Results: All the COVID patients had high glucose levels (&amp;gt;300 mg/dl). The migratory function of CD14 cells show moderately increased migration compared to similar hematopoietic cells from nondiabetic COVID negative patients. In general, CD14 cells showed low expression of ACE2 and/or TMPRSS2 in all samples. For our in-vitro experiments using cultured HUVEC mRNA expression of ACE2 and/or TMPRSS2 receptors were higher in HG compared to NG at day 7. No expression was observed at day 3 either in NG or HG. An elevated gene expression of inflammatory marker, IL-6 and apoptosis marker, P53 were observed in HG at both days 3 and 7. Conclusions: A low expression of SARS-CoV-2 receptors were observed on CD14 macrophage cells from patients which may indicate poor recognition of the virus particle by CD14 cells. Higher expression of the SARS-CoV-2 receptors were noted when HUVEC cells were cultured in HG for 7 days. Conclusion: Longer exposure of human endothelial cells in high glucose condition increase the SARS-CoV-2 receptor expression. Disclosure S. Nandula: None. R. Janapala: None. S. Sen: None. Funding Clinical and Translational Science Institute at Children’s National

Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation.

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

Long-term exposure of human endothelial cells to metformin modulates miRNAs and isomiRs

Increasing evidence suggest that the glucose-lowering drug metformin exerts a valuable anti-senescence role. The ability of metformin to affect the biogenesis of selected microRNAs (miRNAs) was recently suggested. MicroRNA isoforms (isomiRs) are distinct variations of miRNA sequences, harboring addition or deletion of one or more nucleotides at the 5′ and/or 3′ ends of the canonical miRNA sequence. We performed a comprehensive analysis of miRNA and isomiR profile in human endothelial cells undergoing replicative senescence in presence of metformin. Metformin treatment was associated with the differential expression of 27 miRNAs (including miR-100-5p, -125b-5p, -654-3p, -217 and -216a-3p/5p). IsomiR analysis revealed that almost 40% of the total miRNA pool was composed by non-canonical sequences. Metformin significantly affects the relative abundance of 133 isomiRs, including the non-canonical forms of the aforementioned miRNAs. Pathway enrichment analysis suggested that pathways associated with proliferation and nutrient sensing are modulated by metformin-regulated miRNAs and that some of the regulated isomiRs (e.g. the 5′ miR-217 isomiR) are endowed with alternative seed sequences and share less than half of the predicted targets with the canonical form. Our results show that metformin reshapes the senescence-associated miRNA/isomiR patterns of endothelial cells, thus expanding our insight into the cell senescence molecular machinery.

Laminar Flow Protects Vascular Endothelial Tight Junctions and Barrier Function via Maintaining the Expression of Long Non-coding RNA MALAT1.

Atherosclerotic plaque preferentially develops in arterial curvatures and branching regions, where endothelial cells constantly experience disturbed blood flow. By contrast, the straight arteries are generally protected from plaque formation due to exposure of endothelial cells to vaso-protective laminar blood flow. However, the role of flow patterns on endothelial barrier function remains largely unclear. This study aimed to investigate new mechanisms underlying the blood flow pattern-regulated endothelial integrity. Exposure of human endothelial cells to pulsatile shear (PS, mimicking the laminar flow) compared to oscillatory shear (OS, mimicking the disturbed flow) increased the expressions of long non-coding RNA MALAT1 and tight junction proteins ZO1 and Occludin. This increase was abolished by knocking down MALAT1 or Nesprin1 and 2. PS promoted the association between Nesprin1 and SUN2 at the nuclear envelopes, and induced a nuclear translocation of β-catenin, likely through enhancing the interaction between β-catenin and Nesprin1. In the in vivo study, mice were treated via intraperitoneal injection with β-catenin agonist SKL2001 or its inhibitor XAV939, and they were then subjected to Evans blue injection to assess aortic endothelial permeability. The aortas exhibited a reduced wall permeability to Evans blue in SKL2001-treated mice whereas an enhanced permeability in XAV939-treated mice. We concluded that laminar flow promotes nuclear localization of Nesprins, which facilitates the nuclear access of β-catenin to stimulate MALAT1 transcription, resulting in increased expressions of ZO1 and Occludin to protect endothelial barrier function.

Knockdown of CCR1 suppresses expression of Intercellular Cell Adhesion Molecule‐1 induced by hypoxia/reoxygenation in endothelial cells and reduces infarct sizes after myocardinal ischemia/reperfusion

It has been reported that CCL15, a CCR1 ligand, induces proatherogenic factors and has been implicated in atherosclerosis. The present study showed that exposure of human endothelial cells (ECs) to hypoxia and reoxygenation (H/R) resulted in a rapid increase in expression of ICAM‐1 and CCR1 ligands. Our results revealed that knockdown of CCR1 attenuated expression of ICAM‐1 in ECs after H/R. Stimulation of ECs with CCL15 increased expression of ICAM‐1 compared with those in non‐stimulated cells predominantly via CCR1. Our data showed that CCL15 increased cell adhesion of monocytoid cells to endothelial cells. Pretreatment of these cells with CCR1 siRNA prevented the CCL15‐induced expression of ICAM‐1 and monocyte adhesion to ECs. We further investigated expression of CCR1 ligands and ICAM‐1 in vivo after myocardinal ischemia and reperfusion (MIR). Enhanced expression of ICAM‐1 and CCR1 ligands has been demonstrated in rat hearts in vivo after MI‐R. Direct injection of CCR1 siRNA onto the ventricular wall reduced expression of ICAM‐1 in rat hearts activated by MI‐R. Staining of hearts revealed that silencing of CCR1 markedly reduced the infarct sizes after MI‐R. Our results indicate that CCR1 and its ligands play important roles in inflammatory responses after MI/R via upregulating expression of cell adhesion molecules.This work was supported by a grant from National Research Foundation of Korea.

Glucose-independent persistence of PAI-1 gene expression and H3K4 tri-methylation in type 1 diabetic mouse endothelium: Implication in metabolic memory

Clinical trials with type 1 and type 2 diabetes have identified a phenomenon known as “metabolic memory” in which previous periods of hyperglycemia result in the long-lasting deleterious impact on cardiovascular events. Emerging evidence shows that transient hyperglycemic exposure of human endothelial cells induces histone 3 lysine 4 mono-methylation (H3K4me1) on the promoter and persistent mRNA expression of RelA and IL-8 genes, suggesting that epigenetic histone modification and chromatin structure remodeling is a key event underlying metabolic memory. This burgeoning hypothesis, however, critically remains to be tested for relevance in the disease process of diabetes in vivo, and for broader applicability to an array of genes involved in endothelial dysfunction. To address this, we used type 1 diabetes mouse model induced by streptozocin to be hyperglycemic for 8weeks, and isolated endothelial cells that were used either freshly after isolation or after 2 to 3-week cell culture in normoglycemic conditions. mRNA expression profiling in diabetic mouse endothelial cells revealed significant and persistent up-regulation of Serpine1 encoding PAI-1, the hypo-fibrinolytic mediator leading to thrombotic diseases in diabetes, along with Rock2, Fn1 and Ccl2, whereas only Serpine 1 was persistently elevated in high glucose-treated mouse endothelial cells. Chromosome immunoprecipitation assay in type 1 diabetic mouse endothelial cells showed predominant enrichment of H3K4 tri-methylation on Serpine1 promoter, suggesting a unique epigenetic regulation in diabetic mice as opposed to high glucose-treated human ECs. Our study demonstrates the importance of combining in vivo models of diabetes with high glucose-treated cell culture to better assess the epigenetic mechanisms relevant to disease.

Amorphous Silica Nanoparticles Promote Monocyte Adhesion to Human Endothelial Cells: Size‐Dependent Effect

There is evidence that nanoparticles can induce endothelial dysfunction. Here, the effect of monodisperse amorphous silica nanoparticles (SiO(2)-NPs) of different diameters on endothelial cells function is examined. Human endothelial cell line (EA.hy926) or primary human pulmonary artery endothelial cells (hPAEC) are seeded in inserts introduced or not above triple cell co-cultures (pneumocytes, macrophages, and mast cells). Endothelial cells are incubated with SiO(2)-NPs at non-cytotoxic concentrations for 12 h. A significant increase (up to 2-fold) in human monocytes adhesion to endothelial cells is observed for 18 and 54 nm particles. Exposure to SiO(2)-NPs induces protein expression of adhesion molecules (ICAM-1 and VCAM-1) as well as significant up-regulation in mRNA expression of ICAM-1 in both endothelial cell types. Experiments performed with fluorescent-labelled monodisperse amorphous SiO(2)-NPs of similar size evidence nanoparticle uptake into the cytoplasm of endothelial cells. It is concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances their adhesive properties. This process is modified by the size of the nanoparticle and the presence of other co-cultured cells.

Rac1-Regulated Endothelial Radiation Response Stimulates Extravasation and Metastasis That Can Be Blocked by HMG-CoA Reductase Inhibitors

Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation.

Dose-Dependent Modulatory Effects of Insulin on Glucose-Induced Endothelial Senescence In Vitro and In Vivo: A Relationship between Telomeres and Nitric Oxide

The elderly are prone to postprandial hyperglycemia that increases their cardiovascular risk. Although insulin therapy is necessary to treat diabetes, high plasma concentrations of insulin may cause the development of atherosclerosis and accelerate endothelial senescence. We assumed that high glucose causes stress-induced premature senescence and replicative senescence and examined the regulatory role of insulin in endothelial senescence and functions under different glucose conditions. Exposure of human endothelial cells to high glucose (22 mM) for 3 days increased senescence-associated-β-galactosidase activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. Physiological concentrations of insulin preserved telomere length and delayed endothelial senescence under high-glucose conditions. The effect of insulin under high-glucose conditions was associated with reduced reactive oxygen species and increased nitric oxide (NO). Small interfering RNA targeting endothelial NO synthase reduced the antisenescence effects of insulin. Physiological concentrations of insulin also reversed high glucose-induced increases in p53 and vascular cell adhesion molecule-1 and decreases in senescence marker protein-30. On the other hand, when insulin was given at any concentrations under normal glucose or at high concentrations under high glucose, its ability to promote cellular senescence was unrelated to endothelial NO. Finally, streptozotocin-induced diabetes showed more senescent cells in the aortic endothelium of aged rats compared with age-matched control and insulin-treated animals. Conclusively, the regulatory effects of insulin on endothelial senescence were modulated by the glucose environment. These data may help explain insulin's complicated roles in atherosclerosis in the elderly.

Protease-activated receptors, cyclo-oxygenases and pro-angiogenic signalling in endothelial cells

COX (cyclo-oxygenase)-2 and members of the PAR (protease-activated receptor) family (PARs 1-4) are highly overexpressed in a number of angiogenesis-dependent pathologies, including advanced atherosclerosis and cancer. An appreciation of the potential role(s) of PARs and COX enzymes in physiological angiogenesis is, however, currently lacking. Exposure of human endothelial cells to serine proteases (e.g. thrombin) or to PAR-selective agonist peptides leads to a wide range of cellular responses, including enhanced expression of COX-2, and we have shown that this induction depends on activation of classic pro-inflammatory signalling elements [e.g. MAPKs (mitogen-activated protein kinases) and NF-kappaB (nuclear factor kappaB)]. Our current studies suggest that COX-2-derived mediators are important autocrine regulators of PAR-stimulated angiogenesis. This mechanism could help us to explain how this novel family of receptors couple vascular inflammation with repair and angiogenesis in health and disease.

Induction of the Cytoprotective Enzyme Heme Oxygenase-1 by Statins Is Enhanced in Vascular Endothelium Exposed to Laminar Shear Stress and Impaired by Disturbed Flow

In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H(2)O(2)-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.

Pharmacological inhibition of poly(ADP-ribose) polymerase activity down-regulates the expression of syndecan-4 and Id-1 in endothelial cells

Poly(ADP-ribose) polymerase (PARP) is a family of nuclear proteins which regulate a number of cell functions, such as DNA repair, transcription, remodelling of chromatin structure, cell division and cell death. We and others have recently demonstrated that down-regulation of cellular PARP activity, using pharmacological inhibitors, impairs a number of endothelial functions and angiogenesis. In the present study, we investigated the potential mechanisms underlying the anti-angiogenic effect exerted by the potent PARP inhibitor GPI 15427, analyzing gene expression in human endothelial cells shortly after treatment with this compound. Analysis of gene and protein expression indicated that a 2-h exposure of human endothelial cells to GPI 15427 induced a rapid decrease of syndecan-4 (SDC-4), a transmembrane protein involved in modulation of cell signalling during angiogenesis that plays a role in endothelial cell migration and adhesion. Moreover, treatment with the PARP inhibitor induced a reduction of a helix-loop-helix transcription factor, the inhibitor of DNA binding-1 (Id-1), also implicated in the control of endothelial functions. We suggest that the inhibitory effect exerted by GPI 15427 on the angiogenic process is likely due to the reduced activity of specific transcription factors, such as Oct-1 and CREB that contribute to the regulation of SDC-4 and Id-1 expression, respectively. In conclusion, these results strongly suggest that PARP activity is capable of modulating molecules required for endothelial cell migration, adhesion, proliferation or differentiation during the angiogenic process.

Short-term biocompatibility of biphasic nanocolloids with potential use as anisotropic imaging probes

Advances in nanotechnology, in particular the development of novel types of nanoparticles, will result in advanced tools for biomedical research and clinical practice. One exciting aspect of future nanomaterial research will be the possibility to combine therapy and imaging in multifunctional nanoparticle designs. In this context, anisotropic particles with subcellular dimensions may offer so far unattainable capabilities, because they could provide access to directional information with respect to nanoparticle–cell interactions. We have recently developed an electrified jetting process, which can produce water-stable polymer particles with two distinct phases. To address the first critical hurdle towards the application of these biphasic nanocolloids as imaging probes, short-term biocompatibility was evaluated using model cell culture systems. Exposure of human endothelial cells and murine fibroblasts to biphasic nanocolloids made of 0.5% polyacrylic acid and 4.5% poly(acrylamide- co-acrylic acid) did not affect cell proliferation as determined by a colorimetric proliferation assay. Moreover, double staining with Annexin V and propidium iodide and subsequent flow cytometric analysis indicated high cell viability, although slightly decreased viability was observed at the highest dose tested (1 mg particles/10 6 seeded cells). Particle internalization as well as surface binding occurred simultaneously for both cell types, as evidenced by confocal laser scanning microscopy. Taken together, these results suggest excellent short-term biocompatibility in physiological systems for wide concentration ranges of the biphasic nanocolloids and open possibilities for future work investigating receptor- or surface marker-mediated targeting.

Rho-Kinase Mediates Hyperglycemia-Induced Plasminogen Activator Inhibitor-1 Expression in Vascular Endothelial Cells

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The induction of PAI-1 expression by hyperglycemia involves oxidative stress and protein kinase C (PKC). However, the mechanism by which hyperglycemia increases PAI-1 expression is unknown. Compared with normoglycemia, exposure of human endothelial cells to hyperglycemia, but not mannitol, increased Rho-kinase activity in a time- and concentration-dependent manner. This increase was inhibited by a PKC inhibitor, GF109203X, and antioxidants N-acetylcysteine (NAC) and reduced form of glutathione (GSH). This correlated with inhibition of hyperglycemia-induced PAI-1 expression by GF109203X, NAC, and GSH. Hyperglycemia-increased PAI-1 mRNA and protein levels were inhibited by Rho-kinase inhibitors hydroxyfasudil and Y27632 and by a dominant-negative mutant of Rho-kinase. The mechanism for this inhibition occurs at the level of gene transcription because Rho-kinase inhibitors repress hyperglycemia-stimulated PAI-1 promoter activity without affecting mRNA stability. Hyperglycemia failed to stimulate Rho-kinase activity and PAI-1 expression in heterozygous ROCK I-knockout murine endothelial cells. Hyperglycemia stimulates Rho-kinase activity via PKC- and oxidative stress-dependent pathways, leading to increased PAI-1 gene transcription. These results suggest that inhibition of ROCK I may be a novel therapeutic target for preventing thromboembolic complications of diabetes and cardiovascular disease.

Adenosine prevents neutrophil adhesion to human endothelial cells after hypoxia/reoxygenation

Background Neutrophil adhesion to vascular endothelium has been implicated in the pathogenesis of myocardial injury after ischaemia/reperfusion (IR) and the “no-reflow” phenomenon. Adenosine and sodium-nitroprusside (SNP) have been used clinically to ameliorate this injury. We set out to establish a human cellular model for the study of IR and to evaluate the effects of adenosine and SNP on neutrophil adhesion in vitro. Methods Cultured human umbilical vein endothelial cells (HUVEC) were exposed to hypoxia (5% CO 2, 95% N 2) or normoxia (room air, 5% CO 2) for 2 h, followed by reoxygenation for 30 min (IR condition). Human neutrophils were then added together with adenosine (50 μM), SNP (10 μM) or no additive (control). After incubation for 1 h, neutrophil adhesion to endothelial cells was quantified via automated cell counts. The experiment was repeated with the adenosine treatment alone, with and without the addition of the adenosine A 2A receptor blocker ZM-241385. Results Compared with baseline neutrophil adhesion after normoxia, hypoxia followed by reoxygenation increased adhesion to 189 ± 43% ( p = 0.01), but this effect was prevented by the addition of adenosine (109 ± 17%, p = NS compared to control conditions). SNP did not affect the increased adhesion caused by hypoxia (166 ± 25%, p = NS). The addition of ZM-241385 did not inhibit the effect of adenosine on neutrophil adhesion after hypoxia/reoxygenation. Conclusions Exposure of human endothelial cells to hypoxia/reoxygenation causes increased neutrophil adhesion. This effect is prevented by adenosine, but not mediated by the A 2A receptor. SNP does not prevent neutrophil adhesion after IR in vitro.

Estrogen-mediated protection against HIV Tat protein-induced inflammatory pathways in human vascular endothelial cells.

It has been proposed that human immunodeficiency virus (HIV) infection-induced inflammatory environment may contribute to the pathogenesis of cardiovascular diseases. Recent studies have also demonstrated the potential role of estrogen as therapeutic agents in the prevention or treatment of cardiovascular diseases. In the present study, we assessed the hypothesis that estrogen may attenuate the HIV Tat protein-induced inflammatory pathways in human vascular endothelium. Expression of inflammatory mediators in human endothelial cells was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Electrophoretic mobility shift assay (EMSA) also was performed to investigate the DNA-binding activities of several transcription factors, which are known to regulate expression of these inflammatory genes. Acute exposure of human endothelial cells to Tat markedly induced the mRNA and protein expression of IL-1beta, MCP-1, VCAM-1, and E-selectin. Tat also stimulated the adherence of inflammatory cells to endothelial cell monolayers. Significant and dose-dependent increases in NF-kappaB DNA-binding activity were observed in human endothelial cells treated with Tat. However, Tat did not affect DNA-binding activities of AP-1, CREB, and STAT1. Pretreatment with 17beta-estradiol dramatically blocked the activation of NF-kappaB in human endothelial cells exposed to Tat. In addition, 17beta-estradiol selectively inhibited the Tat-induced expression of IL-1beta. Our results suggest that estrogen may protect against Tat-induced inflammatory reactions in human vascular endothelium via blocking the NF-kappaB-mediated molecular signaling pathways. These data may contribute to understanding the pathogenesis of cardiovascular complications and development of therapeutic strategies for HIV-infected patients.