Role In Endothelial Cell Dysfunction Research Articles

The role of microvascular obstruction and intramyocardial hemorrhage in reperfusion injury to the heart

Microvascular obstruction (MVO) of coronary arteries promotes an increase in mortality and major adverse cardiac events in patients with acute myocardial infarction (AMI) and percutaneous coronary intervention (PCI). Intramyocardial hemorrhage (IMH) is observed in 41–50 % of patients with ST-segment elevation myocardial infarction and PCI and is accompanied by inflammation. There is evidence that microthrombi are not involved in the development of MVO, which is associated with infarct size, the duration of ischemia, and myocardial edema. However, there is no conclusive evidence that the latter plays an important role in the development of MVO. There is evidence that platelets, inflammation, Ca2+ overload, neuropeptide Y, and endothelin-1 could be involved in the pathogenesis of MVO. The role of endothelial cell dysfunction in MVO formation in patients with AMI and PCI remains under question. It is unclear whether nitric oxide production is decreased in patients with MVO. It was obtained only indirect evidence on the involvement of inflammation in the development of MVO. The role of reactive oxygen species, necroptosis and pyroptosis in the pathogenesis of MVO is also not studied. The participation of thromboxane A2, vasopressin, and prostacyclin in the formation of MVO is unknown. It was not obtained conclusive evidence on the involvement of coronary artery spasm in the development of MVO. Dual antiplatelet therapy improves the efficacy of PCI in prevention of MVO. It is unknown whether epinephrine or L-type Ca2+-channel blockers improves coronary blood flow in patients with MVO.

Autophagy Behavior in Endothelial Cell Dysfunction

Abstract: Autophagy regulates endothelial cell homeostasis. Autophagy is a catabolic process involving degradation of intracellular components. Dysregulation of autophagy induces endothelial cell dysfunction. Endothelial cell dysfunction is a multifactorial pathophysiological change that occurs at the cellular and subcellular levels. Lipophagy and mitophagy are hallmarks of the pathogenesis of endothelial cell dysfunction. The regulation of the autophagy mechanism involved amino acids, growth factors, hormones, myo-inositol-1,4,5-triphosphate, calpain, calcium, bcl-2, reactive oxygen species, BNIP3, DRAM, p19ARF, FADD and TRAIL. Down-regulation of autophagy reduces endothelial cell resistance to stressful conditions such as shear stress, deprivation of oxidative stress, nutrients deprivation, and hypoxemia. Autophagy optimizes endothelial cell function, increases longevity, slows senescence, and prevents endothelial cell transdifferentiation. Pathophysiologically, autophagy is inhibited in endothelial cells due to mTORC1 repression release. Also, AMPK expression repression downregulates autophagy and subsequently endothelial dysfunction. The paper provides state of art on the current advances in the autophagy role in endothelial cell dysfunction.

The Role of Endothelial Cell Dysfunction in Hemorrhagic Fevers

Endothelial cells (ECs) that line the inner surface of blood vessels play a crucial role in maintaining healthy blood vessels and regulating consistent blood flow. EC dysfunction (ECD) is the improper functioning of ECs as a result of any modification or damage. Numerous health issues such as cardiovascular disease, hypertension, lightheadedness, exhaustion, diabetes, and the like could result from this. Both extrinsic and intrinsic factors may cause ECD. Many virus-induced hemorrhagic fevers, including dengue fever, Ebola, and Lassa fever, exhibit ECD. ECs maintain barrier functions by regulating immune cell interactions, homeostasis, and capillary permeability, and in viral infections, viral interaction alters these factors. These pathogens destroy ECs, leading to a loss of integrity in the blood vessels and increased permeability. Hemorrhagic fever can cause a wide range of symptoms such as fever, headache, muscle pain, weakness, exhaustion, bruises, bleeding, shock, and occasionally even the emergence of additional issues such as organ failure. Hemorrhagic fevers can be life-threatening if not treated promptly and correctly. Considering that ECD can be a silent condition, consultation with physicians about individuals’ risk factors and regular screenings to detect and prevent this condition is highly important. There is no specific antiviral treatment for most hemorrhagic fevers, but research is ongoing to develop effective treatments. This review will discuss the ECD due to viral interactions to cause hemorrhagic fevers, available treatments, and challenges associated with such viral hemorrhagic fevers (VHFs).

Endothelial Cell Dysfunction and Nonalcoholic Fatty Liver Disease (NAFLD): A Concise Review

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. It is strongly associated with obesity, type 2 diabetes (T2DM), and other metabolic syndrome features. Reflecting the underlying pathogenesis and the cardiometabolic disorders associated with NAFLD, the term metabolic (dysfunction)-associated fatty liver disease (MAFLD) has recently been proposed. Indeed, over the past few years, growing evidence supports a strong correlation between NAFLD and increased cardiovascular disease (CVD) risk, independent of the presence of diabetes, hypertension, and obesity. This implies that NAFLD may also be directly involved in the pathogenesis of CVD. Notably, liver sinusoidal endothelial cell (LSEC) dysfunction appears to be implicated in the progression of NAFLD via numerous mechanisms, including the regulation of the inflammatory process, hepatic stellate activation, augmented vascular resistance, and the distortion of microcirculation, resulting in the progression of NAFLD. Vice versa, the liver secretes inflammatory molecules that are considered pro-atherogenic and may contribute to vascular endothelial dysfunction, resulting in atherosclerosis and CVD. In this review, we provide current evidence supporting the role of endothelial cell dysfunction in the pathogenesis of NAFLD and NAFLD-associated atherosclerosis. Endothelial cells could thus represent a “golden target” for the development of new treatment strategies for NAFLD and its comorbid CVD.

Framework Nucleic Acids Enabled Pulmonary Artery Endothelial Cell Growth Inhibition by Targeting microRNA-152.

Pulmonary artery vascular endothelial dysfunction plays a pivotal role in the occurrence and progression of pulmonary vascular remodeling (PVR). To address this, aberrantly expressed non-coding microRNAs (miRNAs) are excellent therapeutic targets in human pulmonary artery endothelial cells (HPAECs). Here, we discovered and validated the overexpression of miRNA-152 in HPAECs under hypoxia and its role in endothelial cell dysfunction. We constructed a framework nucleic acid nanostructure that harbors six protruding single-stranded DNA segments that can fully hybridize with miRNA-152 (DNT-152). DNT-152 was efficiently taken up by HPAECs with increasing time and concentration; it markedly induced apoptosis, and inhibited HPAEC growth under hypoxic conditions. Mechanistically, DNT-152 silenced miRNA-152 expression and upregulated its target gene Meox2, which subsequently inhibited the AKT/mTOR signaling pathway. These results indicate that miRNA-152 in HPAECs may be an excellent therapeutic target against PVR, and that framework nucleic acids with carefully designed sequences are promising nanomedicines for noncancerous cells and diseases.

Reelin Depletion Protects Against Atherosclerosis by Decreasing Vascular Adhesion of Leukocytes.

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Role of endothelial cell receptors in the context of SARS-CoV-2 infection (COVID-19)

Endothelial cell (EC) dysfunction contributes to COVID-19–associated vascular inflammation and coagulopathy, and the angiotensin-converting enzyme 2 (ACE2) receptor plays a role in EC dysfunction in COVID-19. To expand the understanding of the role of the ACE2 receptor relative to EC dysfunction, this review addresses (1) tissue distribution of the ACE2 protein and its mRNA expression in humans, (2) susceptibility of the capillary ECs to SARS-CoV-2 infection, and (3) the role of EC dysfunction relevant to ACE2 and nuclear factor-κB in COVID-19.

Endothelial Dysfunction in Pulmonary Hypertension: Cause or Consequence?

Pulmonary arterial hypertension (PAH) is a rare, complex, and progressive disease that is characterized by the abnormal remodeling of the pulmonary arteries that leads to right ventricular failure and death. Although our understanding of the causes for abnormal vascular remodeling in PAH is limited, accumulating evidence indicates that endothelial cell (EC) dysfunction is one of the first triggers initiating this process. EC dysfunction leads to the activation of several cellular signalling pathways in the endothelium, resulting in the uncontrolled proliferation of ECs, pulmonary artery smooth muscle cells, and fibroblasts, and eventually leads to vascular remodelling and the occlusion of the pulmonary blood vessels. Other factors that are related to EC dysfunction in PAH are an increase in endothelial to mesenchymal transition, inflammation, apoptosis, and thrombus formation. In this review, we outline the latest advances on the role of EC dysfunction in PAH and other forms of pulmonary hypertension. We also elaborate on the molecular signals that orchestrate EC dysfunction in PAH. Understanding the role and mechanisms of EC dysfunction will unravel the therapeutic potential of targeting this process in PAH.

IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome

Cytokine release syndrome (CRS) is a life-threatening complication induced by systemic inflammatory responses to infections, including bacteria and chimeric antigen receptor T cell therapy. There are currently no immunotherapies with proven clinical efficacy and understanding of the molecular mechanisms of CRS pathogenesis is limited. Here, we found that patients diagnosed with CRS from sepsis, acute respiratory distress syndrome (ARDS), or burns showed common manifestations: strikingly elevated levels of the four proinflammatory cytokines interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and IL-10 and the coagulation cascade activator plasminogen activator inhibitor-1 (PAI-1). Our in vitro data indicate that endothelial IL-6 trans-signaling formed an inflammation circuit for robust IL-6, IL-8, and MCP-1 production and promoted PAI-1 production; additionally, an IL-6 signaling blockade by the human monoclonal antibody tocilizumab blunted endothelial cell activation. Plasma from severe COVID-19 patients similarly exhibited increased IL-6, IL-10, and MCP-1 levels, but these levels were not as high as those in patients with CRS from other causes. In contrast, the PAI-1 levels in COVID-19 patients were as highly elevated as those in patients with bacterial sepsis or ARDS. Tocilizumab treatment decreased the PAI-1 levels and alleviated critical illness in severe COVID-19 patients. Our findings suggest that distinct levels of cytokine production are associated with CRS induced by bacterial infection and COVID-19, but both CRS types are accompanied by endotheliopathy through IL-6 trans-signaling. Thus, the present study highlights the crucial role of IL-6 signaling in endothelial dysfunction during bacterial infection and COVID-19.

Glucose-6-phosphate dehydrogenase deficiency increases cell adhesion molecules and activates human monocyte-endothelial cell adhesion: Protective role of l-cysteine

Glucose-6-phosphate dehydrogenase is a major enzyme that supplies the reducing agent nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), which is required to recycle oxidized/glutathione disulfide (GSSH) to reduced glutathione (GSH). G6PD-deficient cells are susceptible to oxidative stress and a deficiency of GSH. Endothelial dysfunction is characterized by the loss of nitric oxide (NO) bioavailability, which regulates leukocyte adhesion to endothelium. G6PD-deficient endothelial cells (EC) demonstrate reduced expression of endothelial nitric oxide synthase (eNOS) and NO levels along with reduced GSH. Whether G6PD deficiency plays any role in EC dysfunction is unknown. The chronic inflammation commonly seen in those with metabolic syndrome, characterized by elevated levels of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1), provided an incentive for investigation of these cytokines as well. A GSH/G6PD-deficient model was created using human umbilical vein endothelial cells (HUVEC) treated with either buthionine sulfoximine (BSO), a pharmacological inhibitor of the rate-limiting enzyme of GSH biosynthesis (γ-glutamylcysteine synthetase), or with 6-aminonicotinamide (6-AN), an inhibitor of G6PD or G6PD siRNA. Normal and G6PD-deficient cells were also treated with pro-atherosclerotic stimuli such as high glucose, TNF, and MCP-1. After inhibiting or knocking down G6PD/GSH, the capacity of endothelial cells for monocyte recruitment was assessed by determining the expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), which was upregulated by G6PD deficiency and accompanied by the presence of the oxidative stress markers NADPH oxidase 4 (NOX4), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS). Treatment with the inhibitors BSO and 6-AN caused increased levels of adhesion molecule mRNA and monocyte-EC adhesion. Following treatment with high glucose, G6PD-deficient cells showed an increase in levels of ICAM-1 and VCAM-1 mRNA, as well as monocyte-EC adherence, compared with results seen in control cells. Treatment with l-cysteine (a precursor of GSH) protected endothelial cells by increasing GSH and attenuating ROS, ICAM-1, VCAM-1, and monocyte-EC adhesion. These results suggest that G6PD/GSH deficiency plays a role in endothelial dysfunction and that supplementation with l-cysteine can restore GSH levels and reduce the EC activation markers in G6PD-deficient conditions.

Association of Soluble TNFR-1 Concentrations with Long-Term Decline in Kidney Function: The Multi-Ethnic Study of Atherosclerosis.

TNF receptor-1 (TNFR-1), which plays a causative role in endothelial cell dysfunction and inflammation, is expressed on the cell surface in glomerular and peritubular capillary endothelium of the kidneys. Higher soluble TNF receptor-1 (sTNFR-1) concentrations are associated with kidney disease progression among persons with established diabetic kidney disease. However, no studies have assessed sTNFR-1's role in long-term kidney function changes in a multiethnic population without cardiovascular disease at baseline. We tested associations between baseline sTNFR-1 concentrations and 10-year decline in eGFR (incident ≥40% decline and annual proportional decline) among 2548 participants in the Multi-Ethnic Study of Atherosclerosis (MESA), a prospective cohort study. Serum creatinine concentrations were determined at enrollment and study years 3, 5, and 10. Mean age of participants was 61 years old, 53% were women, and mean baseline eGFR was 79 ml/min per 1.73 m2. Serum sTNFR-1 was inversely associated with baseline eGFR. Over median follow-up of 9.3 years, 110 participants developed ≥40% decline in eGFR; each SD higher concentration of sTNFR1 was associated with higher risk of 40% eGFR decline (adjusted hazard ratio, 1.43; 95% confidence interval [95% CI], 1.16 to 1.77; P<0.001). The highest sTNFR-1 tertile was associated with adjusted annualized decline in eGFR of 1.94% (95% CI, 1.79 to 2.09). Associations persisted across subgroups defined by demographics, hypertension, diabetes, and baseline CKD status. Elevated serum sTNFR-1 concentrations are associated with faster declines in eGFR over the course of a decade in a multiethnic population, independent of previously known risk factors for kidney disease progression.

The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells.

Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase. These findings support a novel role for P2X7 and P2X4 coupled to induction of inflammatory molecules in modulating high glucose and palmitate-induced endothelial cell activation and dysfunction.

Renal Endothelial Dysfunction in Diabetic Nephropathy

Endothelial dysfunction has been posited to play an important role in the pathogenesis of diabetic nephropathy (DN). Due to the heterogeneity of endothelial cells (ECs), it is difficult to generalize about endothelial responses to diabetic stimuli. At present, there are limited techniques fordirectly measuring EC function in vivo, so diagnosis of endothelial disorders still largely depends on indirect assessment of mediators arising from EC injury. In the kidney microcirculation, both afferent and efferent arteries, arterioles and glomerular endothelial cells (GEnC) have all been implicated as targets of diabetic injury. Both hyperglycemia per se, as well as the metabolic consequences of glucose dysregulation, are thought to lead to endothelial cell dysfunction. In this regard, endothelial nitric oxide synthase (eNOS) plays a central role in EC dysfunction. Impaired eNOS activity can occur at numerous levels, including enzyme uncoupling, post-translational modifications, internalization and decreased expression. Reduced nitric oxide (NO) bioavailability exacerbates oxidative stress, further promoting endothelial dysfunction and injury. The injured ECs may then function as active signal transducers of metabolic, hemodynamic and inflammatory factors that modify the function and morphology of the vessel wall and interact with adjacent cells, which may activate a cascade of inflammatory and proliferative and profibrotic responses in progressive DN. Both pharmacological approaches and potential regenerative therapies hold promise for restoration of impaired endothelial cells in diabetic nephropathy.

Nitric Oxide, Oxidative Stress, andp66ShcInterplay in Diabetic Endothelial Dysfunction

Increased oxidative stress and reduced nitric oxide (NO) bioavailability play a causal role in endothelial cell dysfunction occurring in the vasculature of diabetic patients. In this review, we summarized the molecular mechanisms underpinning diabetic endothelial and vascular dysfunction. In particular, we focused our attention on the complex interplay existing among NO, reactive oxygen species (ROS), and one crucial regulator of intracellular ROS production, p66Shc protein.

The Role of Placental Oxidative Stress and Lipid Peroxidation in Preeclampsia

Preeclampsia is a complex multisystem disorder exclusively seen in human species that is characterized by hypertension and proteinuria. This disorder has the highest maternal and fetal morbidity and mortality of all pregnancy-related complications. Growing evidence suggests that placental oxidative stress is involved in the etiopathogenesis of preeclampsia. Reduced perfusion as a result of abnormal placentation leads to ischemia reperfusion injury to the placenta. Placental oxidative stress, which results from the ischemia reperfusion injury, is being increasingly reported to be involved in the etiopathogenesis of preeclampsia. It has been proposed as a promoter of lipid peroxidation and the endothelial cell dysfunction that is commonly seen in this condition. Although preeclampsia is characterized by increased lipid peroxidation and diminished antioxidant capacity, there is no consensus regarding causality of lipid peroxidation in preeclampsia. In this article, we address the question of the biologic association of lipid peroxidation and preeclampsia. Lipid peroxidation and leukocyte activation may play a pivotal role in endothelial cell dysfunction. We also review the different factors that have been proposed to cause endothelial cell dysfunction in preeclampsia, trials investigating the role of antioxidant supplementation in preeclampsia, and the lack of consensus among the trials. Additional longitudinal studies are necessary to determine if the various oxidative stress biomarkers estimated early in pregnancy can be narrowed to a single marker for predicting preeclampsia. Obstetricians & Gynecologists, Family Physicians. After completion of this article, the reader should be able to recall that placental oxidative stress is involved in the etiopathogenesis of preeclampsia, state that placental oxidative stress results from ischemic reperfusion injury, and explain that ischemic reperfusion injury is a promoter of lipid peroxidation and endothelial cell dysfunction seen in preeclampsia.

Establishment of an immortalized PARP‐1−/− murine endothelial cell line: A new tool to study PARP‐1 mediated endothelial cell dysfunction

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a critical role in endothelial cell dysfunction associated with various pathophysiological conditions. To elucidate PARP-1 pathways involved in endothelial cell dysfunction, it is essential to establish "in vitro" experimental models using isolated endothelial cells. So far, two approaches have been used: primary endothelial cells from PARP-1-/- mice which have a limited life-span, being a major handicap if large quantities of cells are required; and pharmacological inhibition of PARP in PARP-1+/+ endothelial cell lines, which is not specific for PARP-1 and would have biological effects different that genetic inhibition. To overcome these limitations, we have established an immortalized PARP-1-/- endothelial cell line (HYKO6) by transfection of primary cells with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. The HYKO6 cell line exhibited endothelial characteristics, such as constitutive expression of CD105, CD31, ICAM-2, VCAM-1, and von Willebrand factor and formation of capillary-like structures (CLS) on Matrigel surface. However, expression of ICAM-1 antigen is lost in the HYKO6 cells. After TNF-alpha treatment, HYKO6 cells exhibited increased expression of E-selectin and VCAM-1. Likewise, NF-kappaB-dependent transcriptional activation was increased in the HYKO6 cell line in response to TNF-alpha at a level similar to that found for primary PARP-1-/- cells. This cell line should provide, for the first time, a valuable tool to study PARP-1 pathways in endothelial cell dysfunction.

Endothelium and valvular diseases of the heart

It has become increasingly evident that the endothelium plays a critical role in the pathogenesis of valvular heart disease. The endothelium helps regulate vascular tone, inflammation, thrombosis, and vascular remodeling. Dysfunction of the endothelial cells has been linked to many vascular disorders including atherosclerosis. Common valvular diseases such as senile degenerative valve disease, myxomatous (or floppy) valves, rheumatic valves, and infective endocarditis valves show changes in the synthetic, morphologic, and metabolic functions of the valvular endothelial cells. These diseases are active processes related to endothelial cell dysfunction. Endothelial cell dysfunction is caused by mechanical forces, bacterial infection, autoantibodies, and circulating modulators of endothelial cell function. This study reviews the role of endothelial cell dysfunction in the more common valvular diseases. Continued research on endothelial cell dysfunction is crucial to our understanding of valvular heart diseases and may elucidate novel treatment and prevention strategies.

Semicarbazide-sensitive amine oxidase in pre-eclampsia: no relation with markers of endothelial cell activation

Background: Semicarbazide sensitive amine-oxidase (SSAO) is an adhesion molecule and thought to play a role in endothelial cell dysfunction (ECD). SSAO has never been associated with markers of ECD. Pre-eclampsia (PE) is, like the early stages of atherosclerosis, characterised by ECD. SSAO could contribute to ECD in PE. Methods: Plasma samples were obtained in 14 pre-eclamptic patients and 14 matched controls. In these SSAO-activity, von Willebrand factor (vWF) levels and ED1 fibronectin levels were determined. Placental tissue was collected of 12 pre-eclamptic pregnancies, 8 preterm deliveries and 12 term controls. In these samples, SSAO activity was assessed. In a subset of these placentas, immunohistochemical staining was performed for SSAO, ICAM-1 and VCAM-1. Results: Plasma SSAO activity was not significantly different between pre-eclamptic subjects and controls. VWF and ED1 fibronectin were both significantly increased in the pre-eclamptic subjects. There was no correlation between SSAO activity and vWF or ED1 fibronectin levels. Placental SSAO activity was not different between pre-eclamptic pregnancies, preterm deliveries and term controls. There was strong staining of SSAO in vascular smooth muscle cells (VSM) and moderate staining of trophoblast in all three groups. Endothelial cell expression of SSAO was only seen in term controls and the placentas of pre-eclamptic patients. There was no association between the expression of SSAO, ICAM-1 or VCAM-1 in the placentas of pre-eclamptic patients. Conclusion: SSAO expression and activity are not related to markers of ECD.

Activation of Protein Phosphorylation by Oxidants in Vascular Endothelial Cells: Identification of Tyrosine Phosphorylation of Caveolin

Oxidants play a significant role in endothelial cell dysfunction through modulation of diverse biochemical reactions and signal transduction pathways. Towards understanding the role of oxidants in vascular injury, we studied the effect of hydrogen peroxide (H 2O 2), vanadate, and pervanadate (V 4+-OOH) on [ 32P i] uptake and protein phosphorylation in bovine pulmonary artery endothelial cells (BPAEC). The incorporation of labelled [ 32P i] into BPAEC was dependent on the concentration of the oxidant employed and time of incubation. Of the oxidants tested, pervanadate (10 μM) induced maximum incorporation of [ 32P i] into cells (two- to threefold over control) followed by H 2O 2 (1 mM) and vanadate (100 μM) and clear differences in labeled protein profiles were noticed between control and oxidant treated cells. The proteins, analyzed by SDS-PAGE, showed distinct increases in labeling patterns ranging from 21–205 kDa, as evidenced by autoradiography. While the majority of the incorporated [ 32P i] was in serine/threonine residues, immunoprecipitation and immunoblotting of cell lysates, using an antiphosphotyrosine antibody, revealed that oxidant treatment resulted in significant increases in total protein tyrosine phosphorylation. Most significantly, immunoprecipitation of cell lysates, from pervanadate treatment showed distinct tyrosine phosphorylation of 22 kDa protein, which was identified as caveolin, a marker of caveolae. Pervanadate-mediated phosphorylation was effectively inhibited by staurosporine (5 μM), while genistein showed only partial attenuation. Furthermore, H 2O 2 treatment resulted in enhanced phosphorylation of 24 kDa protein, which was attenuated by genistein. In addition, oxidant-treated cells exhibited increased tyrosine kinase activity and decreased phosphatase activity. These data show differences in labeling profiles of proteins in response to different oxidants, suggesting differential modulation of distinct protein kinases/phosphatases. Copyright © 1996 Elsevier Science Inc.

Preservation of endothelium-dependent relaxation in atherosclerotic rabbit aorta by probucol.

Oxidized low-density lipoproteins (LDL), which may play an important role in atherogenesis, inhibit endothelium-dependent relaxations of normal arteries in vitro. The effects of probucol, an inhibitor of LDL oxidation, on endothelium-dependent relaxations in thoracic aorta of rabbits that received a cholesterol-rich (0.5%) or standard diet for 10 weeks were determined. In some rabbits in each group, the diet was supplemented with probucol (1%) for the last 6 weeks. The cholesterol-rich diet markedly increased plasma cholesterol and resulted in increased plasma lipid peroxides. Probucol prevented the increase in lipid peroxides, but had no effect on plasma cholesterol. Rings of aorta were mounted in organ chambers for measurement of isometric tension and contracted with phenylephrine. Endothelium-dependent relaxations to acetylcholine and A23187 were significantly impaired in aortic rings from cholesterol-fed rabbits. Aortic rings from rabbits fed cholesterol and treated with probucol relaxed normally to both vasodilators. Relaxations to acetylcholine and A23187 were not significantly changed in rings from rabbits that received probucol-supplemented standard diet. Endothelium-independent relaxations to sodium nitroprusside (SNP) were not influenced by the cholesterol diet or probucol. Thus, probucol preserves endothelium-dependent relaxations of hypercholesterolemic rabbit aorta without reducing plasma cholesterol. As demonstrated by the reduction in plasma lipid peroxides, the effect of probucol may be related to its antioxidant properties and may imply that oxidized lipids have a role in endothelial cell dysfunction of atherosclerotic arteries in vivo.