Endothelial cells in physiology and in the pathophysiology of vascular disorders.
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Animals↗
Biomedical subjects
J Loscalzo
Publications and source records attributed to J Loscalzo.
Homocysteine and atherothrombosis.
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Amino Acid Metabolism, Inborn Errors↗
Prothrombotic Consequences of the Oxidation of Fibrinogen and their Inhibition by Aspirin.
Oxidant stress leads to covalent oxidative modification of several plasma proteins, chief among which is fibrinogen. Aspirin can nonenzymatically acetylate fibrinogen's lysine residues, the functional groups most susceptible to oxidative modification. Because oxidation of fibrinogen may occur in the atheromatous environment, we studied the effects of oxidative modification on fibrinogen function and the consequences of acetylation by aspirin on fibrinogen's susceptibility to oxidation and functional properties. We exposed fibrinogen to Fe(3+) ascorbate for 1 hour and showed that the carbonyl/protein molar ratio increased from 0.71 +/- 0.18 to 2.86 +/- 0.50 mol carbonyl/mol protein (P < 0.02) with an accompanying reduction in the alpha-helical content of the protein from 34% to 29%. Exposure of fibrinogen to aspirin led to acetylation of lysine residues and inhibition of oxidation. Oxidized fibrinogen was more readily able to form fibrin, and acetylation prevented this enhancement of clot formation. Oxidized fibrinogen also supported platelet aggregation better than did native, unoxidized fibri ogen, and aretylation of fibrinogen prior to oxidation prevented the enhanced platelet aggregation. Oxidized fibrinogen was less effective in stimulating plasminogen activation by tissue-type plasminogen activator (tPA), with a catalytic efficiency that was reduced by 88% compared with native, unoxislized fibrinogen; metylated fibrinogen, by contrast, enhanced plasminogen activation by t-PA with a catalytic efficiency that was increased by 18% compared with native, anoxidized fibrinogen (P < 0.05) and was increased by 51% compared with oxidized fibrinogen (P < 0.05). Acetylation prevented the reduction in catalytic efficiency induced by oxidation. These data show that oxidized fibrinogen manifests prothrombotic effects that can be prevented by acetylation and suggest that inhibition of fibrinogen oxidation may be an additional antithrombotic benefit of aspirin therapy.
Journal Article↗
The red cell transfusion trigger: has a sin of commission now become a sin of omission?
The benefits of a Hct range of 30 to 35 percent include improved oxygen delivery and enhanced hemostasis, which help minimize complications in patients at high risk for ischemia and perioperative nonsurgical bleeding. In these settings, the conservative transfusion practice of using a lower Hct range should be replaced with a more aggressive approach. The known risks of blood transfusion would appear to be sufficiently low and the benefits sufficiently high to justify maintaining a Hct of at least 30 percent. An even higher Hct, of 35 percent, may be desirable in patients who have overt cardiopulmonary disease or who are at high risk for myocardial ischemia. Many retrospective studies have been conducted to persuade us that a conservative transfusion trigger is a safe and prudent practice, but retrospective studies are not what we need. What we need is a series of well-designed, prospective, randomized trials to evaluate the impact of a more aggressive transfusion policy on perioperative mortality, morbidity, and nonsurgical bleeding in patients with known cardiopulmonary disease or who are at high risk for myocardial and cerebrovascular ischemia.
Anemia↗
Nitric Oxide Alters Human Microvascular Endothelial Cell Response to Cyclic Strain.
BACKGROUND: Nitric oxide (NO), a potent vasodilator and inhibitor of platelet function, is elaborated constitutively by endothelial cells through the oxidation of l-arginine by endothelial NO synthase. Although several biochemical agonists, such as bradykinin, have been shown to stimulate NO production by the endothelium, the effects of physical factors have been less well characterized. We have previously examined the shear-stress-dependent induction of NO production by the endothelium, and others have examined the effects of transmural pressure on NO production. In the current study, we analyzed the effects of cyclic strain or load in the presence and absence of an NO agonist on endothelial cell proliferation. METHODS AND RESULTS: Subconfluent human microvascular endothelial cells were grown on deformable culture plates in media containing 10% fetal bovine serum and 1 mM l-arginine, and then subjects to either 0, 11, 18, or 27% cyclic strain at a frequency of 1 Hz. Nitrogen oxides (S-nitrosothiols and free NO) in media were measured by photolysis-chemiluminescence at the end of 24 hours, as were cell number, [(3)H]thymidine incorporation, and total cell protein. In the absence of an NO agonist, 11% cyclic strain produced a 73 +/- 17% increase in nitrogen oxides compared with control (P <.05), whereas 18 and 27 cyclic strain produced no significant increase in nitrogen oxides compared with control. However, cells subjected to 27% strain increased cell number (by 98 +/- 17%) and [(3)H]thymidine incorporation (by 16 +/- 4%, P <.05) compared with control. In an attempt to examine further the effects of endogenous NO on the cells' proliferation response to increasing levels of cyclic strain, we incubated human microvascular endothelial cells with 1 µM bradykinin and subjected them to 27% cyclic strain. In the presence of bradykinin and 27% cyclic strain, production of nitrogen oxides and endothelial NO synthase activity were increased by 110 +/- 37% and 135 +/- 7%, respectively (P <.05 compared with control); however, we observed no significant increase in cell number (5 +/- 5%), despite an 86 +/- 7% increase in [(3)H]thymidine incorporation (P <.05). CONCLUSION: These results suggest that endogenous NO production by human microvascular endothelial cells regulates their growth response to cyclic strain. Modulation of NO production in vivo in areas of the vasculature that are subjected to increased levels of strain may represent one potential mechanism by which to alter endothelial cell proliferation.
Journal Article↗
Pulmonary hypertension.
Pulmonary hypertension can be caused by an increase in pulmonary blood flow, an increase in left-heart impedance or an increase in pulmonary vascular resistance. Although there are many specific causes that have been identified, clinical presentation and natural history are common to all etiologies. Therapeutic options remain limited to correcting underlying causes whenever possible, as well as supplemental oxygen and, in the setting of severely reduced cardiac output, anticoagulation. Vasodilatory therapy with calcium channel blockers, intravenous epoprostenol or inhaled nitric oxide has also met with some success in patients with primary pulmonary hypertension. Lung or heart-lung transplantation remains the only viable therapeutic option for patients with severe pulmonary hypertension. There have been significant advances in our understanding of the pathobiology and treatment of this uncommon but serious class of disorders. Yet, much remains to be learned in our efforts to improve the lot of these unfortunate patients.
Journal Article↗
S-Transnitrosation reactions are involved in the metabolic fate and biological actions of nitric oxide.
S-Nitrosothiols are a group of potent, bioactive compounds that form through the reaction of nitric oxide (NO) with thiols in the presence of oxygen. These compounds are naturally occurring in vivo, stabilize NO and potentiate its biological effects. S-Nitrosoglutathione is the most abundant intracellular S-nitrosothiol, and the kinetics for its formation favors de novo synthesis. In this analysis, we studied the formation of S-nitrosothiols by S-transnitrosation, or exchange of -NO for -H between sulfur atoms; we synthesized S-nitroso-glutathionyl-Sepharose 4B beads (SNO-4B) as a reagent with which to measure S-transnitrosation reactions. We detected a maximum of 1.57 +/- 0.24 pmol NO/bead (n = 5) after S-nitrosation of the beads with acidified nitrite. The stability of the S-NO bond was dependent on temperature, but not pH over the 5 to 9 range (except at pH 9 at 37 degrees ), with an estimated t1/2 of 30 hr at 22 degrees C and of approximately 2 wk at 4 degrees C. We demonstrated that SNO-4B transfers -NO to glutathione and to cysteine rapidly and in a pH-dependent manner. The initial rate of transfer of -NO from SNO-4B to glutathione at room temperature was 0.53, 3.03 and 5.14 microM/min at pH 5.0, 7.4 and 9.0, respectively (P < .05). Under the same conditions, the initial rate of -NO transfer to cysteine was 0. 72, 3.71 and 4.69 microM/min at pH 5.0, 7.4 and 9.0, respectively (P < .05). There was no appreciable S-transnitrosation between SNO-4B and bovine serum albumin. We further demonstrated that SNO-4B evokes significant vasodilator and platelet inhibitory responses in plasma-free systems and activates platelet soluble guanylyl cyclase. These data suggest a mechanism by which to explain the metabolic fate and distribution of NO among thiol pools in the vasculature, and implicate S-transnitrosation at the cell surface in NO signal transduction.
Animals↗
Reduced glutathione prevents nitric oxide-induced apoptosis in vascular smooth muscle cells.
The control of medial and neointimal growth, in which vascular smooth muscle (VSM) plays a central role, is most important to the development of hypertension and atherosclerosis, respectively. Growth of vascular smooth muscle cells is regulated by a number of factors, including the vasodilator nitric oxide (NO). In addition, NO modulates intracellular thiol redox states and the thiol redox state of the cell influences NO production. We, therefore, examined the nature of the effect of NO on growth of VSM cells and its modulation by cellular glutathione content. Here, we report that NO, either generated by NO donors or synthesized by iNOS in VSM cells, inhibited DNA synthesis and induced apoptosis in this cell type. NO-induced apoptosis was associated with a significant decrease in the intracellular concentration of reduced glutathione and with an increase in the level of the tumor suppressor gene p53 mRNA. Moreover, addition of glutathione monoethylester to the culture restored the level of reduced glutathione in VSM cells, and prevented the NO-induced increase in p53 expression and programmed cell death. Our findings suggest a role for reduced glutathione in protecting VSM cells exposed to NO from apoptosis.
Animals↗
Stimulation of endothelial nitric oxide production by homocyst(e)ine.
Hyperhomocyst(e)inemia, characterized by accelerated atherosclerosis, is believed to induce endothelial cell injury and promote atherothrombosis by supporting the generation of hydrogen peroxide. Earlier observations in our laboratory demonstrated that in vitro nitrosation of homocyst(e)ine (HCY) prevents the generation of hydrogen peroxide. We, therefore, hypothesized that stimulating the production of nitric oxide (NO) by endothelial cells would detoxify HCY by forming the corresponding S-nitrosothiol, S-nitroso-homocysteine. In an attempt to prove this hypothesis, media containing 1 mM L-arginine, 1 microM bradykinin, a known NO agonist, and one of the biologically relevant thiols (HCY, cysteine, or glutathione) at concentrations of 0, 0.05, 0.5 and 5.0 mM were incubated with bovine aortic endothelial cells (BAEC) for 0.5, 1 and 4 h. S-nitrosothiol (RSNO) concentrations were measured by photolysis-chemiluminescence. Nitric oxide synthase (eNOS or isoform 3) activity and Nos 3 steady-state mRNA levels were determined by the conversion of [3H]L-arginine to [3H]L-citrulline and Northern analysis, respectively. Results demonstrate that increasing concentrations of HCY, and not cysteine or glutathione, in the presence of bradykinin at 0.5, 1, and 4 h led to significant (P < 0.05 by ANOVA) time- and dose-dependent increases in RSNO produced by BAEC. Cells exposed to 1 microM calcium ionophore A23187 in the presence of 5.0 mM HCY also produced a time-dependent increase in RSNO compared to control (P < 0.05 by ANOVA). In an attempt to determine if de novo synthesis was occurring, BAEC were treated with bradykinin following a 4 h pretreatment with HCY. Pretreatment with HCY followed by stimulation also led to a time- and dose-dependent increase in RSNO production (P < 0.05 by ANOVA). Using high performance liquid chromatography with electrochemical detection, S-nitroso-homocysteine was identified following treatment of BAEC with HCY and bradykinin. The increase in RSNO production in the presence of bradykinin and HCY at 4 h occurred concomitantly with a 78% increase in eNOS activity and a 58% increase in steady-state Nos 3 mRNA, with no change in Nos 3 mRNA half-life, compared to control. A partial explanation for HCY's unique ability to support an increase in NO production was demonstrated by showing that the t1/2 of HCY in media was greater than that of cysteine or glutathione. These data show that, in the presence of an NO agonist, HCY increases RSNO production in a time- and dose-dependent fashion that is reflected by an increase in eNOS activity and Nos 3 transcription. These results suggest that stimulation of endogenous NO, or provision of an exogenous NO donor, may ameliorate endothelial cell injury and thereby decrease the atherothrombotic risk of hyperhomocyst(e)inemic states.
Animals↗
Nitric oxide released from activated platelets inhibits platelet recruitment.
Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.
Adenosine Diphosphate↗
Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase.
Hyperhomocyst(e)inemia is believed to injure endothelial cells in vivo through a number of mechanisms, including the generation of hydrogen peroxide (H2O2). Earlier in vitro studies demonstrated that homocyst(e)ine (Hcy) decreases the biological activity of endothelium-derived relaxing factor and that this decrease can be reversed by preventing the generation of hydrogen peroxide. Here we show that Hcy treatment of bovine aortic endothelial cells leads to a dose-dependent decrease in NOx (p = 0.001 by one-way analysis of variance) independent of endothelial nitric-oxide synthase activity or protein levels and nos3 transcription, suggesting that Hcy affects the bioavailability of NO, not its production. We hypothesized that, in addition to increasing the generation of H2O2, Hcy decreases the cell's ability to detoxify H2O2 by impairing intracellular antioxidant enzymes, specifically the intracellular isoform of glutathione peroxidase (GPx). To test this hypothesis, confluent bovine aortic endothelial cells were treated with a range of concentrations of Hcy, and intracellular GPx activity was determined. Compared with control cells, cells treated with Hcy showed a significant reduction in GPx activity (up to 81% at 250 microM Hcy). In parallel with the decrease in GPx activity, steady-state GPx mRNA levels were also significantly decreased compared with control levels after exposure to Hcy, which appeared not to be a consequence of message destabilization. These data suggest a novel mechanism by which Hcy, in addition to increasing the generation of hydrogen peroxide, may selectively impair the endothelial cell's ability to detoxify H2O2, thus rendering NO more susceptible to oxidative inactivation.
Animals↗
Homocysteine, oxidative stress, and vascular disease.
First recognized in patients with rare inborn errors of metabolism, the association of elevated plasma homocysteine concentrations with atherosclerosis and thrombosis now seems relevant to the general population as well. The mechanism of injury appears to involve oxidative damage to endothelial cells. Vitamin supplementation can normalize homocysteine levels and may lower the incidence of atherothrombotic vascular disease.
Age Factors↗
Hyperhomocyst(e)inemia and atherothrombosis.
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Animals↗
High-dose heparin decreases nitric oxide production by cultured bovine endothelial cells.
BACKGROUND: Abrupt cessation of heparin therapy can lead to a recrudescence of thrombosis and acute ischemia. Endothelial NO is an important endogenous inhibitor of platelet-mediated thrombosis, yet biochemical studies examining the effect of heparin on NO production by the endothelium have heretofore been lacking. METHODS AND RESULTS: In an attempt to address the effect of heparin on endothelial cell production of NO, confluent bovine aortic endothelial cells (BAECs) on microcarrier beads were incubated in the presence or absence of heparin. Results indicate that BAECs incubated with heparin were less able to inhibit platelet aggregation than control cells (P<.005 by ANOVA) and that this effect correlated with a decrease in NO production (36% decrease for heparin compared with control, P<.05). Dextran sulfate evoked the same response (67% decrease, P<.0001 compared with control), suggesting that the decrease in NO after heparin treatment is secondary to its negative charge rather than to a specific polysaccharide sequence. The decrease in NO production by heparin was accompanied by a 72% decrease in steady-state Nos 3 mRNA as well as a 49% decrease in immunodetectable endothelial NO synthase (eNOS) protein. CONCLUSIONS: These data show that high-dose heparin at concentrations achieved in some acute cardiovascular settings increases in vitro platelet aggregation in media conditioned by endothelial cells by decreasing endothelial NO production through a mechanism that involves a decrease in steady-state Nos 3 mRNA and eNOS protein. These observations suggest a possible mechanism by which to explain in part the prothrombotic effects of heparin.
Animals↗
Detection of S-nitrosothiols and other nitric oxide derivatives by photolysis-chemiluminescence spectrometry.
We have developed a method for the detection of biologically relevant derivatives of NO. that couples photolysis to chemiluminescence. S-Nitrosothiols, N-nitroso-L-tryptophan, and iron-nitrosyl derivatives can all be detected by this methodology, and these derivatives can be distinguished from free NO. by making measurements with and without photolysis. The limits of detection of this system are in the 10 nM range, with an intraassay variability of less than 2% and an interassay variability of less than 1.5%. The use of cutoff filters and alterations in pH and flow rate through the photolysis cell provide additional useful parameters with which to characterize specific NO. derivatives based on their intrinsic stability. This methodology is a useful addition to the growing armamentarium of techniques used to characterize the biochemistry and metabolism of NO..
Chelating Agents↗
Pulmonary hypertension: newer concepts in diagnosis and management.
Pulmonary hypertension comprises a family of disorders occurring as a primary disease or as a complication of a large number of respiratory and cardiac diseases. Pulmonary hypertension is present when pulmonary artery pressure or mean pressure exceeds 30 mmHg or 20 mmHg, respectively. Underlying the hemodynamic changes that result in pulmonary hypertension, whether from hypoxia, acidosis, increased pulmonary blood flow, increased shear stress, or idiopathic causes, is a dysfunctional vascular endothelium. In this review, the role of the history and physical examination in the initial assessment is emphasized. Newer diagnostic modalities, such as subselective pulmonary angiography and ultrafast computed tomography scanning, are reviewed. Low-flow oxygen, anticoagulation, and calcium-channel blockade are presented as accepted therapeutic modalities. Inhaled nitric oxide and prostacyclin infusion are presented as newer therapies that may be useful given the limited availability of donor organs for hear-lung transplantation. Future therapeutic strategies are likely to develop from advances in vascular biology.
Blood Viscosity↗
Nitric oxide binding and the adverse effects of cell-free hemoglobins: what makes us different from earthworms.
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Animals↗
The Narrow Therapeutic Index of Thrombin Inhibition: Implications for Newer Antithrombotic Therapies.
Recent trials have compared direct thrombin inhibitors with heparin as adjunctive therapy with thrombolysis for acute myocardial infarction or as antithrombotic therapy following coronary angioplasty. The results suggest that these agents are comparable to heparin in terms of efficacy, and can be safely administered; however, like that of heparin, the therapeutic index of direct thrombin inhibitors is narrow. Thus, one must excercise caution in trial design and data interpretation from studies of these antithrombotic agents in patients with acute coronary syndromes. The potential applicability of these agents to patients with acute coronary syndromes, the appropriate dosing regimen, and the patient population in whom the therapeutic index is optimal all await further study.
Journal Article↗