Characteristic disease defects in circulating endothelial cells isolated from patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary arterial pressures that can lead to right heart failure and death. No cure exists for this disease, but therapeutic advancements have extended its median survival from 2 to 7 years. Mechanistic research in PAH has been limited by factors including that a) animal models do not fully recapitulate the disease or provide insights into its pathogenesis, and b) cellular material from PAH patients is primarily obtained from donor lungs during autopsy or transplantation, which reflect end-stage disease.

Cutaneous iontophoresis of vasoactive medications in patients with scleroderma-associated pulmonary arterial hypertension.

Background: It remains unknown whether the cutaneous microvascular responses are different between patients with scleroderma-associated pulmonary arterial hypertension (SSc-PAH) and SSc without pulmonary hypertension (PH).

Methods: We included 59 patients with SSc between March 2013 and September 2019. We divided patients into 4 groups: (a) no PH by right heart catheterization (RHC) (n = 8), (b) no PH by noninvasive screening tests (n = 16), (c) treatment naïve PAH (n = 16), and (d) PAH under treatment (n = 19).

Disease-specific platelet signaling defects in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a rapidly progressive disease with several treatment options. Long-term mortality remains high with great heterogeneity in treatment response. Even though most of the pathology of IPAH is observed in the lung, there is systemic involvement.

Specific O-GlcNAc modification at Ser-615 modulates eNOS function.

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and devastating disease characterized by vascular smooth muscle and endothelial cell proliferation leading to a narrowing of the vessels in the lung. The increased resistance in the lung and the higher pressures generated result in right heart failure. Nitric Oxide (NO) deficiency is considered a hallmark of IPAH and altered function of endothelial nitric oxide synthase (eNOS), decreases NO production.

Platelet glycolytic metabolism correlates with hemodynamic severity in pulmonary arterial hypertension.

Group 1 pulmonary hypertension (PH), i.e., pulmonary arterial hypertension (PAH), is associated with a metabolic shift favoring glycolysis in cells comprising the lung vasculature as well as skeletal muscle and right heart.

A pilot study on the kinetics of metabolites and microvascular cutaneous effects of nitric oxide inhalation in healthy volunteers.

Rationale: Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked.

Objectives: We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response.

O-linked β-N-acetylglucosamine transferase directs cell proliferation in idiopathic pulmonary arterial hypertension.

Background: Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated.

Function and distribution of apolipoprotein A1 in the artery wall are markedly distinct from those in plasma.

Background: Prior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall.

Disulfide bond as a switch for copper-zinc superoxide dismutase activity in asthma.

Aim: Loss of superoxide dismutase (SOD) activity is a defining biochemical feature of asthma. However, mechanisms for the reduced activity are unknown. We hypothesized that loss of asthmatic SOD activity is due to greater susceptibility to oxidative inactivation.

Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains.

In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c.

Abnormal platelet aggregation in idiopathic pulmonary arterial hypertension: role of nitric oxide.

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease. Several processes are believed to lead to the fatal progressive pulmonary arterial narrowing seen in IPAH including vasoconstriction, cellular proliferation inflammation, vascular remodeling, abnormalities in the lung matrix, and in situ thrombosis. Nitric oxide (NO) produced by NO synthases (NOS) is a potent vasodilator and plays important roles in many other processes including platelet function.

GAPDH regulates cellular heme insertion into inducible nitric oxide synthase.

Heme proteins play essential roles in biology, but little is known about heme transport inside mammalian cells or how heme is inserted into soluble proteins. We recently found that nitric oxide (NO) blocks cells from inserting heme into several proteins, including cytochrome P450s, hemoglobin, NO synthases, and catalase. This finding led us to explore the basis for NO inhibition and to identify cytosolic proteins that may be involved, using inducible NO synthase (iNOS) as a model target.

Glucose-modulated tyrosine nitration in beta cells: targets and consequences.

Hyperglycemia, key factor of the pre-diabetic and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in islets of Langerhans and insulinoma beta cells. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations.

Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase.

Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded).

Glucose-mediated tyrosine nitration in adipocytes: targets and consequences.

Hyperglycemia, a key factor in insulin resistance and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in 3T3-L1 adipocytes. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations.

Versatile regulation of neuronal nitric oxide synthase by specific regions of its C-terminal tail.

The C-terminal tail (CT) of neuronal nitric oxide synthase (nNOS) is a regulatory element that suppresses nNOS activities in the absence of bound calmodulin (CaM). A crystal structure of the nNOS reductase domain (nNOSr) (Garcin, E. D.

A connecting hinge represses the activity of endothelial nitric oxide synthase.

In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved.

Abnormalities in nitric oxide and its derivatives in lung cancer.

Rationale: A cellular prooxidant state promotes cells to neoplastic growth, in part because of modification of proteins and their functions. Reactive nitrogen species formed from nitric oxide (NO) or its metabolites, can lead to protein tyrosine nitration, which is elevated in lung cancer.

Objective: To determine the alteration in these NO derivatives and the role they may play in contributing to lung carcinogenesis.

Immunohistochemical detection and Western blot analysis of nitrated protein in stored human corneal epithelium.

While the production of nitric oxide by human corneas in storage has recently been demonstrated, protein nitration as a result of this production has not been demonstrated. In this study, nitrated protein accumulation in the epithelium of stored human corneas was assessed. One half of five donor corneas maintained in storage media for 3 days were prepared for immunohistochemical studies.

Proteomic method for identification of tyrosine-nitrated proteins.

Biologic nitration of protein tyrosine (to form 3-nitrotyrosine) is a recently described phenomenon that is associated with many diseases. We have devised a proteomic methodology to identify these modified proteins. This utilizes protein fractionation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), partial transfer onto polyvinylidene difluoride (PVDF) membranes, and Western blot analysis using an antinitrotyrosine antibody to identify the proteins.

The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain.

Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding.

Rapid and selective oxygen-regulated protein tyrosine denitration and nitration in mitochondria.

Growing evidence connects a cumulative formation of 3-nitrotyrosyl adducts in proteins as a marker for oxidative damage with the pathogenesis of various diseases and pathological conditions associated with oxidative stress. A physiological signaling role for protein nitration has also been suggested. Controlled "denitration" would be essential for such a contribution of protein nitration to cellular regulatory processes.

Inhibition of nitric oxide synthesis in corneas in storage media.

The nitrate/nitrite content in storage media was determined after nitric oxide synthase inhibition by adding 400 microl of 100 mm N(G)-monomethyl-l-arginine (LMMA) to four chambers of Optisol GS corneal storage media, each containing one viable human cornea. The companion corneas in storage media without LMMA served as controls. Four hundred microlitre aliquots obtained at baseline (day 0) and at one-day intervals for 20 more days for both groups were analyzed for nitrate and nitrite (breakdown products of nitric oxide) concentration levels using a spectrophotometric method based on the Greiss reaction.

Tyrosine nitration impairs mammalian aldolase A activity.

Protein tyrosine nitration increases in vivo as a result of oxidative stress and is elevated in numerous inflammatory-associated diseases. Mammalian fructose-1,6-bisphosphate aldolases are tyrosine nitrated in lung epithelial cells and liver, as well as in retina under different inflammatory conditions. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we now show that aldolase A is nitrated in human skin fibroblasts.