Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.

Background: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH.

Redox Regulation, Oxidative Stress, and Inflammation in Group 3 Pulmonary Hypertension.

Group 3 pulmonary hypertension (PH), which occurs secondary to hypoxia lung diseases, is one of the most common causes of PH worldwide and has a high unmet clinical need. A deeper understanding of the integrative pathological and adaptive molecular mechanisms within this group is required to inform the development of novel drug targets and effective treatments. The production of oxidants is increased in PH Group 3, and their pleiotropic roles include contributing to disease progression by promoting prolonged hypoxic pulmonary vasoconstriction and pathological pulmonary vascular remodeling, but also stimulating adaptation to pathological stress that limits the severity of this disease.

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans.

Background: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans.

Methods: In mouse studies, phospholemman knock-in mice (PLM; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements.

Complex interrelationships between nitro-alkene-dependent inhibition of soluble epoxide hydrolase, inflammation and tumor growth.

Nitro-oleate (10-nitro-octadec-9-enoic acid), which inhibits soluble epoxide hydrolase (sEH) by covalently adducting to C521, increases the abundance of epoxyeicosatrienoic acids (EETs) that can be health promoting, for example by lowering blood pressure or their anti-inflammatory actions. However, perhaps consistent with their impact on angiogenesis, increases in EETs may exacerbate progression of some cancers. To assess this, Lewis lung carcinoma (LLc1) cells were exposed to oleate or nitro-oleate, with the latter inhibiting the hydrolase and increasing their proliferation and migration in vitro.

Oxidation of PKGIα mediates an endogenous adaptation to pulmonary hypertension.

Chronic hypoxia causes pulmonary hypertension (PH), vascular remodeling, right ventricular (RV) hypertrophy, and cardiac failure. Protein kinase G Iα (PKGIα) is susceptible to oxidation, forming an interprotein disulfide homodimer associated with kinase targeting involved in vasodilation. Here we report increased disulfide PKGIα in pulmonary arteries from mice with hypoxic PH or lungs from patients with pulmonary arterial hypertension.

Redox-dependent dimerization of p38α mitogen-activated protein kinase with mitogen-activated protein kinase kinase 3.

The kinase p38α MAPK (p38α) plays a pivotal role in many biological processes. p38α is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38α.

Examining a role for PKG Iα oxidation in the pathogenesis of cardiovascular dysfunction during diet-induced obesity.

Background: Protein kinase G (PKG) Iα is the end-effector kinase that mediates nitric oxide (NO)-dependent and oxidant-dependent vasorelaxation to maintain blood pressure during health. A hallmark of cardiovascular disease is attenuated NO production, which in part is caused by NO Synthase (NOS) uncoupling, which in turn increases oxidative stress because of superoxide generation. NOS uncoupling promotes PKG Iα oxidation to the interprotein disulfide state, likely mediated by superoxide-derived hydrogen peroxide, and because the NO-cyclic guanosine monophosphate (cGMP) pathway otherwise negatively regulates oxidation of the kinase to its active disulfide dimeric state.

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response.

The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation.

Deficient angiogenesis in redox-dead Cys17Ser PKARIα knock-in mice.

Angiogenesis is essential for tissue development, wound healing and tissue perfusion, with its dysregulation linked to tumorigenesis, rheumatoid arthritis and heart disease. Here we show that pro-angiogenic stimuli couple to NADPH oxidase-dependent generation of oxidants that catalyse an activating intermolecular-disulphide between regulatory-RIα subunits of protein kinase A (PKA), which stimulates PKA-dependent ERK signalling. This is crucial to blood vessel growth as 'redox-dead' Cys17Ser RIα knock-in mice fully resistant to PKA disulphide-activation have deficient angiogenesis in models of hind limb ischaemia and tumour-implant growth.

Quantification of microcirculatory blood flow: a sensitive and clinically relevant prognostic marker in murine models of sepsis.

Sepsis and sepsis-associated multiorgan failure represent the major cause of mortality in intensive care units worldwide. Cardiovascular dysfunction, a key component of sepsis pathogenesis, has received much research interest, although research translatability remains severely limited. There is a critical need for more comprehensive preclinical sepsis models, with more clinically relevant end points, such as microvascular perfusion.

Protein kinase G Iα oxidation paradoxically underlies blood pressure lowering by the reductant hydrogen sulfide.

Dysregulated blood pressure control leading to hypertension is prevalent and is a risk factor for several common diseases. Fully understanding blood pressure regulation offers the possibility of developing rationale therapies to alleviate hypertension and associated disease risks. Although hydrogen sulfide (H2S) is a well-established endogenous vasodilator, the molecular basis of its blood-pressure lowering action is incompletely understood.

Biochemical methods for monitoring protein thiol redox states in biological systems.

Oxidative post-translational modifications of proteins resulting from events that increase cellular oxidant levels play important roles in physiological and pathophysiological processes. Evaluation of alterations to protein redox states is increasingly common place because of methodological advances that have enabled detection, quantification and identification of such changes in cells and tissues. This mini-review provides a synopsis of biochemical methods that can be utilized to monitor the array of different oxidative and electrophilic modifications that can occur to protein thiols and can be important in the regulatory or maladaptive impact oxidants can have on biological systems.

Protection from hypertension in mice by the Mediterranean diet is mediated by nitro fatty acid inhibition of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is inhibited by electrophilic lipids by their adduction to Cys521 proximal to its catalytic center. This inhibition prevents hydrolysis of the enzymes' epoxyeicosatrienoic acid (EET) substrates, so they accumulate inducing vasodilation to lower blood pressure (BP). We generated a Cys521Ser sEH redox-dead knockin (KI) mouse model that was resistant to this mode of inhibition.

Experimental hyperleptinemia in neonatal rats leads to selective leptin responsiveness, hypertension, and altered myocardial function.

The prevalence of obesity among pregnant women is increasing. Evidence from human cohort studies and experimental animals suggests that offspring cardiovascular and metabolic function is compromised through early life exposure to maternal obesity. Previously, we reported that juvenile offspring of obese rats develop sympathetically mediated hypertension associated with neonatal hyperleptinemia.

Anti-proliferative actions of T-type calcium channel inhibition in Thy1 nephritis.

Aberrant proliferation of mesangial cells (MCs) is a key finding in progressive glomerular disease. TH1177 is a small molecule that has been shown to inhibit low-voltage activated T-type Ca(2+) channels (TCCs). The current study investigates the effect of TH1177 on MC proliferation in vitro and in vivo.

Protein kinase G oxidation is a major cause of injury during sepsis.

Sepsis is a common life-threatening clinical syndrome involving complications as a result of severe infection. A cardinal feature of sepsis is inflammation that results in oxidative stress. Sepsis in wild-type mice induced oxidative activation of cGMP-dependent protein kinase 1 alpha (PKG Iα), which increased blood vessel dilation and permeability, and also lowered cardiac output.

cGMP-dependent activation of protein kinase G precludes disulfide activation: implications for blood pressure control.

Protein kinase G (PKG) is activated by nitric oxide (NO)-induced cGMP binding or alternatively by oxidant-induced interprotein disulfide formation. We found preactivation with cGMP attenuated PKG oxidation. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) blockade of cGMP production increased disulfide PKG to 13 ± 2% and 29±4% of total in aorta and mesenteries, respectively.

Nitroglycerin fails to lower blood pressure in redox-dead Cys42Ser PKG1α knock-in mouse.

Background: Although nitroglycerin has remained in clinical use since 1879, the mechanism by which it relaxes blood vessels to lower blood pressure remains incompletely understood. Nitroglycerin undergoes metabolism that generates several reaction products, including oxidants, and this bioactivation process is essential for vasodilation. Protein kinase G (PKG) mediates classic nitric oxide-dependent vasorelaxation, but the 1α isoform is also independently activated by oxidation that involves interprotein disulfide formation within this homodimeric protein complex.

Single atom substitution in mouse protein kinase G eliminates oxidant sensing to cause hypertension.

Blood pressure regulation is crucial for the maintenance of health, and hypertension is a risk factor for myocardial infarction, heart failure, stroke and renal disease. Nitric oxide (NO) and prostacyclin trigger well-defined vasodilator pathways; however, substantial vasorelaxation in response to agents such as acetylcholine persists when the synthesis of these molecules is prevented. This remaining vasorelaxation activity, termed endothelium-derived hyperpolarizing factor (EDHF), is more prevalent in resistance than in conduit blood vessels and is considered a major mechanism for blood pressure control.

Increased cardiovascular reactivity to acute stress and salt-loading in adult male offspring of fat fed non-obese rats.

Diet-induced obesity in rat pregnancy has been shown previously to be associated with consistently raised blood pressure in the offspring, attributed to sympathetic over-activation, but the relative contributions to this phenotype of maternal obesity versus raised dietary fat is unknown. Sprague-Dawley female rats were fed either a control (4.3% fat, n = 11) or lard-enriched (23.

Nitrosative protein oxidation is modulated during early endotoxemia.

Formation of nitric oxide and its derivative reactive nitrogen species during endotoxemia has been implicated in the pathogenesis of the associated cardiovascular dysfunction. This stress can promote nitrosative post-translational modifications of proteins that may alter their activity and contribute to dysregulation. We utilized the ascorbate-dependent biotin-switch method to assay protein S-nitrosylation and immunoblotted for tyrosine nitration to monitor changes in nitrosative protein oxidation during endotoxemia.