Joshua P. Fessel

Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension

Free radicals have been implicated in the pathogenesis of an increasing number of diseases. Lipids, which undergo peroxidation, are major targets of free radical attack. We report the discovery of a pathway of lipid peroxidation that forms a series of isomers in vivo that are characterized by a substituted tetrahydrofuran ring structure, termed isofurans (IsoFs). We have proposed two distinct pathways by which IsoFs can be formed based on 18O2 and H218O labeling studies. Measurement of F2-isoprostanes (IsoPs), prostaglandin F2-like compounds formed nonenzymatically as products of lipid peroxidation, is considered one of the most reliable approaches for assessing oxidative stress status in vivo. However, one limitation with this approach is that the formation of IsoPs becomes limited at high oxygen tension. In contrast, the formation of IsoFs becomes increasingly favored as oxygen tension increases. IsoFs are present at readily detectable levels in normal fluids and tissues, and levels increase dramatically in CCl4-treated rats, an animal model of oxidant injury. The ratio of IsoFs to IsoPs in major organs varies according to normal steady-state tissue oxygenation. In addition, IsoFs show a marked increase early in the course of hyperoxia-induced lung injury, whereas IsoPs do not significantly increase. We propose that combined measurement of IsoFs and IsoPs should provide a more reliable index of oxidant stress severity than quantification of either alone because of the opposing modulation of the two pathways by oxygen tension, which can vary widely in different organs and disease states.

Oxidative Mediated Lipid Peroxidation Recapitulates Proarrhythmic Effects on Cardiac Sodium Channels

Sudden cardiac death attributable to ventricular tachycardia/fibrillation (VF) remains a catastrophic outcome of myocardial ischemia and infarction. At the same time, conventional antagonist drugs targeting ion channels have yielded poor survival benefits. Although pharmacological and genetic models suggest an association between sodium (Na+) channel loss-of-function and sudden cardiac death, molecular mechanisms have not been identified that convincingly link ischemia to Na+ channel dysfunction and ventricular arrhythmias. Because ischemia can evoke the generation of reactive oxygen species, we explored the effect of oxidative stress on Na+ channel function. We show here that oxidative stress reduces Na+ channel availability. Both the general oxidant tert-butyl-hydroperoxide and a specific, highly reactive product of the isoprostane pathway of lipid peroxidation, E2-isoketal, potentiate inactivation of cardiac Na+ channels in human embryonic kidney (HEK)-293 cells and cultured atrial (HL-1) myocytes. Furthermore, E2-isoketals were generated in the epicardial border zone of the canine healing infarct, an arrhythmogenic focus where Na+ channels exhibit similar inactivation defects. In addition, we show synergistic functional effects of flecainide, a proarrhythmic Na+ channel blocker, and oxidative stress. These data suggest Na+ channel dysfunction evoked by lipid peroxidation is a candidate mechanism for ischemia-related conduction abnormalities and arrhythmias.

The Biochemistry of the Isoprostane, Neuroprostane, and Isofuran Pathways of Lipid Peroxidation

Isoprostanes are prostaglandin‐like compounds that are formed non‐enzymatically by free radical‐catalyzed peroxidation of arachidonic acid (C20:4ω6). Intermediates in the pathway of the formation of isoprostanes are labile prostaglandin H2‐like bicyclic endoperoxides (H2‐isoprostanes). H2‐isoprostanes are reduced to form F‐ring isoprostanes (F2‐isoprostanes), but they also undergo chemical rearrangement in vivo to form E2‐ and D2‐isoprostanes, isothromboxanes, and highly reactive acyclic γ‐ketoaldehdyes (isoketals). E2‐ and D2‐isoprostanes also undergo dehydration in vivo to form cyclopentenone A2‐ and J2‐isoprostanes. Docosahexaenoic acid (C22:6ω3) is highly enriched in neurons in the brain and is highly susceptible to oxidation. Free radical‐catalyzed oxidation of docosahexaenoic acid results in the formation of isoprostane‐like compounds (neuroprostanes). F4‐,D4‐,E4‐,A4‐,and J4‐neuroprostanes and neuroketals have all been shown to be produced in vivo. In addition, we recently discovered a new pathway of lipid peroxidation that forms compounds with a substituted tetrahydrofuran ring (isofurans). Oxygen concentration differentially modulates the formation of isoprostanes and isofurans. As oxygen concentrations increase, the formation of isofurans is favored whereas the formation of isoprostanes becomes disfavored.

Orthostatic hypertension: when pressor reflexes overcompensate

Orthostatic hypertension—a rise in blood pressure upon assuming upright posture—is an underappreciated and understudied clinical phenomenon. There is currently no widely agreed-upon definition of clinical orthostatic hypertension, the current definitions being operational within the context of particular studies. The underlying pathophysiology is thought to involve activation of the sympathetic nervous system, but the actual etiology is poorly understood. Orthostatic hypertension is observed in association with a variety of other clinical conditions, including essential hypertension, dysautonomias, and type 2 diabetes mellitus. Orthostatic hypertension has been associated with increased occurrence of silent cerebrovascular ischemia and possibly with neuropathy in type 2 diabetes. So, appreciation of the true incidence of orthostatic hypertension, elucidation of the underlying pathophysiology, and an understanding of potentially effective treatment approaches and their associated risks and benefits might all have major clinical significance. Orthostatic hypertension is an aspect of hypertension that is in need of further focused investigation.

Isofurans, but not F2-isoprostanes, are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease

F2‐isoprostanes (F2‐IsoPs) are well‐established sensitive and specific markers of oxidative stress in vivo. Isofurans (IsoFs) are also products of lipid peroxidation, but in contrast to F2‐IsoPs, their formation is favored when oxygen tension is increased in vitro or in vivo. Mitochondrial dysfunction in Parkinsons disease (PD) may not only lead to oxidative damage to brain tissue but also potentially result in increased intracellular oxygen tension, thereby influencing relative concentrations of F2‐IsoPs and IsoFs. In this study, we attempted to compare the levels of F2‐IsoPs and IsoFs esterified in phospholipids in the substantia nigra (SN) from patients with PD to those of age‐matched controls as well as patients with other neurodegenerative diseases, including dementia with Lewy body disease (DLB), multiple system atrophy (MSA), and Alzheimers disease (AD). The results demonstrated that IsoFs but not F2‐IsoPs in the SN of patients with PD and DLB were significantly higher than those of controls. Levels of IsoFs and F2‐IsoPs in the SN of patients with MSA and AD were indistinguishable from those of age‐matched controls. This preferential increase in IsoFs in the SN of patients with PD or DLB not only indicates a unique mode of oxidant injury in these two diseases but also suggests different underlying mechanisms of dopaminergic neurodegeneration in PD and DLB from those of MSA.

Metabolomic analysis of bone morphogenetic protein receptor type 2 mutations in human pulmonary endothelium reveals widespread metabolic reprogramming

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease of the lung vasculature for which the molecular etiologies are unclear. Specific metabolic alterations have been identified in animal models and in PAH patients, though existing data focus mainly on abnormalities of glucose homeostasis. We hypothesized that analysis of the entire metabolome in PAH would reveal multiple other metabolic changes relevant to disease pathogenesis and possible treatment. Layered transcriptomic and metabolomic analyses of human pulmonary microvascular endothelial cells (hPMVEC) expressing two different disease-causing mutations in the bone morphogenetic protein receptor type 2 (BMPR2) confirmed previously described increases in aerobic glycolysis but also uncovered significant upregulation of the pentose phosphate pathway, increases in nucleotide salvage and polyamine biosynthesis pathways, decreases in carnitine and fatty acid oxidation pathways, and major impairment of the tricarboxylic acid (TCA) cycle and failure of anaplerosis. As a proof of principle, we focused on the TCA cycle, predicting that isocitrate dehydrogenase (IDH) activity would be altered in PAH, and then demonstrating increased IDH activity not only in cultured hPMVEC expressing mutant BMPR2 but also in the serum of PAH patients. These results suggest that widespread metabolic changes are an important part of PAH pathogenesis, and that simultaneous identification and targeting of the multiple involved pathways may be a more fruitful therapeutic approach than targeting of any one individual pathway.

Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension

Dysregulation of vascular stiffness and cellular metabolism occurs early in pulmonary hypertension (PH). However, the mechanisms by which biophysical properties of the vascular extracellular matrix (ECM) relate to metabolic processes important in PH remain undefined. In this work, we examined cultured pulmonary vascular cells and various types of PH-diseased lung tissue and determined that ECM stiffening resulted in mechanoactivation of the transcriptional coactivators YAP and TAZ (WWTR1). YAP/TAZ activation modulated metabolic enzymes, including glutaminase (GLS1), to coordinate glutaminolysis and glycolysis. Glutaminolysis, an anaplerotic pathway, replenished aspartate for anabolic biosynthesis, which was critical for sustaining proliferation and migration within stiff ECM. In vitro, GLS1 inhibition blocked aspartate production and reprogrammed cellular proliferation pathways, while application of aspartate restored proliferation. In the monocrotaline rat model of PH, pharmacologic modulation of pulmonary vascular stiffness and YAP-dependent mechanotransduction altered glutaminolysis, pulmonary vascular proliferation, and manifestations of PH. Additionally, pharmacologic targeting of GLS1 in this model ameliorated disease progression. Notably, evaluation of simian immunodeficiency virus-infected nonhuman primates and HIV-infected subjects revealed a correlation between YAP/TAZ-GLS activation and PH. These results indicate that ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis and anaplerosis, and thereby link mechanical stimuli to dysregulated vascular metabolism. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in PH.

Evidence for Right Ventricular Lipotoxicity in Heritable Pulmonary Arterial Hypertension

RATIONALE Shorter survival in heritable pulmonary arterial hypertension (HPAH), often due to BMPR2 mutation, has been described in association with impaired right ventricle (RV) compensation. HPAH animal models are insulin resistant, and cells with BMPR2 mutation have impaired fatty acid oxidation, but whether these findings affect the RV in HPAH is unknown. OBJECTIVES To test the hypothesis that BMPR2 mutation impairs RV hypertrophic responses in association with lipid deposition. METHODS RV hypertrophy was assessed in two models of mutant Bmpr2 expression, smooth muscle-specific (Sm22(R899X)) and universal expression (Rosa26(R899X)). Littermate control mice underwent the same stress using pulmonary artery banding (Low-PAB). Lipid content was assessed in rodent and human HPAH RVs and in Rosa26(R899X) mice after metformin administration. RV microarrays were performed using human HPAH and control subjects. RESULTS RV/(left ventricle + septum) did not rise directly in proportion to RV systolic pressure in Rosa26(R899X) but did in Sm22(R899X) (P < 0.05). Rosa26(R899X) RVs demonstrated intracardiomyocyte triglyceride deposition not present in Low-PAB (P < 0.05). RV lipid deposition was identified in human HPAH RVs but not in controls. Microarray analysis demonstrated defects in fatty acid oxidation in human HPAH RVs. Metformin in Rosa26(R899X) mice resulted in reduced RV lipid deposition. CONCLUSIONS These data demonstrate that Bmpr2 mutation affects RV stress responses in a transgenic rodent model. Impaired RV hypertrophy and triglyceride and ceramide deposition are present as a function of RV mutant Bmpr2 in mice; fatty acid oxidation impairment in human HPAH RVs may underlie this finding. Further study of how BMPR2 mediates RV lipotoxicity is warranted.

A potential role for insulin resistance in experimental pulmonary hypertension

Patients with pulmonary arterial hypertension have increased prevalence of insulin resistance. We aimed to determine whether metabolic defects are associated with bone morphogenic protein receptor type 2 (Bmpr2) mutations in mice, and whether these may contribute to pulmonary vascular disease development. Metabolic phenotyping was performed on transgenic mice with inducible expression of Bmpr2 mutation, R899X. Phenotypic penetrance in Bmpr2R899X was assessed in a high-fat diet model of insulin resistance. Alterations in glucocorticoid responses were assessed in murine pulmonary microvascular endothelial cells and Bmpr2R899X mice treated with dexamethasone. Compared to controls, Bmpr2R899X mice showed increased weight gain and demonstrated insulin resistance as assessed by the homeostatic model assessment insulin resistance (1.0±0.4 versus 2.2±1.8) and by fat accumulation in skeletal muscle and decreased oxygen consumption. Bmpr2R899X mice fed a high-fat diet had strong increases in pulmonary hypertension penetrance (seven out of 11 versus three out of 11). In cell culture and in vivo experiments, Bmpr2 mutation resulted in a combination of constitutive glucocorticoid receptor activation and insensitivity. Insulin resistance is present as an early feature of Bmpr2 mutation in mice. Exacerbated insulin resistance through high-fat diet worsened pulmonary phenotype, implying a possible causal role in disease. Impaired glucocorticoid responses may contribute to metabolic defects.

Plasma biomarkers of oxidant stress and development of organ failure in severe sepsis.

We hypothesized that circulating levels of lipid peroxidation products in patients with severe sepsis are associated with the development of pulmonary, renal, hepatic, circulatory, and coagulation failure. Plasma levels of F2-isoprostanes and isofurans were measured by mass spectroscopy on intensive care unit day 2 in 50 critically ill patients with severe sepsis. Plasma F2-isoprostane levels were higher in patients who developed renal failure compared with those who did not (65 pg/mL [interquartile range {IQR} 44-112] vs. 44 pg/mL [IQR 29-54], P = 0.009) as were isofuran levels (1,223 pg/mL [IQR 348-2,531] vs. 329 pg/mL [IQR 156-1,127], P = 0.009). Plasma F2-isoprostane levels were higher in patients who developed hepatic failure compared with those who did not (72 pg/mL [IQR 44-112] vs. 44 pg/mL [IQR 30-65], P = 0.023), and there was also a trend for higher isofuran levels (1,411 pg/mL [IQR 298-1,965] vs. 525 pg/mL [IQR 160-1,223], P = 0.14). Coagulation failure (thrombocytopenia) was associated with higher isofuran levels. Circulatory failure and acute lung injury were not associated with elevated levels of isoprostanes or isofurans. Patients with isoprostane levels above the 25th percentile had higher mortality (42%) compared with patients with levels below the 25th percentile (8%, P = 0.03). Plasma levels of F2-isoprostanes and isofurans are associated with renal, hepatic, and coagulation failure, but not with circulatory or pulmonary failure in severe sepsis, suggesting that lipid peroxidation is a prominent feature of septic multisystem organ failure. Plasma isoprostanes and isofurans may be useful for monitoring oxidative stress in treatment trials of antioxidant therapies in severe sepsis.ABBREVIATIONS: APACHE II-Acute Physiology And Chronic Health Evaluation II; ICU-intensive care unit; IQR-interquartile range; SAPS II-Simplified Acute Physiology Score II; VALID Study-Validating Acute Lung Injury biomarkers for Diagnosis Study