Jeffrey Baust

The roles of iNOS in liver ischemia-reperfusion injury.

To determine the contribution of the inducible nitric oxide synthase (iNOS) to hepatic injury following warm ischemia-reperfusion, we developed a model of partial hepatic ischemia-reperfusion in mice and studied the injury response in iNOS knockout (KO) mice. Compared with wild types, iNOS KO animals exhibited lower plasma transaminase levels after 1 and 6 h of reperfusion following 1 h of ischemia. At the 3-h time point, enzyme levels were not different between the two groups. iNOS mRNA was not detectable in the ischemic hepatic lobes of wild-type mice until 3 h of reperfusion; however, perfusion studies identified a significant delay in reperfusion of the ischemic lobe in the iNOS KO mice at the 1-h time point with similar perfusion rates at 3 and 6 h compared with wild type. By way of comparison, mice deficient in the endothelial NOS (eNOS) were also assessed for the degree of hepatic damage 3 h post-reperfusion. Plasma transaminase levels were significantly increased in eNOS KO animals compared with wild-type controls. These data suggest that systemic as well as local sources of iNOS regulate reperfusion, and local iNOS contributes to hepatic injury, while eNOS is protective in warm hepatic ischemia-reperfusion.

Fatty Acid Nitroalkenes Ameliorate Glucose Intolerance and Pulmonary Hypertension in High Fat Diet-Induced Obesity

AIMS Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions. METHODS AND RESULTS It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels. CONCLUSIONS These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.

Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development.

Fibroblast growth factor receptors (Fgfrs) have critical roles in kidney development. FgfrIIIb is thought to act in epithelium, while FgfrIIIc functions in mesenchyme. We aimed to determine roles of Fgfr2IIIc in kidney development. Mice with deletion of Fgfr2IIIc (Fgfr2IIIc−/−) had normal kidneys. Combination of Fgfr2IIIc−/− with conditional deletion of Fgfr1 in metanephric mesenchyme (MM) (Fgfr1Mes−/−Fgfr2IIIc−/−) had small but identifiable MM at embryonic day (E) 10.5, expressing mesenchymal markers including Eya1, Six2, Pax2, and Gdnf (unlike Fgfr1/2Mes−/− mice that have no obvious MM). E11.5 Fgfr1Mes−/−Fgfr2IIIc−/− mice had rudimentary MM expressing only Eya1. Control, Fgfr2IIIc−/−, and Fgfr1Mes−/−Fgfr2IIIc−/− kidney mesenchymal tissues also express Fgfr2IIIb. In ureteric lineages, E10.5 Fgfr1Mes−/−Fgfr2IIIc−/− embryos had ureteric outgrowth (sometimes multiple buds); however, by E11.5 Gdnf absence lead to no ureteric elongation or branching (similar to Fgfr1/2Mes−/− mice). Beyond E12.5, Fgfr1Mes−/−Fgfr2IIIc−/− mice had no renal tissue. In conclusion, Fgfr2IIIc and Fgfr1 in kidney mesenchyme (together) are critical for normal early renal development. Developmental Dynamics, 2011.

Testosterone-dependent sex differences in red blood cell hemolysis in storage, stress, and disease

Red blood cell (RBC) hemolysis represents an intrinsic mechanism for human vascular disease. Intravascular hemolysis releases hemoglobin and other metabolites that inhibit nitric oxide signaling and drive oxidative and inflammatory stress. Although these pathways are important in disease pathogenesis, genetic and population modifiers of hemolysis, including sex, have not been established.

Nox-derived ROS are acutely activated in pressure overload pulmonary hypertension: indications for a seminal role for mitochondrial Nox4.

Pulmonary arterial hypertension is a severe progressive disease with marked morbidity and high mortality in which right ventricular (RV) failure is the major cause of death. Thus knowledge of the mechanisms underlying RV failure is an area of active interest. Previous studies suggest a role of NADPH oxidase in cardiomyocyte dysfunction in the left heart. Here we postulate that acute pressure overload induced by pulmonary artery banding (PAB) leads to a Nox4-initiated increase in reactive oxygen species (ROS) in mouse RV that may lead to feed-forward induction of Nox2. To test our hypothesis, ROS production was measured in RV and left ventricle homogenates. The data show that hydrogen peroxide (H2O2), but not superoxide anion (O2(·-)), was increased in the early phases (within 6 h) of PAB in RV and that this increase was diminished by catalase and diphenyleneiodonium chloride but not by SOD, N(ω)-nitro-l-arginin methyl ester, febuxostat, or indomethacin. H2O2 production in RV was not attenuated in Nox2 null mice subjected to 6 h PAB. Moreover, we observed an upregulation of Nox4 mRNA after 1 h of PAB and an increase in mitochondrial Nox4 protein 6 h post-PAB. In contrast, we observed an increase in Nox2 mRNA 1 day post-PAB. Expression of antioxidant enzymes SOD, catalase, and glutathione peroxidase did not change, but catalase activity increased 6 h post-PAB. Taken together, these findings show a role of mitochondria-localized Nox4 in the early phase of PAB and suggest an involvement of this isozyme in early ROS generation possibly contributing to progression of RV dysfunction and failure.

Cardiac CD47 Drives Left Ventricular Heart Failure Through Ca2+‐CaMKII‐Regulated Induction of HDAC3

Background Left ventricular heart failure (LVHF) remains progressive and fatal and is a formidable health problem because ever‐larger numbers of people are diagnosed with this disease. Therapeutics, while relieving symptoms and extending life in some cases, cannot resolve this process and transplant remains the option of last resort for many. Our team has described a widely expressed cell surface receptor (CD47) that is activated by its high‐affinity secreted ligand, thrombospondin 1 (TSP1), in acute injury and chronic disease; however, a role for activated CD47 in LVHF has not previously been proposed. Methods and Results In experimental LVHF TSP1‐CD47 signaling is increased concurrent with up‐regulation of cardiac histone deacetylase 3 (HDAC3). Mice mutated to lack CD47 displayed protection from transverse aortic constriction (TAC)‐driven LVHF with enhanced cardiac function, decreased cellular hypertrophy and fibrosis, decreased maladaptive autophagy, and decreased expression of HDAC3. In cell culture, treatment of cardiac myocyte CD47 with a TSP1‐derived peptide, which binds and activates CD47, increased HDAC3 expression and myocyte hypertrophy in a Ca2+/calmodulin protein kinase II (CaMKII)‐dependent manner. Conversely, antibody blocking of CD47 activation, or pharmacologic inhibition of CaMKII, suppressed HDAC3 expression, decreased myocyte hypertrophy, and mitigated established LVHF. Downstream gene suppression of HDAC3 mimicked the protective effects of CD47 blockade and decreased hypertrophy in myocytes and mitigated LVHF in animals. Conclusions These data identify a proximate role for the TSP1‐CD47 axis in promoting LVHF by CaKMII‐mediated up‐regulation of HDAC3 and suggest novel therapeutic opportunities.

TSP1–CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction

Aims Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1−/− mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1–CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. Methods and results We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1–CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47−/− mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47−/− pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1–CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. Conclusions In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1–CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Development of a Mouse Model of Metabolic Syndrome, Pulmonary Hypertension, and Heart Failure with Preserved Ejection Fraction

&NA; Pulmonary hypertension (PH) associated with heart failure with preserved ejection fraction (PH‐HFpEF; World Health Organization Group II) secondary to left ventricular (LV) diastolic dysfunction is the most frequent cause of PH. It is an increasingly recognized clinical complication of the metabolic syndrome. To date, no effective treatment has been identified, and no genetically modifiable mouse model is available for advancing our understanding for PH‐HFpEF. To develop a mouse model of PH‐HFpEF, we exposed 36 mouse strains to 20 weeks of high‐fat diet (HFD), followed by systematic evaluation of right ventricular (RV) and LV pressure‐volume analysis. The HFD induces obesity, glucose intolerance, insulin resistance, hyperlipidemia, as well as PH, in susceptible strains. We observed that certain mouse strains, such as AKR/J, NON/shiLtJ, and WSB/EiJ, developed hemodynamic signs of PH‐HFpEF. Of the strains that develop PH‐HFpEF, we selected AKR/J for further model validation, as it is known to be prone to HFD‐induced metabolic syndrome and had low variability in hemodynamics. HFD‐treated AKR/J mice demonstrate reproducibly higher RV systolic pressure compared with mice fed with regular diet, along with increased LV end‐diastolic pressure, both RV and LV hypertrophy, glucose intolerance, and elevated HbA1c levels. Time course assessments showed that HFD significantly increased body weight, RV systolic pressure, LV end‐diastolic pressure, biventricular hypertrophy, and HbA1c throughout the treatment period. Moreover, we also identified and validated 129S1/SvlmJ as a resistant mouse strain to HFD‐induced PH‐HFpEF. These studies validate an HFD/AKR/J mouse model of PH‐HFpEF, which may offer a new avenue for testing potential mechanisms and treatments for this disease.

Pharmacological Inhibition of mTOR Kinase Reverses Right Ventricle Remodeling and Improves Right Ventricle Structure and Function in Rats

Abstract Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling, increased pulmonary artery (PA) pressure, right‐heart afterload and death. Mechanistic target of rapamycin (mTOR) promotes smooth muscle cell proliferation, survival, and pulmonary vascular remodeling via two functionally distinct mTOR complexes (mTORCs)‐1 (supports cell growth) and ‐2 (promotes cell survival), and dual mTORC1/mTORC2 inhibition selectively induces pulmonary arterial hypertension PA vascular smooth muscle cell apoptosis and reverses pulmonary vascular remodeling. The consequences of mTOR inhibition on right ventricle (RV) morphology and function are not known. Using SU5416/hypoxia rat model of pulmonary hypertension (PH), we report that, in contrast to activation of both mTORC1 and mTORC2 pathways in small remodeled PAs, RV tissues had predominant up‐regulation of mTORC1 signaling accompanied by cardiomyocyte and RV hypertrophy, increased RV wall thickness, RV/left ventricle end‐diastolic area ratio, RV contractility and afterload (arterial elastance), and shorter RV acceleration time compared with controls. Treatment with mTOR kinase inhibitor, PP242, at Weeks 6‐8 after PH induction suppressed both mTORC1 and mTORC2 in small PAs, but only mTORC1 signaling in RV, preserving basal mTORC2‐Akt levels. Vehicle‐treated rats showed further PH and RV worsening and profound RV fibrosis. PP242 reversed pulmonary vascular remodeling and prevented neointimal occlusion of small PAs, significantly reduced PA pressure and pulmonary vascular resistance, reversed cardiomyocyte hypertrophy and RV remodeling, improved max RV contractility, arterial elastance, and RV acceleration time, and prevented development of RV fibrosis. Collectively, these data show a predominant role of mTORC1 versus mTORC2 in RV pathology, and suggest potential attractiveness of mTOR inhibition to simultaneously target pulmonary vascular remodeling and RV dysfunction in established PH.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor

&NA; Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high‐fat diet (HFD) in some inbred mouse strains. We conducted a genome‐wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD‐induced PH. Mice from 36 inbred and wild‐derived strains were fed with regular diet or HFD for 20 weeks beginning at 6‐12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end‐systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest‐path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH‐related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high‐fat feeding. Network‐based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest‐path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high‐fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network‐based approach adds evidence for a role for Egfr in murine PH.