Krystyna M. Shioura

The sphingosine kinase 1/sphingosine-1-phosphate pathway in pulmonary arterial hypertension

RATIONALE Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis. OBJECTIVES We aimed to examine whether SphKs and their product, S1P, play a role in the development of pulmonary arterial hypertension (PAH). METHODS SphK1(-/-), SphK2(-/-), and S1P lyase heterozygous (Sgpl1(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S1P receptor 2 (S1PR2) antagonist (JTE013) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S1P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured. MEASUREMENTS AND MAIN RESULTS mRNA and protein levels of SphK1, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S1P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK1(-/-) mice were protected against HPH, whereas Sgpl1(+/-) mice were more susceptible to HPH. Pharmacologic SphK1 and S1PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK1 and stimulation with S1P potentially via ligation of S1PR2 promoted PASMC proliferation in vitro, whereas SphK1 deficiency inhibited PASMC proliferation. CONCLUSIONS The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

Sex-related changes in cardiac function following myocardial infarction in mice

Recent awareness of cardiovascular diseases as a number one killer of the middle-aged women has prompted interest in sex differences leading to heart failure (HF). Therefore, we evaluated cardiac function in female and male mice following myocardial infarction (MI) using the Millar pressure-volume (P-V) conductance system in vivo, at time points corresponding to early (2 wk), late compensatory hypertrophy (4 wk), and decompensation (10 wk) to HF. A significant deterioration of the load dependent and independent hemodynamic measurements occurred in both female and male mice during the early phase of hypertrophy. Later, compensatory hypertrophy was marked by a normalization of volumes to control levels in females compared with males. The most notable differences between sexes occurred in the measurements of cardiac contractility during the decompensation to HF. In females, there was a significant improvement in contractility compared with males, which was apparent in the load-independent measurements of preload recruitable stroke work (10 wk post-MI, female=48.7+/-8.0 vs. male=25.2+/-1.8 mmHg, P<0.05) and maximum dP/dt vs. maximum end-diastolic volume (10 wk post-MI, female=359+/-58 vs. male=149+/-28 mmHg.s(-1).microl(-1), P<0.05). Despite these differences, there were no differences in the heart weight to body weight ratio and infarct size between the sexes. These data demonstrate that compensatory hypertrophy is associated with an improvement in contractility and a delayed decompensation to HF in females. However, compensatory hypertrophy in males appears to be undermined by a steady decline in contractility associated with decompensation to HF.

Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and is a Therapeutic Target in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension. Methods: Plasma, mRNA, and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension and in the lungs of rodent models of pulmonary hypertension. Nampt+/− mice were exposed to 10% hypoxia and room air for 4 weeks, and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen hypoxia models of pulmonary hypertension. The effects of NAMPT activity on proliferation, migration, apoptosis, and calcium signaling were tested in human pulmonary artery smooth muscle cells. Results: Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension, as well as in lungs of rodent models of pulmonary hypertension. Nampt+/− mice were protected from hypoxia-mediated pulmonary hypertension. NAMPT activity promoted human pulmonary artery smooth muscle cell proliferation via a paracrine effect. In addition, recombinant NAMPT stimulated human pulmonary artery smooth muscle cell proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Last, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia–induced pulmonary hypertension in rats. Conclusions: Our data provide evidence that NAMPT plays a role in pulmonary vascular remodeling and that its inhibition could be a potential therapeutic target for pulmonary arterial hypertension.

Myofilament calcium sensitization delays decompensated hypertrophy differently between the sexes following myocardial infarction

Contractile dysfunction is common to many forms of cardiovascular disease. Approaches directed at enhancing cardiac contractility at the level of the myofilaments during heart failure (HF) may provide a means to improve overall cardiovascular function. We are interested in gender-based differences in cardiac function and the effect of sarcomere activation agents that increase contractility. Thus, we studied the effect of gender and time on integrated arterial-ventricular function (A-V relationship) following myocardial infarction (MI). In addition, transgenic mice that overexpress the slow skeletal troponin I isoform were used to determine the impact of increased myofilament Ca(2+) sensitivity following MI. Based on pressure-volume (P-V) loop measurements, we used derived parameters of cardiovascular function to reveal the effects of sex, time, and increased myofilament Ca(2+) sensitivity among groups of post-MI mice. Analysis of the A-V relationship revealed that the initial increase was similar between the sexes, but the vascular unloading of the heart served to delay the decompensated stage in females. Conversely, the vascular response at 6 and 10 wk post-MI in males contributed to the continuous decline in cardiovascular function. Increasing the myofilament Ca(2+) sensitivity appeared to provide sufficient contractile support to improve contractile function in both male and female transgenic mice. However, the improved contractile function was more beneficial in males as the concurrent vascular response contributed to a delayed decompensated stage in female transgenic mice post-MI. This study represents a quantitative approach to integrating the vascular-ventricular relationship to provide meaningful and diagnostic value following MI. Consequently, the data provide a basis for understanding how the A-V relationship is coupled between males and females and the enhanced ability of the cardiovascular system to tolerate pathophysiological stresses associated with HF in females.

Administration of a Synthetic Peptide Derived from the E-domain Region of Mechano-Growth Factor Delays Decompensation Following Myocardial Infarction.

Insulin like growth factor-I (IGF-1) isoforms differ structurally in their E-domain regions and their temporal expression profile in response to injury. We and others have reported that Mechano-growth factor (MGF), which is equivalent to human IGF-1c and rodent IGF-1Eb isoforms, is expressed acutely following myocardial infarction (MI) in the mouse heart. To examine the function of the E-domain region, we have used a stabilized synthetic peptide analog corresponding to the unique 24 amino acid region E-domain of MGF. Here we deliver the human MGF E-domain peptide to mice during the acute phase (within 12 hours) and the chronic phase (8 weeks) post-MI. We assessed the impact of peptide delivery on cardiac function and cardiovascular hemodynamics by pressure-volume (P-V) loop analysis and gene expression by quantitative RT-PCR. A significant decline in both systolic and diastolic hemodynamics accompanied by pathologic hypertrophy occurred by 10 weeks post-MI in the untreated group. Delivery of the E-domain peptide during the acute phase post-MI ameliorated the decline in hemodynamics, delayed decompensation but did not prevent pathologic hypertrophy. Delivery during the chronic phase post-MI significantly improved systolic function, predominantly due to the effects on vascular resistance and prevented decompensation. While pathologic hypertrophy persisted there was a significant decline in atrial natriuretic factor (ANF) expression in the E-domain peptide treated hearts. Taken together our data suggest that administration of the MGF E-domain peptide derived from the propeptide form of IGF-1Ec may be used to facilitate the actions of IGF-I produced by the tissue during the progression of heart failure to improve cardiovascular function.

ID: 72: INHIBITION OF NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT) ATTENUATES EXPERIMENTAL PULMONARY HYPERTENSION

Rationale We have previously shown that Nampt, which regulates intracellular NAD levels and cellular redox state, regulates histone deacetylases and inhibits apoptosis, is significantly upregulated in patients with pulmonary arterial hypertension (PAH). The aims of this study were to determine (1) whether Nampt+/− mice are protected from hypoxia-mediated pulmonary hypertension (HPH), (2) whether pharmacological inhibition of Nampt could attenuate monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. In addition, we hypothesized that Nampt secreted from pulmonary artery endothelial cells (PAECs) or overexpressing Nampt in pulmonary artery smooth muscle cells (PASMCs) may promote PASMC proliferation via upregulation of calcium signaling pathway, which plays a role in cell proliferation and vascular constriction. Methods Nampt+/− mice and their WT siblings (male, 7-wk old) were exposed to a hypoxia chamber with 10% O2 for four weeks. Male Sprague-Dawley rats (n=6 per group) received one dose of MCT (60 mg/kg), IP. They were administrated with FK866 (an inhibitor of Nampt enzymatic activity) (2.5 mg/kg, IP, twice daily for 2wks) two weeks after MCT. Right ventricular systolic pressure (RVSP) was determined with a pressure transducer catheter. The right ventricle: left ventricle+septum (RV/LV+S) ratio was calculated. In a cell culture model, hPASMCs were stimulated with recombinant Nampt (25 mg/ml) for 6 hrs and 48 hrs. [Ca2+]cyt was measured in PASMC loaded with flura-2/AM (4mM) in a fluorescence microscope and cyclepiazonic acid (CPA, a specific Ca2+-ATPase inhibitor) was used to induce store-operated calcium entry (SOCE). In addition, BrdU assays were conducted to examine rNampt or overexpressing Nampt can promote PASMC proliferation or Nampt secreted from PAECs isolated from PAH patients stimulates more PASMC proliferation than from healthy controls. Results Administration of FK866 reversed established PH (RVSP [mm Hg] 19.77±0.80 [control] vs 51.24±4.35 [MCT] vs 34.45±3.49 [MCT+FK866], p<0.05 ) and RVH (0.25±0.0013 vs 0.60±0.019 vs 0.43±0.022, p<0.01). In PASMCs, short (6 hrs) and long (48 hrs) treatment with recombinant PBEF enhanced SOCE which is involved in sustained pulmonary vasoconstriction and PASMC proliferation. rNampt promotes PASMC proliferation in a dose dependent manner. PAECs from PAH patients secreted more Nampt which stimulates more PASMC proliferation compared to healthy controls. Overexpressed Nampt promotes PASMC proliferation. Inhibition of Nampt via FK866 attenuates rNampt-, Nampt overexpressed or PAEC-secreted Nampt – mediated PASMC proliferation. Conclusion Inhibition of Nampt attenuates hypoxia-mediated PH in mice or MCT-induced PH in rats. Nampt may play a role in vascular remodeling via regulation of calcium signaling pathway. These data suggest that Nampt inhibition could be a potential therapeutic target for PH.