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Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased mean pulmonary arterial pressure. Elevated plasma and lung concentrations of oxidized lipids, including 15-hydroxyeicosatetraenoic acid (15-HETE), have been demonstrated in patients with PAH and animal models. We previously demonstrated that feeding mice with 15-HETE is sufficient to induce pulmonary hypertension, but the mechanisms remain unknown. RNA sequencing data from the mouse lungs on 15-HETE diet revealed significant activation of pathways involved in both antigen processing and presentation and T cell–mediated cytotoxicity. Analysis of human microarray from patients with PAH also identified activation of identical pathways compared with controls. We show that in both 15-HETE–fed mice and patients with PAH, expression of the immunoproteasome subunit 5 is significantly increased, which was concomitant with an increase in the number of CD8/CD69 (cluster of differentiation 8 / cluster of differentiation 69) double-positive cells, as well as pulmonary arterial endothelial cell apoptosis in mice. Human pulmonary arterial endothelial cells cultured with 15-HETE were more prone to apoptosis when exposed to CD8 cells. Cultured intestinal epithelial cells secreted more oxidized lipids in response to 15-HETE, which is consistent with accumulation of circulating oxidized lipids in 15-HETE–fed mice. Administration of an apoA-I (apolipoprotein A-I) mimetic peptide, Tg6F (transgenic 6F), which is known to prevent accumulation of circulating oxidized lipids, not only inhibited pulmonary arterial endothelial cell apoptosis but also prevented and rescued 15-HETE–induced pulmonary hypertension in mice. In conclusion, our results suggest that (1) 15-HETE diet induces pulmonary hypertension by a mechanism that involves oxidized lipid-mediated T cell–dependent pulmonary arterial endothelial cell apoptosis and (2) Tg6F administration may be a novel therapy for treating PAH.

Background Recently, we and others have reported a causal role for oxidized lipids in the pathogenesis of pulmonary hypertension ( PH ). However, the role of low‐density lipoprotein receptor ( LDL ‐R) in PH is not known. Methods and Results We examined the role of LDL ‐R in the development of PH and determined the efficacy of high‐density lipoprotein mimetic peptide 4F in mitigating PH . Explanted human lungs and plasma from patients with PH and control subjects were analyzed for gene expression, histological characteristics, and lipoprotein oxidation. Male LDL ‐R null ( LDL ‐R knockout) mice (12–15 months old) were fed chow, Western diet ( WD ), WD with 4F, and WD with scramble peptide for 12 weeks. Serial echocardiography, cardiac catheterization, oxidized LDL assay, real‐time quantitative reverse transcription–polymerase chain reaction, and histological analysis were performed. The effect of LDL ‐R knockdown and oxidized LDL on human pulmonary artery smooth muscle cell proliferation was assessed in vitro. LDL ‐R and CD 36 expression levels were significantly downregulated in the lungs of patients with PH. Patients with PH also had increased lung lipid deposits, oxidized LDL , E06 immunoreactivity, and plasma oxidized LDL / LDL ratio. LDL ‐R knockout mice on WD developed PH , right ventricular hypertrophy, right ventricular dysfunction, pulmonary vascular remodeling, fibrosis, and lipid deposition in lungs, aortic atherosclerosis, and left ventricular dysfunction, which were prevented by 4F. Interestingly, PH in WD group preceded left ventricular dysfunction. Oxidized LDL or LDL ‐R knockdown significantly increased proliferation of human pulmonary artery smooth muscle cells in vitro. Conclusions Human PH is associated with decreased LDL ‐R in lungs and increased oxidized LDL in lungs and plasma. WD‐ fed LDL ‐R knockout mice develop PH and right ventricular dysfunction, implicating a role for LDL ‐R and oxidized lipids in PH .

Pulmonary hypertension secondary to pulmonary fibrosis (PF‐PH) is one of the most common causes of PH, and there is no approved therapy. The molecular signature of PF‐PH and underlying mechanism of why pulmonary hypertension (PH) develops in PF patients remains understudied and poorly understood. We observed significantly increased vascular wall thickness in both fibrotic and non‐fibrotic areas of PF‐PH patient lungs compared to PF patients. The increased vascular wall thickness in PF‐PH patients is concomitant with a significantly increased expression of the transcription factor Slug within the macrophages and its target prolactin‐induced protein (PIP), an extracellular matrix protein that induces pulmonary arterial smooth muscle cell proliferation. We developed a novel translational rat model of combined PF‐PH that is reproducible and shares similar histological features (fibrosis, pulmonary vascular remodeling) and molecular features (Slug and PIP upregulation) with human PF‐PH. We found Slug inhibition decreases PH severity in our animal model of PF‐PH. Our study highlights the role of Slug/PIP axis in PF‐PH.

Background We have previously shown that intralipid (lipid emulsion) protects the heart against ischemia/reperfusion injury and bupivacaine-induced cardiotoxicity. However, the precise underlying mechanisms are not fully understood. Here we explored the hypothesis that free fatty acid receptor-1 or G-protein–coupled receptor 40 is expressed in the heart and that cardioprotective effects of lipid emulsion are mediated through G-protein–coupled receptor 40 in two animal models of ischemia/reperfusion injury and bupivacaine-induced cardiotoxicity. Methods Langendorff-perfused male mouse hearts were subjected to ischemia/reperfusion with lipid emulsion alone (1%) or with G-protein–coupled receptor 40 antagonist (GW1100, 10 µM). Additionally, cardiotoxicity was achieved in male rats with bupivacaine bolus (10 mg/kg, IV) followed by lipid emulsion alone (20%, 5 ml/kg bolus, and 0.5 ml · kg–1 · min–1 maintenance, IV) or with GW1100 pretreatment (2.5 mg/kg, IV). Results G-protein–coupled receptor 40 is expressed in rodent hearts. GW1100 abolished lipid emulsion-induced cardioprotection against ischemia/reperfusion in mice because rate pressure product and left ventricular developed pressure were lower than lipid emulsion alone (rate pressure product: 2,186 ± 1,783 [n = 7] vs. 11,607 ± 4,347 [n = 8]; left ventricular developed pressure: 22.6 ± 10.4 vs. 63.8 ± 20; P < 0.0001). Lipid emulsion + GW1100 also demonstrated reduced LV dP/dtmax and LV dP/dtmin (dP/dtmax = 749 ± 386 vs. 2,098 ± 792, P < 0.001; dP/dtmin = −443 ± 262 vs. −1,447 ± 546, P < 0.001). In bupivacaine-induced cardiotoxicity rat model, GW1100 pretreatment had no significant effect on heart rate (HR) and ejection fraction after 30 min (HR: 302 ± 17 vs. 312 ± 38; ejection fraction: 69 ± 3% vs. 73 ± 4%). GW1100 pretreatment, however, prevented lipid-rescue, with no recovery after 10 min. In the control group, lipid emulsion improved HR (215 ± 16 at 10 min) and fully rescued left ventricle function at 10 min (ejection fraction = 67 ± 8%, fractional shortening = 38 ± 6%). Conclusions G-protein–coupled receptor 40 is expressed in the rodent heart and is involved in cardioprotection mediated by lipid emulsion against ischemia/reperfusion injury and bupivacaine-induced cardiotoxicity.

Background: Pregnant patients routinely get neuraxial anesthesia. Bupivacaine is one of the most commonly used local anesthetics in obstetric anesthesia. Studies have suggested that pregnancy increases Bupivacaine cardiotoxicity. We and others have previously shown that Intralipid (ILP) rescues the heart from Bupivacaine cardiotoxicity in male rats. ILP mediates its cardioprotective effects via glycogen synthase kinase-3b (GSK-3b) phosphorylation. We aimed to investigate Bupivacaine cardiotoxicity and potential for ILP’s rescue in pregnant rats, that has not been studied before. Methods: Late pregnant rats (200-300 g, n=12) at day 20 of pregnancy were used for the study. A group of pregnant rats received Bupivacaine bolus (10 mg/kg, IV over ~20 seconds, n=7) to induce asystole under anesthesia. Resuscitation with ILP 20% (5 ml/kg bolus, and 0.5 ml/kg/min maintenance, n=7) and chest compressions were initiated. The ejection-fraction (EF%), and heart rate (HR) were calculated from echocardiography and EKG at baseline and at 1, 5, and 10 min after ILP treatment. A group of pregnant rats that did not undergo Bupivacaine-induced cardiac arrest and ILP rescue served as sham controls (n=5). Hearts were collected and Western blot was performed to assess GSK-3b phosphorylation. Results: All 7 late pregnant rats developed cardiac arrest a few seconds after Bupivacaine. Interestingly, 6 out of the 7 rats were not rescued with ILP using the same dose as in male rats. In these 6 rats, the average baseline EF and HR were 67.344.47% and 34130.44 bpm. One, five and ten minutes after ILP administration, the EF and HR were 0. Western blots showed that pregnant rats which were not rescued by ILP failed to have GSK-3b activation (pGSK/GSK=0.80±0.51 vs. 1.00±0.56 in late pregnant sham controls; p=0.578). In the late pregnant rat that recovered with ILP, baseline EF and HR (66.302.99% and 31034 bpm), were not different than the rats that did not recover. Conclusions: ILP failed to rescue Bupivacaine-induced cardiac arrest in pregnant rats. This failure of rescue is most likely attributed to the inability of ILP to activate GSK-3b, a known mediator of ILP’s cardioprotection.

Background: The hearts of pregnant women and rodents undergo physiological hypertrophy with preserved function. Recently, we have demonstrated that the hearts of late pregnant (LP) rodents are more prone to ischemia reperfusion injury compared to non-pregnant (NP) rodents. Currently, measurements such as ejection fraction (EF) are not sensitive enough to determine subclinical myocardial dysfunction. However, speckle-tracking echocardiography (STE), a novel imaging tool, has been demonstrated to assess myocardial function with greater sensitivity. Tumor Necrosis Factor Receptor 2 (TNFR2), a membrane receptor that has been implicated in cardioprotection and has been demonstrated as marker for subclinical myocardial dysfunction in patients. Thus, we hypothesized that global circumferential strain (GCS) measured by STE can detect subclinical myocardial dysfunction in pregnancy that is associated with a decrease in TNFR2 in rats. Methods: LP (20 days pregnant) and NP female Sprague-Dawley rats were used for the study (n=14). Left ventricular EF (LVEF, %), fractional shortening (FS, %), heart rate (HR, bpm), and GCS (%) were obtained using echocardiography (Visualsonics, Vevo 2100) (n=7/group). GCS was obtained with STE and was measured in triplicates. Total ventricular weight (VW) was recorded and LV was isolated to measure LV hypertrophy (LV/VW) (n=6/group). TNFR2 expression was measured using qPCR. Results: LP rats showed slightly reduced but insignificant LVEF (LP: 67.65+/-1.41% vs. NP: 71.35+/-1.52%, P=0.10) and FS (39.07+/-0.90 % vs. 42.643+/-1.76 %, P=0.09) compared to NP rats. Additionally, no difference in HR between LP and NP rats were found (352.50+/-16.36 bpm vs. 333.75+/-4.78 bpm, respectively, p=0.314).However, hearts from LP rats had evidence of hypertrophy evident by an increase in LV/VW compared to NP (0.57+/-0.03 g vs. 0.48+/-0.02 g). LP rats had a significantly reduced GCS compared to NP rats (-27.50+/-1.40 % vs. -34.375+/-1.95 %, p Conclusions: Our results demonstrate that late pregnancy is associated with a decrease in GCS with no significant change in EF and a decrease in TNFR2, which may be a subclinical marker for myocardial dysfunction.

Background: Exparel is a formulation of bupivacaine encapsulated in multi-vesicular liposomes, developed for post-surgical anesthesia. The liposomes have been shown to increase the drug’s stability and extend its duration of action. Exparel may prevent accumulation of bupivacaine in blood and/or tissue; thus, decreasing risk of central nervous or cardiovascular toxicities. However, administration of Exparel has its risks. The maximum dosage of Exparel for adults is ~266 mg, but the maximum mg/kg dosing limit is unknown. The role of Intralipid (ILP, 20%) for reversal of Exparel toxicity is not known. Our aim is to determine toxic dose of Exparel in mg/kg and if ILP can reverse its cardiotoxic effects in rats. Methods: Female rats (~250 gm, n=6) were used for the study. Asystole was attempted with different IV doses of Exparel (25 mg/kg, 15 mg/kg, 7.5mg/kg over 10 seconds). For the second part of the study, asystole was induced with Exparel (15 mg/kg over 10 sec, IV), and resuscitation with ILP (5 ml/kg bolus and 0.5 ml/kg/min maintenance) was started immediately along with chest compressions. Heart rate (HR, beats per min) and ejection fraction (EF, %) were measured using echo at 1, 5, and 10 min. Results: We found that a dose of 25 mg/kg of Exparel caused cardiac arrest immediately. Next we decreased the dose of Exparel to 15 mg/kg, that also caused immediate cardiovascular collapse. Exparel 7.5 mg/kg did not induce cardiac arrest; instead, it caused wide complex tachycardia that self resolved in 5 min. ILP rescue of Exparel cardiotoxicity was found to be unpredictable (n=3). EF, HR and EKG improved after cardiac arrest the most at 1 min after ILP. At 1 min, EF was 42.8+/-15% and HR was 169.3 +/- 25 bpm. ILP rescue of Exparel cardiotoxicity was not sustained contrary to ILP’s rescue of Bupivacaine cardiotoxicity in rats. EF and HR continued to deteriorate when measured at 5 and 10 min, with 2 out of 3 rats with EF=0 at 5 and 10 min. Conclusion: We found a dose of Exparel that reliably induced cardiotoxicity in rats. ILP is unreliable in rescuing Exparel induced cardiotoxicity at the dosage regimen typically used for rats with Bupivacaine cardiotoxicity. More experiments are needed to determine ILP’s dosage that can reliably reverse Exparel cardiotoxicity.

Background: Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling that leads to right ventricular failure and death. In PH, vascular remodeling is driven by hyper-proliferative and anti-apoptotic pulmonary arterial smooth muscle cells (PASMC). Protein Tyrosine Phosphatase 1B (PTP1B), is a negative regulator for apoptosis and proliferation of vascular smooth muscle cells through down-stream mediators such as BCL-2. While BCL-2 is increased in PH patients, the pathways for regulating BCL-2 are poorly understood. Therefore, our aim is to investigate if PTP1B has a role in PASMC proliferation and resistance to apoptosis in human PASMCs and in the angioproliferative Sugen/Hypoxia/Normoxia (SuHx) PH rat model. Method: Male Sprague-Dawley rats were treated with a single intraperitoneal dose of a vascular endothelial growth factor antagonist (SU5416, 20 mg/kg, n=4) and kept in hypoxia for 3 weeks followed by normoxia for 2 weeks. Saline treated controls were kept in normoxia for 5 weeks (n=4). RV systolic pressure (RVSP) was recorded by RV catheterization and hearts were isolated to assess RV hypertrophy (RV/LV + IVS). PTP1B and BCL-2 expression was determined using qPCR. Healthy hPASMCs were incubated with a PTP1B inhibitor (Ethyl-3,4-dephostatin) at IC50=0.58ug/ml for 24hrs and stained with Ki67 to assess for proliferation. Results: SuHx rats had severe PH, evident by elevated RVSP compared to control (88.97+/- 13.67 vs. 28.47+/- 2.22 mmHg, p Conclusion: Severe Angioproliferative PH in rats is associated with a downregulation of PTP1B and increased expression of BCL-2. Additionally, PTP1B inhibition resulted in hPASMC proliferation, suggesting a role for PTP1B in the pathogenesis of PH.

Epidemiologic studies have previously suggested that premenopausal females have reduced incidence of cardiovascular disease (CVD) when compared to age-matched males, and the incidence and severity of CVD increases postmenopause. The lower incidence of cardiovascular disease in women during reproductive age is attributed at least in part to estrogen (E2). E2 binds to the traditional E2 receptors (ERs), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ), as well as the more recently identified G-protein-coupled ER (GPR30), and can exert both genomic and non-genomic actions. This review summarizes the protective role of E2 and its receptors in the cardiovascular system and discusses its underlying mechanisms with an emphasis on oxidative stress, fibrosis, angiogenesis, and vascular function. This review also presents the sexual dimorphic role of ERs in modulating E2 action in cardiovascular disease. The controversies surrounding the clinical use of exogenous E2 as a therapeutic agent for cardiovascular disease in women due to the possible risks of thrombotic events, cancers, and arrhythmia are also discussed. Endogenous local E2 biosynthesis from the conversion of testosterone to E2 via aromatase enzyme offers a novel therapeutic paradigm. Targeting specific ERs in the cardiovascular system may result in novel and possibly safer therapeutic options for cardiovascular protection.

Background: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling that leads to an increase in pulmonary arterial pressure resulting in right ventricle failure and death. PAH is driven by pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. Protein Tyrosine Phosphatase 1B (PTP1B), a negative regulator for platelet-derived growth factor (PDGF) and BCL-2, has recently been implicated in PAH in humans. While PDGF and BCL-2 are increased in PAH patients, the pathway for regulating BCL-2 and PDGF is poorly understood. We aim to investigate if PTP1B has a role in proliferation and resistance to apoptosis in PAH in human PACMCs and in the Sugen/Hypoxia/Normoxia (Su/Hx/Nx) PH rat model. Method: Adult male Sprague-Dawley rats were treated with single intraperitoneal dose of SU5416 (20 mg/kg) and kept in Hx for 3 weeks followed by Nx for 2 weeks. Saline treated rats kept in Nx for 5 weeks served as control (n=4/group). RV catheterization was performed terminally for recording RV systolic pressure (RVSP). RV, LV, and interventricular septum (IVS) were isolated for Fulton index (FI, RV/IVS+LV). We analyzed gene expression in lungs via qPCR. Healthy hPASMCs were incubated with a PTP1B inhibitor (Ethyl-3,4-dephostatin) at IC50=0.58ug/ml for 24hrs under Nx conditions and cells were stained with Ki67 to assess proliferation. Results: Su/Hx/Nx rats had severe PH evidenced by a significantly elevated RVSP compared to control (88.97+/- 13.67 vs 28.47+/- 2.22 mmHg, p Conclusion: Severe angioproliferative PH in rats is associated with a downregulation of PTP1B and increased expression of BCL-2 and PASMC proliferation.

Pulmonary arterial hypertension (PAH) is a deadly disease characterized by increased pulmonary arterial pressure and pulmonary vascular occlusion. Recently, we and others demonstrated a robust increase in oxidized lipids, including 15-hydroxyeicosatetraenoic acids (15-HETE), in the lungs and plasma of PAH patients and animal models of pulmonary hypertension (PH). We hypothesized that diets rich in 15-HETE are sufficient to cause PH in wild type mice. We also examined whether 15-HETE or its metabolites are required to cause PH by comparing the effect of 15-HETE with 15-HETE methyl ester, which is a stable form of 15HETE that is not easily metabolized. C57BL/6 male mice were fed for 3 weeks with 15-HETE diet (5μg/day), 15-HETE methyl ester (15-HETE-ME, 5μg/day), or regular chow diet (n=8-21 mice/group). PH development was followed via weekly serial echocardiography. Right ventricular systolic pressure (RVSP) was measured via direct heart catheterization. RV hypertrophy index (RV/[IVS+LV]) was measured. Lung morphology and lipid accumulation were assessed using H&E and Oil red O staining. Echocardiography revealed the first sign of PH in mice on 15HETE diet as early as one week and a significant decrease in the pulmonary arterial acceleration time after 2 weeks of treatment (16.6±1.9 vs. 21.2±1.4 msec, p

Anthracyclines are highly effective against a variety of malignancies. However, their dose-dependent cardiotoxic effects can potentially limit their use. In the past decade, serum biomarkers have been used to diagnose, monitor, predict, and prognosticate disease. Biomarkers such as cardiac troponin and natriuretic peptides have some predictive value, but still lack reliability in this patient population. Novel biomarkers such as galectin-3, soluble ST-2 proteins, myeloperoxidase, and fibrocytes are being explored as potential biomarkers to reliably predict the onset of cardiotoxicity. Leveraging multiomics technology to map highly sensitive biomarkers in an integrated approach through pattern deconvolution may better define those at highest risk of developing cardiotoxicity and further the goal of precision medicine. In this work, we aim to provide a brief overview of traditional serum biomarkers, summarize current investigations on novel circulating biomarkers, and discuss a systems-based approach to anthracycline-induced cardiotoxicity through “omics” technology.

BackgroundApolipoprotein E (ApoE) is a multifunctional protein, and its deficiency leads to the development of atherosclerosis in mice. Patients with pulmonary hypertension (PH) have reduced expression of ApoE in lung tissue. ApoE is known to inhibit endothelial and smooth muscle cell proliferation and has anti-inflammatory and anti-platelet aggregation properties. Young ApoE-deficient mice have been shown to develop PH on high fat diet. The combined role of female sex and aging in the development of PH has not been investigated before. Here, we investigated the development of PH in young and middle-aged (MA) female ApoE-deficient mice and explored the role of exogenous estrogen (E2) replacement therapy for the aging females.MethodsWild type (WT) and ApoE-deficient female mice (Young and MA) were injected with a single intraperitoneal dose of monocrotaline (MCT, 60 mg/kg). Some ApoE-deficient MA female mice that received MCT were also treated with subcutaneous E2 pellets (0.03 mg/kg/day) from day 21 to 30 after MCT injection. Direct cardiac catheterization was performed terminally to record right ventricular systolic pressure (RVSP). Right ventricular (RV), left ventricular (LV), and interventricular septum (IVS) were dissected and weighed. Lung sections were examined using trichrome and immunofluorescence staining. Western blot analyses of lung and RV lysates were performed.ResultsIn WT female mice, the severity of PH was similar between young and MA mice as RVSP was not significantly different (RVSP = 38.2 ± 1.2 in young vs. 40.5 ± 8.3 mmHg in MA, p < 0.05). In ApoE-deficient mice, MA females developed significantly severe PH (RVSP = 63 ± 10 mmHg) compared to young females (RVSP; 36 ± 3 mmHg, p < 0.05 vs. MA female). ApoE-deficient MA females also developed more severe RV hypertrophy compared to young females (RV hypertrophy index (RV/[LV + IVS]) = 0.53 ± 0.06 vs. 0.33 ± 0.01, p < 0.05). ApoE-deficient MA female mice manifested increased peripheral pulmonary artery muscularization and pulmonary fibrosis. E2 treatment of MA female ApoE-deficient mice resulted in a significant decrease in RVSP, reversal of pulmonary vascular remodeling, and RV hypertrophy. In MA female ApoE-deficient mice with PH, only the expression of ERβ in the lungs, but not in RV, was significantly downregulated, and it was restored by E2 treatment. The expression of ERα was not affected in either lungs or RV by PH. GPR30 was only detected in the RV, and it was not affected by PH in MA female ApoE-deficient mice.ConclusionsOur results suggest that only aging female ApoE-deficient but not WT mice develop severe PH compared to younger females. Exogenous estrogen therapy rescued PH and RV hypertrophy in aging female ApoE-deficient mice possibly through restoration of lung ERβ.