Arterial Medial Thickness Research Articles

Effect of caspase inhibitors on hemodynamics and inflammatory factors in ARDS model rats

To study the effects of caspase inhibitors on hemodynamics and inflammatory factors in acute respiratory distress syndrome (ARDS) model rats. Sixty healthy male Wistar rats were randomly divided into three groups, namely, the control group, ARDS group and ARDS + Caspase inhibitor group, with 20 rats in each group. The control group was intraperitoneally injected with 2 mL/kg saline, and the ARDS model group was established by intraperitoneally injecting 4 mg/kg Lipopolysaccharide (LPS), ARDS + Caspase inhibitor group was adminstered 20 mg/kg caspase inhibitor after intraperitoneal LPS injection. Changes in pulmonary arterial pressure (PAP) and mean arterial pressure (MAP) at 6 and 12 h before and after administration were recorded. Moreover, arterial blood gas was evaluated with a blood gas analyzer and changes in the partial pressure of O2 (PaO2), partial pressure of CO2 (PaCO2), partial pressure of O2/fraction of inspired O2 (PaO2/FiO2) were evaluated. In addition, the lung wet/dry weight (W/D) ratio and inflammatory factor levels in lung tissue were determined. Finally, pathological sections were used to determine the pulmonary artery media thickness (MT), MT percentage (MT%), and the degree of muscle vascularization. The pulmonary arterial pressure of rats was determined at several time points. Compared with the control group, the model group had a significantly increased pulmonary arterial pressure at each time point (P < 0.01), and the mean arterial pressure significantly increased at 6 h (P < 0.05). Compared with that of rats in the model group, the pulmonary arterial pressure of rats in drug administration group was significantly reduced at each time point after administration (P < 0.01), and the mean arterial pressure was significantly reduced at 6 h (P < 0.05). The arterial blood gas analysis showed that compared with those in the control group, PaO2, PaCO2 and PaO2/FiO2 in the model group were significantly reduced (P < 0.01), and PaO2, PaCO2 and PaO2/FiO2 were significantly increased after caspase inhibitor treatment (P < 0.05 or 0.01). The levels of the inflammatory mediators tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the model group were significantly higher than those in the control group (P < 0.01), and they were significantly decreased after caspase inhibitor treatment (P < 0.01). In the model group, pulmonary artery MT, MT% and the degree of muscle vascularization were significantly increased (P < 0.05 or 0.01), and pulmonary artery MT and the degree of muscle vascularization were significantly reduced after caspase inhibitor treatment (P < 0.05 or 0.01). Apoptosis Repressor with a Caspase Recuitment Domain (ARC) can alleviate the occurrence and development of pulmonary hypertension (PH) by affecting hemodynamics and reducing inflammation.

Early Alteration of Right Ventricle-Pulmonary Artery Coupling in Experimental Heart Failure With Preserved Ejection Fraction.

Pulmonary hypertension and right ventricular (RV) dysfunction are major prognostic determinants in patients with heart failure with preserved ejection fraction (HFpEF). The underlying pathomechanisms remain unknown. In this context, we sought to study the pathogenesis of pulmonary hypertension and RV dysfunction in a rat model of obesity-associated HFpEF. HFpEF was induced in obesity-prone rats fed a high-fat diet (n=13) and compared with obesity-resistant rats fed with standard chow (n=9). After 12 months, the animals underwent echocardiographic and hemodynamic evaluation followed by tissue sampling for pathobiological assessment. HFpEF rats presented mild RV pressure overload (with increased RV systolic pressure and pulmonary vascular resistance). No changes in pulmonary artery medial thickness and exvivo vasoreactivity (to acetylcholine and endothelin-1) were observed and RNA sequencing analysis failed to identify gene clustering in HFpEF lungs. However, released nitric oxide levels were decreased in HFpEF pulmonary artery, while lung expression of preproendothelin-1 was increased. In HFpEF rats, RV structure and function were altered, with RV enlargement, decreased RV fractional area change and free wall longitudinal fractional shortening, together with altered right ventricle-pulmonary artery coupling (estimated by tricuspid annular plane systolic excursion/systolic pulmonary artery pressure). Hypertrophy and apoptosis (evaluated by transferase biotin- dUTP nick-end labeling staining) were increased in right and left ventricles of HFpEF rats. There was an inverse correlation between tricuspid annular plane systolic excursion/systolic pulmonary artery pressure and RV apoptotic rate. Plasma levels of soluble suppression of tumorigenicity-2, interleukin-1β, -6 and -17A were increased in HFpEF rats. Obesity-associated HFpEF in rats spontaneously evolves to pulmonary hypertension-HFpEF associated with impaired right ventricle-pulmonary artery coupling that appears disproportionate to a slight increase in RV afterload.

Ethanol-induced lung and cardiac right ventricular inflammation and remodeling underlie progression to pulmonary arterial hypertension.

Current research on ethanol-induced cardiovascular anomalies has focused on left ventricular (LV) function and blood pressure. To extend this area of research, we sought to determine whether ethanol-induced alterations in the structure and function of the right cardiac ventricle (RV) and pulmonary artery (PA) lead to pulmonary arterial hypertension (PAH). Two groups of male Sprague-Dawley rats received a balanced liquid diet containing 5% ethanol (w/v) or a pair-fed isocaloric liquid diet for 8 weeks. Weekly echocardiography was conducted to evaluate cardiopulmonary function, and lung and RV tissues were collected for exvivo histological and molecular studies. The ethanol-treated rats exhibited: (1) Elevated mean pulmonary arterial pressure and decreased pulmonary artery acceleration time/ejection time; (2) Pulmonary vascular remodeling comprising intrapulmonary artery medial layer thickening; and (3) RV hypertrophy along with increased RV/LV + septum, RV diameter, RV cardiomyocyte cross-sectional area, and LV mass/body weight ratio. These responses were associated with increased lung and RV pro-inflammatory markers, endothelin-1 (ET-1), TNF-α, and IL-6 levels and higher ET-1,ET-1 type A/B receptor ratio, and downregulation of the cytoprotective protein, bone morphogenetic protein receptor 2 (BMPR2), in the lungs. These findings show that moderate ethanol-induced cardiopulmonary changes underlie progression to PAH via an upregulated proinflammatory ET1-TNFα-IL6 pathway and suppression of the anti-inflammatory BMPR2.

Phase 3 Anticancer Agent, RRx-001, Ameliorates Hypoxia-Induced Pulmonary Hypertension

Background: The hallmark features of pulmonary artery hypertension (PAH), for which hypoxia triggers, are vascular remodeling, elevated pulmonary blood pressure, oxidative stress, and right ventricular hypertrophy. RRx-001 is a minimally toxic Phase 3 anticancer agent with anti-oxidative, anti-inflammatory, nitric oxide-donating, and anti-proliferative properties. This study aimed to investigate the effect of RRx-001 on intermittent hypoxia-induced PAH in rats. Methods: Male Wistar rats (180-220 g) were divided into four groups (n=6 per group): normoxic control, hypoxic with vehicle treatment (2 weeks breathing an atmosphere containing 10% oxygen), hypoxic treated with oral endothelin-1 receptor inhibitor, bosentan, 50 mg/kg per day for 2 weeks during hypoxia and one intravenous dose of 5mg/kg RRx-001 given the day before the start of the hypoxia. After sacrifice, hearts and lungs were removed and harvested for analysis. Results: Both RRx-001 and bosentan treated animals, separately and to a similar degree, significantly attenuated hypoxia-induced increases in right ventricular systolic pressure and right ventricular hypertrophy compared to control animals. RRx-001 and bosentan, separately and to a similar degree, also significantly decreased pulmonary artery media thickness. RRx-001 treatment reduced vasocostriction of pulmonary arterioles and the wall thickness/external diameter ratio of the pulmonary arteries compared to vehicle treated animals. Conclusions: RRx-001 reduces right ventricular systolic pressure and pulmonary vascular remodeling in a rat model of PAH, potentially making it a new therapeutic option for this disease. The combination of bosentan and RRx-001 will be attempted as a next step. Commercial and private foundation grants This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Establishment of a canine model of pulmonary arterial hypertension induced by dehydromonocrotaline and ultrasonographic study of right ventricular remodeling

ABSTRACT Objective Pulmonary arterial hypertension (PAH) means high blood pressure in the lungs. We aimed to observe the right ventricular size, wall thickness and characteristic functional changes and their associations with PAH in an established model of beagle dogs, and to explore convenient, reliable and sensitive ultrasound indicators for assessing right ventricular remodeling. Methods Twenty healthy beagle dogs (8–10 kg) were randomly divided into control group (N-dimethylformamide, n = 10) and dehydromonocrotaline (DHMCT) group (DHMCT, n = 10). N-dimethylformamide or DHMCT was injected through a catheter into the right atrium, and then right heart catheterization, routine echocardiography and two-dimensional speckle tracking imaging (2D-STI) were performed before modeling (0 weeks) and 8, 14 weeks after modeling. Hemodynamic parameters and right ventricular function-related ultrasound data were acquired. At the end of the experiment, the animals were killed and the lung tissues were taken for HE staining. Left and right ventricular walls were separated and weighed respectively, and right ventricular hypertrophy index (RVHI) was measured. The associations of the routine ultrasound data and 2D-STI data at each time point with hemodynamic parameters and RVHI were analyzed. Results At 0, 8 and 14 weeks, gradual decreases in the right ventricular global longitudinal strain (RVLS) were found in DHMCT group. RVH occurred in DHMCT group, and DHMCT group had a significantly higher RVHI than that of control group (49.83 ± 4.83% vs. 39.80 ± 1.40%, P < .001) and larger pulmonary artery media thickness. RVLS had significant positive correlations with RVSP (r = 0.74, P < .001), mRVP (r = 0.72, P < .001), PASP (r = 0.75, P < .001), mPAP (r = 0.72, P < .001) and PVR (r = 0.68, P < .001). There was a significant positive correlation between RVLS and RVHI (r = 0.74, P < .001). Conclusion The right ventricular function in PAH can be effectively assessed by echocardiography, and RVLS measured by 2D-STI sensitively reflects right ventricular remodeling following PAH.

Ameliorative Effects of Beetroot Juice Supplementation on Monocrotaline-Induced Pulmonary Hypertension in Rats

Beetroot is a nitrate-rich vegetable with cardiovascular benefits. This study examined whether ingestion of beetroot juice (BRJ) protects against pulmonary hypertension (PH). Rats were injected subcutaneously with 60 mg/kg monocrotaline (MCT) and randomized to receive either drinking water, low-dose BRJ (BRJ-L, nitrate content: 1.4 mmol/L), or high-dose BRJ (BRJ-H, nitrate content: 3.5 mmol/L), which was started 1 week after MCT injection and continued until the end of the experiment. Four weeks after MCT injection, right ventricle (RV) hypertrophy, right ventricular systolic pressure (RVSP) elevation, and pulmonary vascular remodeling were observed. These PH symptoms were less severe in rats supplemented with BRJ-L (Fulton index, p = 0.07; RVSP, p = 0.09, pulmonary arterial medial thickening, p &lt; 0.05), and the beneficial effects were more pronounced than those of BRJ-H supplementation. Plasma and RV nitrite and nitrate levels did not change significantly, even when BRJ-L and BRJ-H were administered. There were no differences in plasma thiobarbituric acid reactive substances (TBARS), a biomarker of oxidative stress, among the groups. BRJ-L supplementation significantly decreased RV TBARS levels compared to MCT alone (p &lt; 0.05), whereas BRJ-H supplementation did not. These findings suggest that starting BRJ supplementation from an early stage of PH ameliorates disease severity, at least partly through the inhibition of local oxidative stress. Habitual ingestion of BRJ may be useful for the management of PH.

Abstract 15374: Somatic Mutations of Dnmt3a Contribute to Pulmonary Vascular Remodeling and Right Ventricular Dysfunction in PAH

Background: Pulmonary Arterial Hypertension (PAH) is an obliterative pulmonary vasculopathy in which structural changes are driven by inflammation, fibrosis, mitochondrial dysfunction, and pathological angiogenesis. While ~10% of cases of PAH have germline BMPR2 mutations (or one of 16 other recognized or putative PAH genes), the genetic basis for the majority of PAH patients remains unexplained. Somatic mutations in hematopoietic stem cells (HSC) are related to the disorders of clonal hematopoiesis of indeterminate potential (CHIP). Tet methylcytosine dioxygenase 2 (TET2) and DNA methyltransferase 3a (DNMT3A) are among the primary genes involved in CHIP. We found in a PAH-Biobank that 0.39% of PAH patients had somatic or germline TET2 mutations. Also, TET2-knockout mice can develop PAH spontaneously. Thus TET2 is a new PAH gene. We now hypothesize that CHIP driven by mutations of DNMT3A can also cause pulmonary vascular remodeling and right ventricular failure (RVF), contributing to PAH. Methods: Studies were performed on 9 month old DNMT3A knockout (-/-) mice and their relative controls (f/f) (N=5/group). The right ventricular (RV) function was assessed via cardiac ultrasound. Pulmonary artery medial wall thickness (PAMWT) and radial alveolar count (RAC) were assessed in lung tissue sections using light microscopy. Results: In DNMT3A -/- mice, there was a reduction in pulmonary artery acceleration time (PAAT) compared to controls ( p= 0.0268). There was also a significant reduction in percent RV free wall thickening (RVFWT %) compared to controls (57.44±9.97 DNMT3A-/-, 118.5±14.98 f/f; p= 0.017). Both Tricuspid annular plane systolic excursion (TAPSE) and cardiac output (CO) were also significantly reduced in DNMT3A -/- mice ( p &lt; 0.0001, p = 0.0135). The medial thickness of small pulmonary arteries was significantly higher in DNMT3A -/- mice compared to controls (56.51% ±1.19 DNMT3A-/-, 39.79% ±2.07 f/f; p= 0.002). RAC was significantly reduced in the DNMT3A -/- mice when compared to controls ( p= 0.018). Conclusions: DNMT3A mutations may contribute to the development of pulmonary vascular disease and RVF in PAH.

Abstract 10715: Therapeutic Efficacy of Drpitor1a, a Novel Dynamin-Related Protein 1 (DRP1) Inhibitor, in Preclinical Pulmonary Arterial Hypertension

Introduction: Dynamin-related protein 1 (DRP1) is a large GTPase that mediates mitotic fission (the division of mitochondria which is coordinated with mitosis, ensuring equitable distribution of mitochondria to daughter cells). In pulmonary arterial hypertension (PAH) pulmonary artery smooth muscle cells (PASMC), mitotic fission, and DRP1 activity are increased, contributing to its hyperproliferative phenotype. We investigated the therapeutic efficacy of Drpitor1a, a novel, small molecule, Drp1 GTPase inhibitor, in a rat PAH model. Methods: A single dose of monocrotaline (MCT, 60mg/kg, SC) or PBS was injected to female Sprague-Dawley rats on day 0 (n=20 for MCT and n=15 for PBS). On day 14, the development of PAH was confirmed by echocardiography and the rats were randomized for treatments. An indwelling catheter was implanted through the left jugular vein on day 15. Drpitor1a (1mg/kg) or normal saline (NS) was administered by IV every 48 hours from day 17 to day 27. Right ventricular (RV) structure and function were assessed on day 28 with echocardiography. Pulmonary hemodynamics was evaluated on day 29 using right heart catheterization (RHC). Results: MCT rats developed PAH with RV dysfunction on day 14. There was no statistical difference between the Drpitor1a and NS groups at randomization. At the endpoint, MCT+Drpitor1a rats, vs. MCT+NS rats, had a significantly reduced severity of pulmonary hypertension evident as longer pulmonary artery acceleration time (PAAT) and lower pulmonary vascular resistance index. Drpitor1a also improved RV function, evident as greater RV free wall thickening (RVFWT%), increased tricuspid annular plane systolic excursion (TAPSE), and increased cardiac index (CI). Drpitor1a regressed pulmonary artery medial thickening and inhibited RV hypertrophy without hepatological, renal or hepatic toxicities. Conclusion: Drpitor1a is a safe and effective treatment for preclinical PAH.

Implications of Ezetimibe in Combination with Low- to Moderate-Intensity Atorvastatin Adjuvant Aspirin Therapy for Cerebrovascular Disease.

Objective To analyze the significance of ezetimibe in combination with low- to moderate-intensity atorvastatin adjuvant aspirin therapy for cerebrovascular disease. Methods 110 patients with cerebrovascular disease treated in our hospital from June 2020 to June 2021 were selected and divided into 55 patients in the control group and 55 patients in the study group according to the lottery method. After a comprehensive examination, patients in the two groups should be given aspirin for treatment; the control group was treated with conventional dose of atorvastatin on top of the above, and the study group was given ezetimibe and medium-low-dose atorvastatin on top of aspirin treatment, activities of daily living (ADL) score, carotid artery intima-media thickness, lipid level, coagulation level, clinical effect, and adverse rate of the two groups which were tested and compared. Results After treatment, ADL score, high-density leptin cholesterol (HDL-C), and ATIII levels increased, while carotid artery media thickness, triglyceride (TG), total cholesterol (TC), low-density leptin cholesterol (LDL-C), DD, PC, and hs-CRP levels decreased (P < 0.05). After treatment, ADL score, HDL-C, and ATIII levels were higher in the study group. The levels of carotid media thickness, TG, TC, LDL-C, DD, PC, and hs-CRP were significantly lower (P < 0.05). The clinical effect of the study group was outstanding (P < 0.05). The defect rate of the study group was lower than that of the control group, but there was no difference (P < 0.05). Conclusion Ezetimibe combined with medium- and low-intensity atorvastatin with aspirin in the treatment of cerebrovascular diseases can effectively improve the coagulation function of patients, reduce the level of inflammatory factors in patients, and improve the level of blood lipids in patients, with high safety and worthy of clinical application.

Chronic Daily House Dust Mite Exposure in Mice is an Effective Model to Quantify the Effect of Pharmacologic Agents on Discrete Stages of Artery Remodeling in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogenous and incurable disease marked by varying degrees of pulmonary vascular remodeling. This vascular remodeling, which includes thickening of the smooth muscle layer (an early finding) and formation of occlusive neointimal lesions (a late finding) in the pulmonary arteries, is a major driver of morbidity and mortality in PH. Available PH therapies consist of vasodilators that do not specifically target lesion formation or expansion and neither prevent progression nor reverse disease. This paucity of curative treatments highlights the need for new drug discovery targeting crucial steps of artery remodeling in PH. The cell dynamics and molecular signals driving neointimal lesion formation have been difficult to elucidate as classic mouse models of PH do not develop neointima. Here, we detail the methods to generate a robust and non-genetic mouse model of PH with medial thickening and neointimal lesion formation in the pulmonary arteries, through chronic exposure to an inflammatory stimulus-house dust mite (HDM). This model rapidly generates human-like pulmonary arterial lesions following a reproducible time course, allowing scrutiny of the cellular and molecular mechanisms controlling each stage of artery remodeling. Further, we outline optimal tissue handling, sectioning, and staining methodologies for detailed quantitative analysis of artery medial thickening and neointimal lesion formation and expansion. Finally, we present a method for staged pharmacologic intervention to identify molecules and pathways required at each step of the pulmonary arterial remodeling process. The advantages of this mouse model of PH over currently available animal models are five-fold. (i) It allows the use of the full range of genetic and single cell tools available in mice to manipulate and study the process of vascular remodeling seen in human disease, including the formation of neointimal lesions in a controlled and cell specific manner. (ii) The vascular lesions develop in a stereotyped manner with predictable timing, allowing for pharmacologic manipulation at discrete stages of vessel remodeling. (iii) It is rapid, with development of PH and vascular remodeling in a timeframe of two to eight weeks. (iv) It uses simple techniques and requires neither surgery, unusual equipment, or extensive personnel training. (v) The staining and quantitation methodologies we present are a significant improvement over those currently in use in the field. We hope that dissemination of this model and the associated detailed methods will speed up the development of novel and more effective PH therapeutics. Graphic abstract: Chronic perivascular inflammation induces medial thickening and neointima formation in pulmonary arteries, following a stereotyped time course, and allowing staged pharmacologic intervention during specific remodeling events, as well as quantitative assessment of vascular changes.

Abstract 8859: Hematopoietic Calr Frameshift Mutation Exacerbates Hypoxia-Induced Pulmonary Hypertension in Mice

Introduction: Calreticulin ( CALR ) frameshift is the second frequent driving mutation in myeloproliferative neoplasms (MPNs). MPNs are complicated with pulmonary hypertension (PH), but the mechanistic relevance of the CALR mutations on PH has not yet been clarified. Methods and Results: We generated a 10-bp deletion knock-in mice with murine Calr exon 9 mimicking the human CALR mutations. To investigate whether the hematopoietic CALR mutation contributes to PH, a bone marrow transplantation (BMT) was applied using the donor BM cells from Calr knock-in mice or control wild-type (WT) mice. Four weeks after BMT, the recipient mice transplanted with BM cells from Calr knock-in mice (Del-R) and WT mice (WT-R) were exposed to chronic hypoxia. Three weeks after starting chronic hypoxia exposure, the right ventricular systolic pressure and right ventricle/left ventricle-plus-septum weight ratio in the Del-R mice were significantly increased compared to the WT-R mice. Lung histology showed an acceleration of pulmonary arterial muscularization and medial wall thickness in Del-R mice after chronic hypoxia. Increases in macrophage infiltration was similarly observed in both WT-R and Del-R mice after chronic hypoxia. Endothelin-1 expression and STAT3 phosphorylation were significantly upregulated in the lung of Del-R mice compared to the WT-R mice in response to chronic hypoxia. Using Calr Del/CAG-EGFP double transgenic mice, we visualized the accumulations of donor-derived macrophages in pulmonary arterial regions. BM-derived macrophages carrying the Calr mutation showed increases in endothelin-1 expression, indicating that BM-derived macrophages with the Calr mutation played important roles in the pulmonary arterial remodeling. Conclusion: The frameshifted Calr mutations presented a novel mechanism of PH through BM-derived macrophages accompanied by upregulation of endothelin-1.

Abstract 13094: Pharmacokinetics and Therapeutic Efficacy of Drpitor1a, a Novel Dynamin-Related Protein 1 Inhibitor, in a Preclinical Model of Pulmonary Arterial Hypertension

Introduction: Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission contributes to the pathogenesis of pulmonary arterial hypertension (PAH). We investigated the pharmacokinetic characteristics of Drpitor1a, a novel, small molecule, ellipticine Drp1 inhibitor (prepared in-house), and tested its therapeutic efficacy in a pilot study using the rat monocrotaline (MCT)-PAH model. Methods: A single dose of Drpitor1a (5mg/kg) was administered IV or PO to male and female rats (n=3/sex). Blood was collected at 0, 5 and 30 minutes, and at 1, 2, 4, 6, 8 and 24 hours post drug administration. Plasma, lung and right ventricle (RV) were collected and tissue concentration of Drpitor1a was measured using HPLC coupled with mass spectrometry. The effects of Drpitor1a on mitochondrial fission and cell proliferation were assessed in human PAH pulmonary artery smooth muscle cells (PASMC). In an MCT-PAH model, a catheter was implanted in the left jugular vein on day 14 post-MCT injection. Drpitor1a (1mg/kg) was administered by IV every 48 hours from day 17 to day 27. On day 28, RV function and pulmonary hemodynamics were assessed. Results: Drpitor1a’s half-life varies by sex (9.9±5.3 hours and 24.3±21.4 hours in male and female rats respectively). Oral bioavailability of Drpitor1a was similar in males (21.1%) and females (19.7%). Lung concentration of Drpitor1a was similar between males and females (0.441±0.015 nM/mg and 0.444±0.581 nM/mg, respectively). In the RV, Drpitor1a level was higher in females (0.102±0.053 nM/mg) than males (0.055±0.015 nM/mg). Drpitor1a inhibited mitochondrial fission and cell proliferation in human PAH PASMC. In addition, Drpitor1a regressed pulmonary artery medial thickening (25.5% vs 34.9%), inhibited RV hypertrophy (Fulton index: 0.33 vs 0.39) and improved RV function (TAPSE: 2.2mm vs 1.8mm) in female MCT-PAH rats. Conclusion: Drpitor1a is a potential therapeutic agent for preclinical PAH and may be more effective in females.

Cold exposure aggravates pulmonary arterial hypertension through increased miR-146a-5p, miR-155-5p and cytokines TNF-α, IL-1β, and IL-6

BackgroundCold temperatures can aggravate pulmonary diseases and promote pulmonary arterial hypertension (PAH); however, the underlying mechanism has not been fully explored. AimTo explore the effect of chronic cold exposure on the production of inflammatory cytokines and microRNAs (miRNAs) in a monocrotaline (MCT)-induced PAH model. MethodsMale Sprague Dawley rats were divided into a Control (23.5 ± 2 °C), Cold (5.0 ± 1 °C for ten days), MCT (60 mg/kg body weight i.p.), and MCT + Cold (ten days of cold exposure after 3 weeks of MCT injection). Hemodynamic parameters, right ventricle (RV) hypertrophy, and pulmonary arterial medial wall thickness were determined. IL-1β, IL-6, and TNF-α levels were determined using western blotting. miR-21-5p and -3p, miR-146a-5p and -3p, and miR-155-5p and -3p and plasma extracellular vesicles (EVs) and mRNA expression of Cd68, Cd163, Bmpr2, Smad5, Tgfbr2, and Smad3 were determined using RT-qPCR. ResultsThe MCT + Cold group had aggravated RV hypertrophy hemodynamic parameters, and pulmonary arterial medial wall thickness. In lungs of the MCT + Cold, group the protein levels of TNF-α, IL-1β, and IL-6 were higher than those in the MCT group. The mRNA expression of Cd68 and Cd163 were higher in the MCT + Cold group. miR-146a-5p and miR-155-5p levels were higher in the plasma EVs and lungs of the MCT + Cold group. Cold exposure promoted a greater decrease in miR-21-5p, Bmpr2, Smad5, Tgfbr2, and Smad3 mRNA expression in lungs of the MCT + Cold group. ConclusionCold exposure aggravates MCT-induced PAH with an increase in inflammatory marker and miRNA levels in the plasma EVs and lungs.

P105 Endocrine, autonomic and vascular function in children with Sleep Disordered Breathing

Abstract SDB is a risk factor for cardiovascular disease and co-exists with chronic endocrine disorders such as type II diabetes and metabolic syndrome. Children with SDB have increased blood flow velocity, an indicator of reduced vascular compliance and early vascular aging. Increased blood flow velocity is positively associated with sympathetic activity, increased arterial sympathetic nerve fibre density and endothelial damage. Whether changes in endocrine function occur concomitantly with altered autonomic and vascular function in children with SDB was assessed. Thirty six children scheduled for tonsillectomy underwent overnight polysomnography (SDB severity), pupil light reflex (autonomic function), fasting brachial artery blood flow assessment (vascular function - Doppler Ultrasound). Leptin and Ghrelin - both hormonal markers associated with sympathetic activity were measured in urine using ELISA and serum using MagPlex. The following dimensions of the dorsal lingual artery (tonsil) were measured – medial thickness, medial area, smooth muscle cell number/layers. We observed a positive correlation between serum and urine leptin and ghrelin concentrations. Increased blood flow velocity and arterial medial thickness were both associated with increased serum and urine leptin and ghrelin concentrations. Pupil light reflex was negatively associated with serum leptin and ghrelin levels. OAHI was positively correlated with leptin and ghrelin concentration (urine and serum) but not blood flow velocity. Blood flow velocity was inversely correlated with SpO2 nadir (REM). Our findings suggest that SDB has a global effect on the autonomic, vascular and endocrine systems.The impact of untreated paediatric SDB on the development of comorbidities in later life needs urgent attention.

O006 Early vascular ageing in children with sleep disordered breathing: evidence of vascular hypertrophy and hyperplasia

Abstract Children with sleep disordered breathing (SDB) have evidence of increased blood flow velocity and sympathetic overactivity. Sympathetic overactivity leads to peripheral vasoconstriction, increased vascular resistance and consequently, increases blood flow velocity. Early vascular ageing involves premature arterial thickening and stiffening that leads to changes in vascular function. Both increased blood flow velocity and sympathetic overactivity are promoters of arterial remodelling and hence, early vascular ageing. No studies have directly histologically investigated arterial wall structure in children with SDB and how it relates to vascular function. Thirty-six children scheduled for tonsillectomy underwent polysomnography to determine SDB severity and resting brachial artery blood flow velocity (velocity time integral and peak systolic velocity) using Doppler ultrasound. The dorsal lingual artery (tonsil) was stained using hematoxylin and eosin techniques to examine arterial wall structures. Increased velocity time integral correlated with increased arterial medial thickness (r = 0.50, P&amp;lt;0.01), arterial smooth muscle cells (r =0.43, P&amp;lt;0.05) and arterial smooth muscle layers (r=0.45, P&amp;lt;0.01). These relationships remained significant after controlling for body-mass index (BMI). Increased BMI was associated with increased velocity time integral (r=0.61, P&amp;lt;0.01), arterial medial thickness (r=0.37, P&amp;lt;0.05) and arterial medial area (r=0.36, P&amp;lt;0.05). SpO2nadir (TST/REM) was inversely associated with arterial medial area (r=-0.35; r=-0.38, P&amp;lt;0.05). These results demonstrate that increased blood flow velocity is associated with changes in arterial wall composition in children with SDB. This suggests that paediatric SDB, a treatable disorder, is potentially a modifiable risk factor for early vascular ageing and resultant cardiovascular disease in adulthood.

Anti-Inflammatory Effect of Allicin Associated with Fibrosis in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling. Recent evidence supports that inflammation plays a key role in triggering and maintaining pulmonary vascular remodeling. Recent studies have shown that garlic extract has protective effects in PAH, but the precise role of allicin, a compound derived from garlic, is unknown. Thus, we used allicin to evaluate its effects on inflammation and fibrosis in PAH. Male Wistar rats were divided into three groups: control (CON), monocrotaline (60 mg/kg) (MCT), and MCT plus allicin (16 mg/kg/oral gavage) (MCT + A). Right ventricle (RV) hypertrophy and pulmonary arterial medial wall thickness were determined. IL-1β, IL-6, TNF-α, NFκB p65, Iκβ, TGF-β, and α-SMA were determined by Western blot analysis. In addition, TNF-α and TGF-β were determined by immunohistochemistry, and miR-21-5p and mRNA expressions of Cd68, Bmpr2, and Smad5 were determined by RT-qPCR. Results: Allicin prevented increases in vessel wall thickness due to TNF-α, IL-6, IL-1β, and Cd68 in the lung. In addition, TGF-β, α-SMA, and fibrosis were lower in the MCT + A group compared with the MCT group. In the RV, allicin prevented increases in TNF-α, IL-6, and TGF-β. These observations suggest that, through the modulation of proinflammatory and profibrotic markers in the lung and heart, allicin delays the progression of PAH.

Preventive effects of nitrate-rich beetroot juice supplementation on monocrotaline-induced pulmonary hypertension in rats.

Beetroot (Beta vulgaris L.) has a high level of nitrate; therefore, its dietary intake could increase nitric oxide (NO) level in the body, possibly preventing the development of pulmonary hypertension (PH). In this study, we examined the effects of beetroot juice (BJ) supplementation on PH and the contribution of nitrate to such effects using a rat model of monocrotaline (MCT, 60 mg/kg s.c.)-induced PH. Rats were injected subcutaneously with saline or 60 mg/kg MCT and were sacrificed 28 days after the injection. In some rats injected with MCT, BJ was supplemented from the day of MCT injection to the day of sacrifice. First, MCT-induced right ventricular systolic pressure elevation, pulmonary arterial medial thickening and muscularization, and right ventricular hypertrophy were suppressed by supplementation with low-dose BJ (nitrate: 1.3 mmol/L) but not high-dose BJ (nitrate: 4.3 mmol/L). Of the plasma nitrite, nitrate, and their sum (NOx) levels, only the nitrate levels were found to be increased by the high-dose BJ supplementation. Second, in order to clarify the possible involvement of nitrate in the preventive effects of BJ on PH symptoms, the effects of nitrate-rich BJ (nitrate: 0.9 mmol/L) supplementation were compared with those of the nitrate-depleted BJ. While the former exerted preventive effects on PH symptoms, such effects were not observed in rats supplemented with nitrate-depleted BJ. Neither supplementation with nitrate-rich nor nitrate-depleted BJ affected plasma nitrite, nitrate, and NOx levels. These findings suggest that a suitable amount of BJ ingestion, which does not affect systemic NO levels, can prevent the development of PH in a nitrate-dependent manner. Therefore, BJ could be highly useful as a therapy in patients with PH.

Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries.

Gestational long-term hypoxia increases the risk of myriad diseases in infants including persistent pulmonary hypertension. Similar to humans, fetal lamb lung development is susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity, which culminates in decreased right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, Western immunoblotting, calcium imaging, and metabolomic analyses on pulmonary arteries isolated from near-term fetal lambs that had been exposed to low- or high-altitude (3,801 m) hypoxia for the latter 110+ days of gestation. Our results demonstrate that the sarcoplasmic reticulum was swollen with high luminal width and distances to the plasma membrane in the hypoxic group. Hypoxic animals were presented with higher endoplasmic reticulum stress and suppressed calcium storage. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g., eicosapentaenoic acid, docosahexaenoic acid, α-linolenic acid, 5-hydroxyeicosapentaenoic acid (5-HEPE), 12-HEPE, 15-HEPE, prostaglandin E3, and 19(20)-epoxy docosapentaenoic acid) and higher levels of some omega-6 metabolites (P < 0.02) including 15-keto prostaglandin E2 and linoleoylglycerol. Collectively, the results reveal broad evidence for long-term hypoxia-induced metabolic reprogramming and phenotypic transformations in the pulmonary arteries of fetal sheep, conditions that likely contribute to the development of persistent pulmonary hypertension.

Structural Thickening of Medial Layer in Coronary Artery With Spasm in Patients With Myocardial Infarction.

BackgroundThe underlying pathophysiology of coronary artery spasm (CAS) remains unclear. We aim to determine whether coronary artery medial layer thickness is associated with CAS using optical coherence tomography.Methods and ResultsA total of 50 patients with previous myocardial infarction underwent optical coherence tomography of the left anterior descending artery: 20 with CAS and 30 without CAS. Intimal and medial layer areas were measured by planimetric analysis of optical coherence tomography images. The medial area/external elastic membrane (EEM) area was significantly greater in patients with than without CAS (0.13±0.01 versus 0.09±0.01, respectively, P<0.01), whereas the intimal area/EEM area was similar in the 2 groups. In patients without CAS, the relationship of intimal area/EEM area with medial area/EEM area and coronary diameter response to intracoronary injection of acetylcholine was characterized by an inverted U‐shaped curve (y=−1.85x 2+0.81x+0.01, R 2=0.43, P<0.001) and a U‐shaped curve (y=2993.2x 2−1359.6x+117.1, R 2=0.53, P<0.001), respectively. Thus, the medial layer became thin and the contractile response became weak in coronary arteries with greater intimal area in the non‐CAS patients. In contrast, in patients with CAS, the intimal area/EEM area had no significant relationship with the medial area/EEM area in either linear correlation analysis or quadratic regression analysis. Thus, even when the intimal layer thickened, the medial layer did not thin in patients with CAS.ConclusionsThe structural thickness of the coronary medial layer was increased in patients with CAS, which may provide mechanistic insight into the pathogenesis of CAS.RegistrationURL: https://www.upload.umin.ac.jp; Unique identifier: UMIN000018432.

DDCI-01, a novel long acting phospdiesterase-5 inhibitor, attenuated monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension is a progressive, malignant heart disease, characterized by pulmonary arteriole remodeling and increased pulmonary vascular resistance, which eventually leads to right heart failure. This study sought to evaluate the effects of a novel long-acting phospdiesterase-5 inhibitor, namely DDCI-01, as an early intervention for monocrotaline-induced pulmonary hypertensive rats. To establish this model, 50 mg/kg of monocrotaline was intraperitoneally injected into rats. At Day 7 after monocrotaline injection, two doses of DDCI-01 (3 or 9 mg/kg/day) or tadalafil (at 3 or 9 mg/kg/day) were intragastrically administered. The rats were anesthetized with pentobarbital for hemodynamic and echocardiographic measurements, at Day 21 after monocrotaline injection. Compared to the monocrotaline group, DDCI-01 at 3 and 9 mg/kg/day (P) reduced the mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure, right ventricular transverse diameter, pulmonary arterial medial wall thickness (WT%), and right ventricle hypertrophy. However, no significant difference in the indices mentioned as above was found between DDCI-01 (3 mg/kg/day) and tadalafil (3 mg/kg/day). In addition, DDCI-01 at 9 mg/kg/day resulted in lower mPAP and WT%, as well as higher cyclic guanosine monophosphate levels in the lung and plasma compared with the same dose of tadalafil (9 mg/kg/day) (all P < 0.05). These findings suggested that DDCI-01 improved monocrotaline-induced pulmonary hypertension in rats, and a dose of DDCI-01 of 9 mg/kg/day might be more effective than the same dose of tadalafil in monocrotaline-induced pulmonary hypertension in rats.