bluuebunny/crossref_metadata_2025 huggingface dataset:
Dendritic cell superoxide and isoketals activate T cells and promote angiotensin II hypertension (1153.2)
Authors: Kirabo Annet , Fontana Vanessa , Davies Sean , Roberts L. Jackson , de Faria Ana , Galindo Cristi , Wu Jing , Bikineyeva Alfiya , Dikalov Sergey , Loperena Roxana , Vinh Antony , Amarnath Venkataraman , Guzik Tomasz , Bernstein Kenneth , Shen Xiao , Moreno Heitor , Harrison David
Published: June 2021

Oxidative injury and inflammation contribute to the genesis of hypertension but the mechanisms involved are not fully understood. We describe a new pathway where angiotensin II promotes dendritic cell (DC) activation of T cells and ultimately hypertension. NADPH oxidase‐dependent superoxide production is increased 5‐fold in DCs from hypertensive mice. This is associated with DC accumulation of protein‐isoketal adducts (isoketals are highly reactive γ‐ketoaldehydes produced via the isoprostane pathway of free radical mediated lipid peroxidation) and production of IL‐6, IL‐1β and IL‐23. DCs from hypertensive but not sham mice promote survival and proliferation of CD8+ T cells. Chemically diverse isoketal scavengers not only prevent activation and immunogenicity of DCs, but also attenuate angiotensin II hypertension (142.59 ± 8.98 mmHg vs. 175.53 ± 5.19 mmHg in controls). Moreover, adaptive transfer of DCs from hypertensive mice prime development of hypertension in response to a subpressor dose of angiotensin II (157.45 ± 33.86 mmHg vs. 119.90 ± 17.33 mmHg in controls). Plasma F2‐isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension when compared to well‐controlled and normotensive subjects (23.2±11.5 vs. 18.3±10.3 vs. 11.6±7.0 pg/mL, p<0.05). We conclude that angiotensin II‐induced hypertension activates DCs, in large part by causing superoxide production and formation of isoketals. These studies define a new mechanism of hypertension and identify a potential new therapeutic approach for this disease.

Grant Funding Source: AHA Post‐Doctoral Fellowship (13POST14440041), R01HL039006, R01HL105294, P01HL095070, P01GM015431
Lysophosphatidic acid signaling is required for NADPH oxidase activation during agonist stimulation in pulmonary microvascular endothelial cells (1153.8)
Authors: Vazquez‐Medina Jose Pablo , Chatterjee Shampa , Fisher Aron
Published: June 2021

The phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) is required for NADPH oxidase 2 (Nox2) activation in pulmonary microvascular endothelial cells (PMVEC; JBC 286:1196, 2011). Lysophosphatidylcholine is a major product of the PLA2 activity of Prdx6 that is readily metabolized to lysophosphatidic acid (LPA). LPA is a bioactive lipid that signals through distinct G protein‐coupled receptors (LPA receptors 1‐5). LPA receptor activation results in the stimulation of signaling pathways regulated by small GTPases, such as the Nox2 cytosolic activator Rac1. We propose that LPA receptor signaling activates Nox2 by stimulating Rac1 in PMVEC. Treatment with Ki16425, a selective antagonist of the LPA receptors 1/3, decreased Rac1 translocation to the plasma membrane during phorbol 12‐myristate 13‐acetate (PMA) stimulation in PMVEC; as demonstrated by in situ proximity ligation assays (Duolink®) and western blotting of isolated plasma membrane proteins. In the same way, LPA receptor blockade decreased superoxide and hydrogen peroxide production in response to PMA stimulation. These results suggest that LPA signaling is required for Nox2 activation during agonist stimulation in PMVEC and that Rac1 is a main target of LPA receptor signaling during this process.

Grant Funding Source: Supported by HL105509
A mitochondrial‐targeting peptide (Bendavia) decreases apoptosis and suppresses cardiac fibrosis in the noninfarcted border zone of infarcted hearts (1154.3)
Authors: Shi Jianru , Dai Wangde , Hale Sharon , Kloner Robert
Published: June 2021

Bendavia is a mitochondrial‐targeting peptide with cardioprotective properties. Apoptosis and cardiac fibrosis contribute to left ventricular remodeling in the post infarct state. We hypothesized that Bendavia decreases apoptosis and attenuates myocardial collagen deposition in the noninfarcted border zone (2 mm noninfarcted tissue on either side of scar). Starting 2 hours after left coronary artery ligation, rats were randomized to receive Bendavia (3 mg/kg/day), water or sham operation. At 6 weeks, TUNEL staining was performed to detect apoptosis. Picrosirius red staining and qRT‐PCR were used to analyze collagen deposition. TUNEL positive cells within the border zone were reduced by Bendavia (32 ± 3% of nuclei, n=12) vs the water group (41 ± 2%, n=12; p=0.029). Bendavia significantly suppressed collagen deposition, assessed by Picrosirius red staining, within the border zone (11 ± 1% of the area of border zone, n=28) vs water group (15 ± 1%, n=26; p=0.03). qRT‐PCR analysis showed that Col1a1, Col1a2 and Col3a1 gene expression were significantly increased by 4.76 fold, p=0.002, 2.85 fold, p=0.01 and 4.71 fold, p=0.019, respectively, in the water group vs sham. Importantly, Bendavia showed a trend of lowering these gene expression levels. We conclude that Bendavia decreases apoptosis and prevents cardiac fibrosis, which may have led to the preservation of cardiac function and structure.
Comparison of two methods to automatically quantify infarct size in rat isolated hearts following global ischemia and reperfusion (1154.6)
Authors: Riess Matthias , Shidham Sushrut , Nabbi Raha , Gaggl Wolfgang , Lerch‐Gaggl Alexandra
Published: June 2021

Infarct size (IS) remains the gold standard to estimate cardiac injury in many animal models. 2,3,5‐triphenyltetrazolium chloride (TTC) staining delineates infarcted from non‐infarcted tissue. Here, we compare manual image analysis to commercially available software using Bayesian differentiation and a newly developed automated freeware algorithm using color thresholding. As part of a study on genome‐dependent cardioprotection we analyzed 855 slices from 156 isolated rat hearts subject to 30/120 min ischemia/reperfusion after different protective treatments. Hearts were cut into 2 mm transverse slices and TTC‐stained, slices scanned on green background and infarcted areas measured manually and automatically by VisiomorphTM and Image J (NIH) whose color threshold T was varied from 125 to 133 to determine the best differentiation for IS. Statistics: linear regression; alpha 0.05. VisiomorphTM correlated well with manual measurements as did Image J where T129 provided the best agreement. IS measurement by either software provides equal results to manual measurements. Both methods enable fast, operator‐independent and reliable IS assessment. Potential shortcomings such as lack of discrimination are offset by significant time‐ and cost‐savings and a better longitudinal resolution by analyzing more slices per heart. Supported by Department of Veterans Affairs (CARA‐026‐10F) and NIH.

Grant Funding Source: Supported by Department of Veterans Affairs and NIH.
Obesity superimposed on aging magnifies the inflammatory and plasma lipid mediator responses following myocardial infarction (1155.1)
Authors: Halade Ganesh , Lopez Elizabeth , Kabarowski Janusz , Lindsey Merry
Published: June 2021

Fatty acids intake has increased over the last 100 years, contributing to an increase in obesity and diabetes. Obesity and aging are both risk factors for myocardial infarction (MI). How obesity interacts with aging to alter the post‐MI response, however, is unclear. We tested the hypothesis that obesity and aging would impair the resolution of post‐MI inflammation. Mice (12 month old C57BL/6J) fed with 10% (w/w) safflower oil (SO) diet for 5 months showed 28% higher fat mass than lean controls (LC). At 17 months of age, mice were subjected to coronary artery ligation to induce MI. At d1 post‐MI, infarct areas were 42±1% and 45±1% in SO and LC, respectively (p=n.s.). Obese mice showed higher neutrophil infiltration into the infarcted area and elevated VCAM‐1 levels compared to LC (both p<0.05). Plasma analysis revealed that monocyte chemotactic protein‐3, macrophage inflammatory protein‐1 and CD40 were increased at d1, while myeloperoxidase was increased at d5 post‐MI, in the SO group compared to LC (all p<0.05). Lipidomic analysis showed higher levels of arachidonic acid in SO group at all‐time points compared to LC (all p<0.05). Further, 12(S) Hydroxyeicosatetraenoic acid was increased at d1 compared to LC (p<0.05). Together, our results indicate that obesity accentuated the post‐MI inflammatory response by stimulating neutrophil trafficking and pro‐inflammatory lipid mediators.
4‐HNE post‐translational modification of TRPV1: implications for diabetic cardiomyopathy (1155.8)
Authors: DelloStritto Daniel , Kmetz John , Klarich Brittany , Chen Yeong‐Renn , Geldenhuys Werner , Bratz Ian
Published: June 2021

Previously our lab has shown TRPV1 ‐dependent regulation of myocardial blood flow (MBF) is disrupted in a db/db mouse model of diabetic cardiomyopathy (DCM). A crucial mechanism behind pathologies seen in diabetes is the imbalance in oxidative stress (OS) leading to increased lipid peroxidation (LPO). The LPO product 4‐hydroxynonenal (4‐HNE) is known to increase oxidative post translational modification (PTM) resulting in altered protein function. Investigation into the implications of this oxidative PTM on TRPV1 has yet to be examined. Accordingly, we hypothesized that 4‐HNE PTM of TRPV1 results in decreased functional expression leading to perfusion impairments in DCM. Initial experiments were performed to evaluate 4‐HNE modification of TRPV1. Pull down experiments on HEK cells constitutively expressing TRPV1, treated with H2O2 (to simulate OS) and 4‐HNE, resulted in increased 4‐HNE modified TRPV1 compared to control with a concomitant decrease in TRPV1 expression. Similar findings were demonstrated in hearts from diabetic animals and controls treated with H2O2 and 4‐HNE. Computer generated modeling identified numerous possible residues capable of undergoing 4‐HNE modification of which several were confirmed using mass spectroscopy. TRPV1 functional analysis using path‐clamp electrophysiology revealed blunted capsaicin‐mediated currents in the presence of 4‐HNE and/or prolonged H2O2 treatment. Similarly, 4‐HNE decreased capsaicin mediated increases in MBF and relaxation in isolated coronary microvessels. These data suggest that increased OS in a diabetic state leads to decreased TRPV1 functional expression resulting in perfusion impairments seen in DCM.
Transcriptome‐wide RNA sequencing analysis of rat skeletal muscle feed arteries (1156.12)
Authors: Jenkins Nathan , Padilla Jaume , Thorne Pamela , Martin Jeffrey , Rector R , Davis J , Laughlin M.
Published: June 2021

We employed next generation RNA sequencing (RNA‐Seq) technology to determine the influence of obesity on global gene expression in skeletal muscle feed arteries. Transcriptional profiles of the gasctrocnemius and soleus muscle feed arteries (GFA and SFA, respectively) and aortic endothelial cell‐enriched samples from obese Otsuka Long Evans Tokushima Fatty (OLETF) and lean Long Evans Tokushima Otsuka (LETO) rats were examined. Obesity produced 282 upregulated and 133 downregulated genes in SFA and 163 upregulated and 77 downregulated genes in GFA (false discovery rate < 10%) with an overlap of 93 genes between the arteries. Within LETO, there were 89 upregulated and 114 downregulated genes in the GFA compared to the SFA. There were 244 upregulated and 275 downregulated within OLETF (FDR < 10%) in the GFA compared to the SFA, with an overlap of 76 differentially expressed genes common to both LETO and OLETF in both the GFA and SFA. A total of 396 transcripts were found to be differentially expressed between LETO and OLETF in aortic endothelial cell‐enriched samples. Overall, we found (i) the existence of heterogeneity in the transcriptional profile of the SFA and GFA within healthy LETO rats, (ii) that this between‐vessel heterogeneity was markedly exacerbated within the hyperphagic, obese OLETF rat, and (iii) a greater number of genes whose expression was altered by obesity in the SFA compared to the GFA. Also, results indicate that in OLETF rats, the GFA takes on a relatively more pro‐atherogenic phenotype compared to the SFA.

Grant Funding Source: Supported by NIH RO1HL036088, T32‐AR048523, VHA‐CDA2 1299‐02
3β‐Diol, an endogenous androgen, elicits relaxation via estrogen receptor activation in rat mesenteric and cerebral arteries (1156.15)
Authors: Gonzales Rayna , Ramirez Angeleah , O'Connor Devin
Published: June 2021

Nongenomic vasorelaxant effects of androgens, such as testosterone and dihydrotestosterone (DHT), have been shown to be beneficial in a variety of vascular beds. These nongenomic vasorelaxant actions have been shown to be independent of androgen receptor (AR) activation. However, the vascular properties and associated mechanisms for endogenous androgen metabolites downstream of these potent AR agonists have not been investigated. Therefore, we studied the direct vasorelaxing effects of the endogenous DHT metabolite, 5α‐androstane 3β, 17β diol (3β‐diol), on contracted mesenteric and basilar arterial rings isolated from male rats via wire myography. Previous studies have demonstrated that the actions of 3β‐diol are mediated by estrogen receptor beta (ERβ). Thus, we confirmed the presence of ERβ expression along with AR and ERα in mesenteric and cerebral arteries using western blot and RT‐PCR. In the contractile studies, 3β‐diol (10‐9 to 10‐5 M) reduced phenylephrine‐induced contractions in mesenteric rings. Similarly, serotonin‐induced contractions in basilar rings were also reduced by the addition of 3β‐diol (10‐9 to 10‐5 M). Vasorelaxing efficacy for 3β‐diol was greater in mesenteric rings (Rmax = 77.34%±17.05) compared to basilar rings (Rmax = 65.92% ±7.74). In both mesenteric and basilar arteries, non‐selective ER inhibition with ICI 182780 and selective ERβ inhibition with PHTPP attenuated 3β‐diol relaxation compared to vehicle. Based on these findings it is concluded that 1) the endogenous androgen metabolite, 3β‐diol, mediates direct vasorelaxation of arterial rings via ERβ and 2) regional differences in vasorelaxation of 3β‐diol exist between the peripheral and cerebral circulations.

Grant Funding Source: Supported by the UA Sarver Heart Center
Skeletal muscle mitochondrial function in severely burned children improves following rehabilitative exercise (1159.13)
Authors: Chao Tony , Suman Oscar , Herndon David , Sidossis Labros , Porter Craig
Published: June 2021

Severe burn injury results in skeletal muscle mitochondrial dysfunction. In this study, we determined in skeletal muscle mitochondrial function in children with severe burns (n=8) at discharge from hospital and following 6 weeks of rehabilitative exercise (REX).

Mitochondrial respiration was determined in permeabilized myofibers following the addition of substrates, ADP, succinate, and oligomycin. After which, the respiratory control ratio (RCR), substrate control ratio (SCR), and coupling control ratio (CCR) were calculated.

Maximum mitochondrial respiration significantly increased after exercise training (12.7±2.1 pmol/mg/sec vs. 38.6±5.4 pmol/mg/sec, p<0.01), RCR (1.0 vs. 1.5) and SCR (1.1 vs. 1.7) increased while the CCR decreased (0.7 vs. 0.4) post REX.

Skeletal muscle mitochondrial function is increased in burn victims following REX. Increased RCR and SCR indicate greater ADP and succinate sensitivity. The decrease in CCR suggests that mitochondria are more coupled post REX. These data suggest that early REX restores skeletal muscle mitochondrial function in burn survivors.

Grant Funding Source: Supported by NIH (P50‐GM60338, R01‐HD049471, R01‐GM056687), and SHC (84090, 84080 and 84085) grants.
Skeletal muscle immobilized in a stretched position does not display characteristics of disuse atrophy (1163.1)
Authors: Kelleher Andrew , Gordon Bradley , Luongo Lauren , Kimball Scot , Jefferson Leonard
Published: June 2021

In a recent report we showed that skeletal muscle immobilized in a shortened position exhibits atrophy in association with reduced rates of protein synthesis and mTORC1 signaling. The objective of the present study was to test the hypothesis that, in contrast to the effects of immobilizing skeletal muscle in a shortened position, muscle immobilized in a stretched position would be resistant to the development of disuse atrophy. To test the hypothesis, male Sprague‐Dawley rats were subjected to unilateral hindlimb immobilization for 3 days with the soleus muscle placed in either a shortened or stretched position. All comparisons were made to the contralateral non‐immobilized muscle. Soleus muscles immobilized in a shortened position exhibited disuse atrophy, attenuated rates of protein synthesis, attenuated phosphorylation of Thr389 on the mTORC1 substrate p70S6K1, and induction of the mTORC1 repressors REDD1 and REDD2. Muscles immobilized in a stretched position exhibited no difference in muscle mass, rates of protein synthesis, p70S6K1 phosphorylation, or REDD1 expression and a slight reduction in REDD2 expression. In conclusion, fixed muscle length plays a major role in immobilization‐induced skeletal muscle atrophy whereby muscle placed in a stretched position exhibits resistance to disuse atrophy.

Grant Funding Source: Supported by NIH Grant DK‐15658
Patients with chronic myosteatosis have a reduction in muscle fiber force production and disrupted myofibril architecture (1163.18)
Authors: Mendias Christopher , Roche Stuart , Davis Max , Lynch Evan , Bedi Asheesh
Published: June 2021

A persistent atrophy of muscle fibers and an accumulation of fat, collectively referred to as myosteatosis, commonly occurs in patients with chronic muscle tears. The rotator cuff muscle group develops substantial myosteatosis after chronic tears, and patients with these injuries often have marked reductions in upper extremity function and quality of life. As the etiology of myosteatosis has not been well characterized in humans, our objective was to determine changes in muscle fiber structure and function in patients with chronic rotator cuff tears. We hypothesized that muscles from patients with chronic rotator cuff tears have reduced muscle fiber force production, disordered myofibrils and an accumulation of inflammatory macrophages.

Compared with control muscles, there was no difference in muscle fiber cross‐sectional area, but torn supraspinatus muscles had a 30% reduction in maximum isometric force production and a 29% reduction in normalized force. Using electron microscopy and histology, disordered sarcomeres were noted, along with an accumulation of lipid‐laden macrophages in the extracellular matrix surrounding muscle fibers. The size of the tear correlated well (R2=0.49, P<0.01) with reductions in force production. The results of the study indicate that patients with myosteatosis have marked reductions in muscle fiber force production, disrupted myofibril architecture and an accumulation of inflammatory macrophages in the muscle extracellular matrix. This work will help to establish future therapies to restore muscle function for patients with myosteatosis.
Effects of a 6‐month trial of weight‐loss diet plus aerobic exercise versus matched (by dose/intensity) exercise alone on glucose tolerance and insulin resistance in subjects with pre‐diabetes (117.2)
Authors: Bales Connie , Starr Kathryn , Granville Esther , Slentz Cris , Bateman Lori , Willis Leslie , Piner Lucy , Kraus William
Published: June 2021

The Diabetes Prevention Program first demonstrated benefits of lifestyle interventions for delaying progression of pre‐diabetes. Our study examines the separate effects of a weight‐loss diet plus exercise or exercise alone on oral glucose tolerance (OGTT) in a randomized 6‐mo. study of participants (age=57.8+7.6 y; BMI 30.6±2.9) with pre‐diabetes (fasting glucose 95‐124 mg/dL). Weight reduction diet + moderate aerobic exercise (D+Ex; n=41) was compared to a matched exercise program with no diet component (Ex; n=36). Baseline age, BMI, fitness level, and gender/race did not differ. D+Ex included a weekly support group and body weights (BW). Compliance with supervised exercise was similar for D+Ex and Ex (87.8% and 84.2%). At 6 mo., BW reductions in D+Ex (‐6.1+5.0 kg) exceeded (p<0.0001) those in Ex (‐0.9±3.3 kg). D+Ex improved (p<0.0001) fasting glucose (‐4.9±8.1 mg/dL), fasting insulin (‐3.5±3.8 mg/dL), 2‐hr glucose (‐20.5±35.9 mg/dL), HOMA‐IR (‐1.0±1.2) and glucose areas under the response curve. Ex, as well as D+Ex, improved (p<0.05) 2‐hr glucose, insulin area under the curve, and Matsuda Index. Weight loss plus exercise was superior for improving glucose tolerance/insulin resistance in this population. Thus, clinical recommendations for prediabetes should emphasize diet‐induced weight loss, along with exercise, as the first line of treatment.

Grant Funding Source: Supported by NIDDK R01‐DK081559 and NIA AG000029.
Mechanisms mediating the muscle metaboreflex pressor response during post‐exercise muscle ischemia are altered in hypertension (1170.2)
Authors: Spranger Marty , Kaur Jasdeep , Sala‐Mercado Javier , Krishnan Abhinav , Alvarez Alberto , O'Leary Donal
Published: June 2021

During dynamic exercise, muscle metaboreflex activation (MMA; induced via partial hindlimb ischemia) markedly increases mean arterial pressure (MAP) and MAP is sustained when the ischemia is maintained following the cessation of exercise (post‐exercise muscle ischemia ‐ PEMI). We previously reported that the sustained pressor response during PEMI in normal individuals is driven by a sustained increase in cardiac output (CO) with no peripheral vasoconstriction. However, we have recently shown that the rise in CO with MMA is significantly blunted in hypertension (HTN). The mechanisms sustaining the pressor response during PEMI in HTN are unknown. In six chronically instrumented canines, hemodynamic responses were observed during rest, mild exercise (3.2 kph), MMA and PEMI in the same animals before and after the induction of HTN (2K1C). In control, MAP, CO and HR increased with MMA (+52 ± 6 mmHg, +2.1 ± 0.3 l/min and +37 ± 7 bpm). After the induction of HTN (MAP at rest increased from 97 ± 3 to 130 ± 4 mmHg), these responses were markedly attenuated (+32 ± 5 mmHg, +0.6 ± 0.2 l/min and +11 ± 3 bpm), yet MAP was similarly sustained during PEMI while CO and peripheral vascular conductance were significantly lower (5.2 ± 0.2 l/min and 32 ± 3 ml/min/mmHg vs. 4.1 ± 0.4 l/min and 26 ± 2 ml/min/mmHg). We conclude that peripheral vasoconstriction significantly contributes to the sustained pressor response during PEMI in HTN.

Grant Funding Source: HL‐55743 and HL‐095819
Salt loading increases the muscle sympathetic nerve activity response to upright tilt: a pilot study (1170.3)
Authors: Best Stuart , Okada Yoshiyuki , Parker Rosemary , Bivens Tiffany , Fu Qi
Published: June 2021

Increased sodium intake reduces resting muscle sympathetic nerve activity (MSNA). We hypothesized that the increase in MSNA during upright tilt would be greater following a high sodium (HS) diet compared to a low sodium (LS) diet. Six healthy women (40±6[SD]yrs) were tested following a 7 day HS (5750mgNa+/day) and 7 day LS diet (1150mgNa+/day) in randomized order during the mid‐luteal phase. Blood pressures (BP, SunTech), heart rate (HR, ECG), cardiac output (Qc, C2H2 rebreathing) and MSNA (microneurography) were measured at rest, 30o and 60o head‐up tilt. Stroke volume (SV) and total peripheral resistance (TPR) were calculated. BP was similar at supine and 30o but higher at 60o following the HS when compared to LS diet. HR, Qc, SV and TPR were similar following each diet. Supine MSNA burst frequency and burst incidence (BI) respectively were lower following the HS (13±8 bursts.min‐1, 22±13 bursts.100beats‐1) than the LS diet (22±10 bursts.min‐1, 36±18 bursts.100beats‐1). The increase in BI from baseline to 60o was greater after the HS (28±4 bursts.100beats‐1) than the LS diet (22±11 bursts.100beats‐1). These preliminary results suggest a HS diet may enhance the MSNA response and increase BP during orthostasis in healthy women. This could be due to greater sympathetic vasoconstrictor reserve and/or alteration of the sympathetic baroreflex following the HS diet.
Inflammatory cytokines trigger SR/ER stress in human airway smooth muscle (1174.3)
Authors: Wallerich Samantha , Delmotte Philippe , Prakash Y.S. , Sieck Gary
Published: June 2021

Airway inflammation is a key feature of asthma, with inflammatory cytokines (e.g., TNFα and IL‐13) triggering hyperreactivity (enhance contractile state) and proliferation and remodeling (enhanced synthetic state) in airway smooth muscle (ASM). We hypothesize that TNFα and IL‐13 induce sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) stress, thereby altering expression of mitochondrial fission/fusion proteins (dynamin‐related protein ‐ Drp1 and mitofusin ‐ Mfn2) with downstream impact on ASM Ca2+ regulation and contractility as well as proliferation. Human ASM cells were isolated from lung specimens obtained incidental to patient surgery. We found that after 24 h exposing ASM cells to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, calnexin and XBP1) increased; 2) Drp1 expression increased Mfn2 expression decreased; 3) mitochondrial fragmentation increased; 4) [Ca2+]cyt and [Ca2+]mito responses to ACh stimulation were uncoupled; 5) net [Ca2+]cyt increased; 6) contractility increased; and 7) cell proliferation increased. Together these results suggest that inflammation induces SR/ER stress that leads to changes in Mfn2 and Drp1 expression, which alter mitochondrial dynamics and Ca2+ regulation resulting in ASM hyperreactivity and remodeling.

Grant Funding Source: Supported by NIH grant HL74309 (GCS)
Chronic hypoxia increases pro‐inflammatory TH17 cells via NFATc3‐induced interleukin 6 production in lung (1175.5)
Authors: Maston Levi , Reinhart Katelyn , Giermakowska Wieslawa , Nitta Carlos , Herbert Lindsay , Jernigan Nikki , Cannon Judy , Resta Thomas , González Bosc Laura
Published: June 2021

We have demonstrated that the adaptive immune system along with nuclear factor of activated T cells isoform c3 (NFATc3) contribute to chronic hypoxia (CH)‐induced pulmonary hypertension (PH). NFATc3 regulates IL‐6 expression in multiple cells types. Considering that IL‐6 contributes to hypoxic PH and is required for polarization of naïve CD4+ T helper cells to the TH17 subset, we hypothesized that NFATc3 drives IL‐6 expression in pulmonary arterial smooth muscle cells (PASMC) contributing to CH‐induced increases in lung TH17 cells. Since TH17 cells are implicated in several cardiovascular diseases, these cells may play an unexplored role in PH. In support of our hypothesis, we found that IL‐6 transcripts as determined by real‐time PCR were increased in cultured mouse PASMC in which NFAT was activated with ionomycin and phorbol 12‐myristate 13‐acetate. This response was prevented by the calcineurin/NFAT inhibitor cyclosporine A and is consistent with our previous report demonstrating that CH activates NFATc3 in PASMC. Furthermore, lung TH17 cells as determined by flow cytometry were increased in male C57BL/6 mice exposed to 5 days of CH (PB=380 mmHg) compared to normoxic mice. This response was prevented by i.p. administration of a selective NFAT inhibitor (A‐285222, AbbVie). Our data suggest that CH‐induced PASMC NFATc3 activation and subsequent IL‐6 production contribute to increases in lung TH17 cells.

Grant Funding Source: Supported by T32 HL07736, R01HL088151, R01 HL088192, R01 AI097202
Purinergic receptor stimulation induces Ca2+ oscillations and smooth muscle contraction in small pulmonary veins (1175.6)
Authors: Henriquez Mauricio , Perez‐Zoghbi Jose
Published: June 2021

The small intrapulmonary veins (SPV) are thought to participate in the control of pulmonary blood flow and its excessive contraction has been associated with the development of pulmonary edema. However, the regulation of smooth muscle contraction by intracellular signaling in SPV is poorly understood. We used rat lung slices with phase‐contrast and confocal microscopy to investigate Ca2+ signaling and contraction during purinergic receptor activation in small (100‐250 µm) pulmonary veins. We found that ATP induced strong, concentration‐dependent, and reversible vein contraction. Vein contraction was accompanied by intracellular Ca2+ oscillations that propagated along the vein smooth muscle cell as Ca2+ waves. In the absence of extracellular Ca2+, ATP induced Ca2+ oscillations that were attenuated in time and transient vein contraction. The InsP3 receptor inhibitor 2‐APB inhibited the Ca2+ oscillations and contraction induced by ATP in absence of extracellular Ca2+. In contrast, Ca2+ oscillations and contraction were resistant to ryanodine. Vein contraction was inhibited by phospholipase Cβ inhibitor U73122 and by purinergic receptor antagonist suramin. These results suggest that ATP is a strong vasoconstrictor of SPV and that ATP activate purinergic P2Y receptors in vein smooth muscle to induce InsP3 receptor‐mediated Ca2+ oscillations and vein contraction.

Grant Funding Source: American Heart Association
The Src family tyrosine kinase, fyn, is a negative regulator of inflammation‐induced apoptosis in the lung (1176.6)
Authors: Jin Yi , Liu Yusen , Chicoine Louis , Chen Bernadette , Nelin Leif
Published: June 2021

Endothelial cell apoptosis is an early event in the development of acute respiratory distress syndrome (ARDS). We hypothesized that the Src‐family tyrosine kinases (STK) would differentially regulate endothelial cell apoptosis. Human pulmonary microvascular endothelial cells (hPMVEC) were transfected with siRNA against fyn or yes and then treated with cytomix (LPS, TNF‐α, IL‐1β and INF‐γ) or vehicle. Following cytomix treatment knock‐down of fyn resulted in greater levels of caspase 3 activity and lower viable cell numbers, while knock‐down of yes resulted in lower levels of caspase 3 activity and greater viable cell numbers, than in scramble treated control hPMVEC. We then tested the hypothesis that mice deficient in fyn will have greater lung injury following exposure to LPS. Wild‐type and fyn‐/‐ mice were given LPS 10 mg/kg or saline before harvesting tissues or measuring static lung compliance. The fyn‐/‐ mice had lower static lung compliance 24 hours after LPS compared to the WT mice. LPS exposure also caused more cleaved‐caspase 3 and p21 expression in the lungs of fyn‐/‐ mice than in WT mice. Real time‐PCR results showed that following LPS treatment the mRNA levels for arginase I, iNOS, and COX‐2 were greater in the fyn knockout mice than in WT mice. Our results demonstrate that fyn is an important negative regulator of LPS‐induced iNOS expression and apoptosis in cultured endothelial cell and in a mouse model of inflammatory lung disease.
Combinatorial treatment with epigenetic modifiers inhibits thrombin‐induced eNOS/RhoA signaling and restores AJ integrity (1176.7)
Authors: Balasubramanian Sathyamoorthy , Thangavel Jayakumar , Dileepan Kottarappat N , Dhanasekaran Anuradha , Dawn Buddhadeb , Johnson Rajasingh
Published: June 2021

Objective: Treatment of acute lung injury (ALI) is to target the pathways that lead to profound dysregulation of the restrictive endothelial barrier, and thereby restore lung microvascular endothelial barrier function and inhibit protein‐rich tissue fluid accumulation. Here we test the hypothesis that epigenetic modifiers TSA (Trichostatin‐A, HDAC inhibitor) and Aza (5‐aza‐2‐deoxycytidine, DNA methyl transferase inhibitor) together inhibit the thrombin induced eNOS/RhoA signaling and restore adherens junctions (AJs) integrity and diminished endothelial hyperpermeability.

Results: The signaling, AJ integrity and endothelial permeability were assessed by RT‐PCR, immunofluoresence, Western analysis, and transendothelial resistance (TER). Our data show that combination of TSA+Aza is more effective in inhibiting thrombin‐induced lung hyperpermeability. We used an in vitro culture system of TER to determine the effect of TSA+Aza on thrombin‐induced change in resistance over the human pulmonary arterial endothelial cells. TSA+Aza treatment ameliorated the thrombin‐induced endothelial barrier disruption. The Western analysis data show that the treatment of TSA+Aza decreases the levels of thrombin induced RhoA and eNos signaling in lung endothelial cells.

Conclusions: Our data indicate, for the first time, that TSA+Aza exert a protective role in preventing lung microvascular hyperpermeability and endothelial barrier disruption in thrombin‐induced lung injury, and thus have great potential as novel therapeutic agents
Respiratory activation of neurons in the central nucleus of the amygdala (CeA) and effect of CeA neuron stimulation on respiration (1178.5)
Authors: Zhou Guannan , Tsai Hsiu‐Wen , Davenport Paul
Published: June 2021

Respiratory disease causes aversive responses, which can elicit panic or anxiety. It is known that the central nucleus of the amygdala (CeA) is involved in fear and anxiety emotion, however, the ventilator pattern modulation by CeA is unclear. We hypothesized that tracheal obstruction would elicit activation of CeA neurons and electrical stimulation of within the CeA would cause specific changes in breathing frequency (BF). Tracheal occlusions were applied to anesthetized rats and those rat brains were subsequently sectioned and stained for c‐Fos expression. c‐Fos expressions were found in CeA in the tracheal occlusion group, which suggests that CeA is activated by the mechanical respiratory sensory afferent from the trachea. Micorelectrode electrical stimulation was systematically applied to sites in CeA. The sites with greatest increase and decrease of the BF were identified. The sites with greatest increased BF were located at the rostral part of the dorsal medial border of CeA. The greatest decreased BF responses were observed in the ventral caudal part of CeA. The BF changes after the electrical stimulations indicate that the specific areas of the CeA facilitate or inhibit the respiratory neural drive. The opposite responses found in different CeA sites suggest that the central nucleus of the amygdala can regulate respiratory pattern via multiple pathways.
Alterations in Janus (JAK2)/signal transducer of activated transcription signaling pathway in human renal cell carcinoma (1179.2)
Authors: Banes‐Berceli Amy , Singhal Sara , Fulton Matthew , Geddes Timothy , Thibodeau Bryan , Wilson George , Hafron Jason
Published: June 2021

RCC accounts for 9 out of 10 cases of kidney cancer. Approximately 65,150 new cases and 13,680 deaths are expected from this disease in 2013. There is urgent clinical need for better treatment options as RCC responds poorly to current chemotherapy. A major limitation to developing new treatments is that the molecular mechanisms responsible for inappropriate cell survival and chemoresistance are unknown. One pathway suggested to be involved in vitro is the JAK2/STAT. We hypothesized that there would be altered expression of the members and regulators of the JAK/STAT pathways in RCC. We utilized samples from normal (n=14), benign (n=6), and histological Fuhrman grades: Grade 1 (n=3), Grade 2 (n=10), Grade 3 (n=14), and Grade 4 (n=3). We analyzed 31 human RCC samples as well as the normal and benign samples with Affymetrix human gene microarrays and Western Blot technology. The samples were all clear cell renal carcinomas verified by a Beaumont Hospital pathologist. We analyzed 22,148 genes and altered gene expression was then detected by ANOVA taking into account histological stage. We found 170 genes that were differentially expressed in all grades compared to both normal and benign kidney samples. We found both JAK1 and JAK2 mRNA to be highly expressed in the RCC samples compared to the controls. We did not observe any decrease in the mRNA levels or protein expression of the regulatory proteins SOCS1, SOCS3 or SHP‐1. We did find an increase in SHP‐1 serine phosphorylation which is inhibitory in the RCC samples. These data suggest that altered function of the regulators not expression may be involved in RCC.

Grant Funding Source: Zafarna Foundation
Lack of adenylyl cyclase 6 causes pseudohypoparathyroidism (1181.1)
Authors: Fenton Robert , Murray Fiona , Dominguez Rieg Jessica , Tang Tong , Levi Moshe , Rieg Timo
Published: June 2021

Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) enhance phosphate (Pi) excretion by the proximal tubule of the kidney via retrieval of the Na+‐dependent Pi transporters (Npt2a & Npt2c). PTH activates adenylyl cyclase (AC) via PTH 1 receptors and stimulates cAMP/PKA signaling. However, the precise role and isoform(s) of AC in Pi homeostasis are unknown. We previously showed that mice lacking AC6 (AC6‐/‐) have increased plasma PTH and FGF23 while having comparable plasma Pi concentrations. Acute activation of the Ca2+ sensing receptor or feeding a zero Pi diet almost completely suppressed plasma PTH in AC6‐/‐ and wild‐type (WT) mice, indicating secondary hyperparathyroidism. We now studied the abundance and localization of Npt2a and Npt2c. Immunofluorescence and quantitative confocal laser scanning microscopy showed a reduced renal Npt2a and Npt2c protein abundance in AC6‐/‐ mice. Immunogold labeling indicated a tendency for a reduced total number of gold particles per proximal tubule cell in AC6‐/‐ mice (510±53 vs 715±119, P=0.09). In contrast to the Npt2a distribution under basal conditions in WT mice (53, 28 and 17% reside in microvilli, lysosomes and intracellular, respectively), in AC6‐/‐ mice the majority of Npt2a resides in lysosomes (12, 77 and 10%, respectively). The distribution of Npt2a gold particles 1h after PTH treatment (200 µg/kg) in WT mice was shifted, with less particles residing in the apical microvilli (24%) compared to lysosomes (40%) and intracellular compartments (40%) whereas the distribution in AC6‐/‐ was unchanged. We conclude that AC6 in the proximal tubule determines Npt2a trafficking and urinary phosphate excretion, such that AC6‐/‐ mice have pseudohypoparathyroidism.

Grant Funding Source: P01 HL66941, HL088426, 1101BX001515, R01DK066029, P30DK079337 and 10SDG2610034
Acid activates ENaC and enhances salt taste in human subjects (1181.10)
Authors: Collier Daniel , Snyder Peter
Published: June 2021

Blood pressure is dependent on the balance between Na+ intake and urinary Na+ excretion. In the kidney, ENaC regulates Na+ excretion. In the tongue, ENaC functions as a salt taste receptor, thereby contributing to the control of dietary Na+ intake. In previous work, we found that protons within the pH range of the kidney (pH 8.5‐5) reversibly stimulate ENaC. In the tongue, ENaC encounters extreme changes in pH—foods we ingest can be highly acidic (e.g. Coca‐cola is pH 2.2). We tested the effect of highly acidic pH (2.5) on ENaC activity. We expressed human ENaC (‐, ‐, and ENaC) in Xenopus oocytes and recorded ENaC Na+ currents. We found that pH 2.5 irreversibly increased ENaC current 2.2‐fold. To determine the mechanism, we performed single channel experiments. Channels treated with pH 2.5 showed an irreversible 2‐fold increase in open probability. Furthermore, we identified two residues in ENaC (Q371 and D376) that are required for acid activation. We hypothesized that activation of ENaC by acid would increase salt taste sensitivity. To test this, we measured the salt taste detection threshold in human subjects before and after lingual application of 10 mM H2PO4 (pH 2.2). We found that acid reduced salt detection threshold by 50%. Together, the data indicate that highly acidic pH irreversibly activates ENaC, resulting in enhanced salt taste. This may provide a physiological mechanism to regulate dietary Na+ intake.

Grant Funding Source: Supported by NIH 2T32HL007121‐36 (DMC) and HL072256 (PMS)
Identifying transcriptional targets of vasopressin using combined ChIP‐seq and RNA‐seq analysis of mpkCCD cells (1181.6)
Authors: Claxton J'Neka , Sandoval Pablo , Lee Jae Wook , Hoffert Jason , Knepper Mark
Published: June 2021

The peptide hormone vasopressin regulates transport in the renal collecting duct in part through regulation of gene expression. To obtain a global profile of genes regulated by vasopressin, we performed ChIP‐seq analysis of RNA polymerase II and acetylated histone H3K27 binding sites associated with transcriptionally active genes. Cultured mpkCCD cells were treated with either the vasopressin analog dDAVP (0.1 nM) or vehicle for 24 hours, and then processed for chromatin IP. DNA‐protein complexes were immunoprecipitated using either RNA pol II or H3K27ac antibodies. After reversal of crosslinks, DNA fragments were sequenced and mapped to a reference genome. ChIP‐seq data analysis allowed for precise mapping of genomic elements that were significantly enriched or de‐enriched with dDAVP treatment including exonic, intronic, intergenic, and promoter regions. We correlated these changes at the genomic level with corresponding changes in RNA using RNA‐seq analysis of ‘Poly(A)’‐enriched libraries from mpkCCD cells treated with or without dDAVP. Targets enriched in ChIP‐seq and RNA‐seq data sets in the presence of dDAVP included the water channel aquaporin‐2 (Aqp2), genes associated with AQP2 trafficking (Mal2) and AQP2 transcriptional regulation (Elf3), and many others. This study presents a wealth of new data needed for modeling the transcriptional response to vasopressin.

Grant Funding Source: Supported by NHLBI
Immunohistochemical evidence of a reduced vasodilatory capacity in vertebrobasilar arteries in pre‐hypertensive spontaneous hypertensive rats (1183.9)
Authors: Roloff Eva , Kasparov Sergey , Paton Julian
Published: June 2021

Our earlier work has shown that vertebrobasilar artery remodelling causing high vascular resistance occurs in SHR prior to the development of hypertension. We hypothesise that a component of this remodelling results from reduced capacity of the vasodilatory parasympathetic innervation and that this contributes to the etiology of neurogenic hypertension via Cushing’s mechanism. Whole mounts of brainstem ventral brain vessels from hypertensive SHR (<12weeks), PH‐SHR (P28‐35) and age‐matched Wistars were immunostained for dopamine‐β hydroxylase (DBH), vesicular acetylcholine transporter (VAChT) and vasoactive intestinal peptide (VIP); the latter is co‐expressed in parasympathetic fibres. We found an age dependent reduction in the parasympathetic (~60‐85%) and sympathetic (10‐40%) innervation density of vertebrobasilar arteries in SHRs compared to Wistar rats (p<0.001). VAChT positive fibre densities were ~30‐50% lower than VIP‐ in all rat groups. In conclusion, SHRs have fewer parasympathetic fibres innervating the vertebrobasilar arteries than Wistars. Given the higher prevalence of both VAChT and VIP in Wistar rats, we speculate that the vasodilatory capacity of these cerebral vessels in SHR will be compromised.

Grant Funding Source: Supported by BHF, NIH
Quantifying autoregulation from estimated model parameters: an optimization approach (1184.8)
Authors: Simpson David , Henriques Berroeta Claudio , Katsogridakis Emmanouil , Panerai Ronney
Published: June 2021

In order to quantify cerebral autoregulation (CA), one of the most commonly used techniques is to fit a linear model to the recorded signals, with arterial blood pressure (ABP) as input and cerebral blood flow velocity (CBFV) as output. Then parameters are extracted from the model to quantify CA: for example average phase or gain in a narrow frequency band, or features of the predicted step‐response. We propose an optimized method to distinguish between CA during normocapnia and hypercapnia. In 27 adults (18‐55 years old) at rest, CBFV and ABP were recorded simultaneously during normocapnia and then hypercapnia induced by breathing 5% CO2 in air. Recordings were repeated on a separate day, giving a total of 108 recordings of approximately 5 minutes each, all resampled at 1 Hz. The interrelationship between ABP and CBFV was modelled using a linear finite‐impulse‐response filter (7 coefficients). Multiple linear regression of these coefficients was used to predict normocapnia (1) or hypercapnia (0), achieving an area under the ROC curve of 0.91, considerably better than ARI (0.81) and slightly higher than for phase‐lead at 0.1 Hz (0.88). The method provides a new, data‐driven and optimized parameter for assessing autoregulation. This work is being extended to include non‐linear classifiers (support vector machines).

Grant Funding Source: Supported by EPSRC (UK) and Central University, Chile
Autofluorescence of lymphocytes induced by manual acupuncture (1186.1)
Authors: Cabrita António , Peixoto Ana , Loureiro Estela , Santos Paulo , Alves Vera , Rosa Manuel
Published: June 2021

The leukocytes are specialized blood cells participating in the defense of the organism against pathologic factors. They produce molecules that modulate humoral and tissular actions. The common mechanism of action includes for most of them the activation of the cell by a specific stimulus. Autofluorescence of Leucocytes has been described and associated with activation and may be used for detection of this cellular phenomena. In this study the autofluorescence in a group of volunteers with no knowledge stimulus of etiologic factors for the activation of these cells was evaluated and submitted to manual acupuncture.

In this study 17 healthy volunteers of both sexes, aged between 19 and 22 years were submitted to the puncture of Dazhu (11UB), Geshu (17UB), Shenshu (23UB) and Xuangzhong (39GB), 3 times a week during 3 weeks, for 15 minutes each session. Before the first puncture and two days after the last one, blood was collected to evaluation of lymphocyte autofluorescence. Autofluorescence has similar excitation and emission of Fluorescein isothiocyanate (FITC) and R‐Phycoerythrin (RPE). Therefore, autofluorescence was evaluated on a FITC/RPE dot plot using a BD FACSCanto II flow cytometer. The volunteers with clinical signs of pathology where excluded.

The values found before the application of Acupuncture was 0.02±0.025, and after the Acupuncture procedure were 0.062± 0.048. In these series there was a significant increase of autofluorescence of the lymphocytes.

The significant increase in the autofluorescence of de lymphocytes is a clear sign of the stimulation of this group of cells. The points selected were based on the indications of Traditional Chinese Medicine.

To better characterize the activation of these cells it is now justified to evaluate the plasmatic level of proteins released by them during the study. This study shows a clear action of these selected points on the activation of the lymphocytes.