bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidation of the structural determinants of ARB binding to the human AT1R (1173.4)
Authors: Taylor Temy , Nguyen Jenny , Liu Tammy , Roshankar Mina , Chen Haixiang , LaCuran Alecander , Oelschlaeger Peter , Nistala Ravi , Luo Yun , Andresen Bradley
Published: June 2021

The angiotensin (Ang) II type 1 receptor (AT1R) is the target for a widely used class of drugs named angiotensin receptor blockers (ARBs). Polymorphisms in the ARB binding site are known to alter affinity. Mutagenesis studies identifying the binding site are limited as the site can be distorted simply by altering helical rotation/position. Previously, we demonstrated that wild‐type (wt) opossum (o) AT1R does not bind ARBs. Therefore, we made a model of the human (h) AT1R and oAT1R based on the structure of the mu‐opioid receptor through an iterative process. The models were optimized using all atomic molecular dynamics (MD) simulation with explicit membrane bilayer and water solvent. Utilizing docking and MD simulations the ARB binding pocket was elucidated; currently, both K199 in the 3rd helix and the backbone near the lipid‐solvent interface appear to be involved in binding. Additionally, we cloned the hAT1R and made individual point mutations reflecting the differences between hAT1R and oAT1R. Each receptor was stably transfected into HEK cells and characterized to determine if there are alterations in Ang II signaling as well as ARB‐mediated inhibition of signaling. Iterations of modeling and wet lab work will refine the ARB binding site allowing for identification of ARB resistant patients.

Grant Funding Source: T. Taylor was supported by an APS Frontiers in Physiology Fellowship.
Genetic blockade of (pro)renin receptor in adipose tissue modulate the mice body weight and glucose homeostasis (1173.6)
Authors: Shamansurova Zulaykho , Ahmed Basma , Kajla Sonia , Michel Catherine , Lavoie Julie
Published: June 2021

Introduction: The (Pro)Renin receptor [(P)RR] local effects realizes by angiotensin depended and independent pathways were shown by many authors. Study the role of (P)RR in adipose tissue in regulation of the body weight and glucose homeostasis present the aim of the presentation. Material and methods: Specific in adipose tissue (P)RR receptor knockout mice (KO) created by crelox technology, where the male mice was homozygous (MHm) and female mice heterozygous (FHt). In adult mice fed high‐fat high‐carbohydrate diet (HF/HC) or regular chow (ND) the body weight (BW) assessed weekly, glucose homeostasis evaluated by oral glucose tolerant (OGTT) and intra‐peritoneal insulin tolerant (IP‐ITT) tests. Results: The BW curve in KO mice was significantly lower and more pronounced in MHm. OGTT test show higher glycemia, AUC of glucose and glucose/insulin ratio at basal and stimulated state in MHm on ND. IP‐ITT results show higher glycemia and AUC of glucose in FHt mice on HF/HC. This results accompanied by lower (P)RR gene (40%, p<0,05) and protein (40%) expression in adipocytes. Conclusion: The (P)RR specific deletion in adipose tissue accompanied by lower BW, glucose intolerance in MHm on ND, insulin insensitivity in FHt on HF/HC.

Grant Funding Source: Suppoerted by CDA, MERK‐FROST
Augmentation of kidney angiotensinogen expression and urinary angiotensinogen excretion by an intracellular angiotensin II fusion protein (1173.8)
Authors: Navar L. Gabriel , Kobori Hiroyuki , Satou Ryosuke , Katsurada Akemi , Zhuo Jia , Li Xiao
Published: June 2021

Long‐term angiotensin II (ANG II) administration significantly increases ANG II levels in the kidney through two major mechanisms; AT1 receptor‐mediated augmentation of angiotensinogen (AGT) expression and uptake of circulating ANG II by the proximal tubule. However, it is not known if intracellular ANG II also stimulates AGT expression and secretion in the proximal tubule of the kidney. In the present study, we performed intrarenal adenoviral transfer of an intracellular cyan fluorescent fusion of ANG II (Ad‐sglt2‐ECFFP/ANG II) selectively in the proximal tubule using the sodium and glucose co‐transporter 2 (sglt2) promoter. AGT mRNA expression in the renal cortex and 24 h urinary AGT excretion were determined 2 and 4 weeks after ECFP/ANG II transfer. ECFP/ANG II was expressed in the proximal tubule in a time‐dependent manner, which was associated with significant increases in blood pressure 4 weeks after ECFP/ANG II transfer (p<0.01). AGT mRNA expression in the cortex was significantly increased at Week 4 by 61 ± 16% (p<0.05). Urinary AGT excretion rates were increased from 48.7 ± 5.7 (n=13) to 102 ± 13.5 (n=13) ng/24 h (p<0.05). However, plasma AGT levels were not altered. These results support the hypothesis that intracellular ANG II stimulates AGT expression in the proximal tubule leading to increased AGT formation and secretion into the tubular fluid.

Grant Funding Source: Supported by P30GM10337 and RO1DK067299
Proline‐rich tyrosine kinase regulates NF‐κB and PPARγ to promote hypoxia‐induced proliferative phenotype of human pulmonary artery smooth muscle cells (1175.1)
Authors: Bijli Kaiser , Kang Bum‐Yong , Adesina Sherry , Murphy Tamara , Kleinhenz Jennifer , Hart C
Published: June 2021

Hypoxia stimulates pulmonary hypertension in part by increasing the proliferation of pulmonary vascular wall cells via sustained activation of MAP kinase ERK 1/2, NF‐κB, and downregulation of PPARγ levels. However, the upstream signaling events that mediate NF‐κB upregulation and PPARγ downregulation remain unknown. We examined the role of Proline‐rich tyrosine kinase 2 (Pyk2) in hypoxia‐induced proliferation of human pulmonary artery smooth muscle cells (HPASMC). Exposure of HPASMC to hypoxia (1% O2) for 72 hrs resulted in the activation of Pyk2. siRNA‐mediated Pyk2 depletion or its pharmacological inhibition attenuated hypoxia‐induced HPASMC proliferation. Pyk2 inhibition attenuated hypoxia‐induced: 1) ERK 1/2 activation, 2) NF‐κB transcriptional activity and 3) reductions in PPARγ mRNA levels. Conversely, siRNA‐mediated PPARγ depletion enhanced, whereas PPARγ overexpression attenuated HPASMC Pyk2 activation. Exposing C57BL/6 mice to 10% O2 for 3 weeks reduced lung PPARγ but increased Pyk2 levels compared to normoxic mice. Also, Pyk2 levels were higher in pulmonary vascular tissues of smooth muscle‐specific PPARγ knockout mice compared to control mice. Taken together, these findings point to a crucial role of Pyk2 in the mechanism that promotes hypoxia‐induced proliferative phenotype of HPASMC.

Grant Funding Source: Supported by: grants from VA Research Service (1I01BX001910, CMH), NIH (HL102167, CMH), and AHA‐SDG
Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation‐elicited subunit dimerization of protein kinase G1α (1175.9)
Authors: Patel Dhara , Kandhi Sharath , Kelly Melissa , Neo Boon Hwa , Wolin Michael
Published: June 2021

The activity of glucose‐6‐phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG1α) by a thiol oxidation‐elicited subunit dimerization. This PKG1α‐activation mechanism appears to contribute to responses of isolated endothelium‐removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Since one of the known actions of DHEA is inhibiting G6PD, we investigated if it promoted relaxation associated with NADPH oxidation, PKG1α dimerization and PKG activation detected by increased vasodilator‐stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG1α dimerization and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild type mice, which was not detected in the PKG knockin mouse model. Thus, PKG1α dimerization is a major contributing factor to the vasodilator actions of DHEA, and perhaps its beneficial effects in treating pulmonary hypertension.

Grant Funding Source: Supported by NIH Grant R01HL115124
Heterogeneous endothelial Ca2+ signal modalities in wild type and TRPC4 knockout rats in pulmonary arterial hypertension (1176.4)
Authors: Francis Christopher , Xu Ningyong , Taylor Mark , Zhou Chun , Alexeyev Mikhail , Stevens Troy
Published: June 2021

Pulmonary arterial hypertension (PAH) is a deadly disease characterized by high pulmonary arterial pressure, and there are currently no effective treatments. Recently, our lab found that rats genetically lacking the canonical transient receptor potential channel 4 (TRPC4), a Ca2+‐permeable channel, exhibit a survival benefit in a model of PAH. Here we tested the hypothesis that heterogeneous endothelial Ca2+ signaling modalities exist in wild type and TRPC4 knockout rats, and that these modalities are altered in PAH. We compared the cytosolic endothelial Ca2+ activity of wild type and knockout rats in both normal and PAH conditions using opened artery preparations and confocal microscopy. We found that the amplitude of basal endothelial Ca2+ activity was significantly greater for knockout (mean F/F0: 1.46) than for wild type (mean F/F0: 1.11) rats. Additionally, both genotypes exhibited considerable Ca2+ signal heterogeneity within opened artery segments, which was altered in response to PAH. Furthermore, we found that Ca2+ responses to Acetylcholine were significantly truncated in knockout (mean duration: 9.69 s) compared to wild type (mean duration: 26.7 s) rats. We conclude that heterogeneous Ca2+ signaling modalities exist in the endothelium of wild type and TRPC4 knockout rats, and that these modalities are altered differentially in PAH. This phenomenon may be a key determinant of PAH mortality.

Grant Funding Source: HL66299, HL60024
Mesenchymal stem cell expressed VEGF‐E protects pulmonary endothelial cells from cigarette smoke toxicity (1176.9)
Authors: Zhai Bo , Zhang Li , Huang Chaoqun , Varshney Rohan , Sivasami Pulavendran , Hinsdale Myron , Liu Lin , Lloyd Pamela
Published: June 2021

Chronic obstructive pulmonary disease (COPD) is a major public health problem worldwide and the fourth leading cause of mortality in the US. COPD patients have compromised lung microvascular endothelial cells (EC) and decreased lung capillarity. Vascular endothelial growth factor (VEGF), a critical survival factor for EC, is reduced in lungs of COPD patients. Mesenchymal stem cells (MSC) are adult stem cells which secrete paracrine cytoprotective factors, including VEGF‐A. However, VEGF‐A is active at both VEGFR‐1 and VEGFR‐2, and VEGFR‐1 is implicated in the pathogenesis of COPD. Thus, we hypothesized that selective VEGFR‐2 activation would better protect lung EC from cigarette smoke induced damage in vitro. VEGF‐E, a VEGFR‐2 selective viral isoform, was expressed using a lentiviral vector in MSC isolated from bone marrow of C57BL/6 mice. Conditioned medium from wtMSC and VEGFE‐MSC was collected and concentrated 10‐fold. Primary human lung microvascular EC (HLMVEC) were then exposed to cigarette smoke extract (CSE,10%) ± wtMSC or VEGFE‐MSC conditioned medium (CM). HLMVEC treated with VEGFE‐MSC CM showed increased viability (MTT assay; absorbance at 570nm 0.295±0.073) after 24 h of CSE exposure , compared to wtMSC CM treated CSE‐exposed HLMVEC (absorbance 0.085±0.031, p<0.001).We next will compare the effects of VEGF‐A and VEGF‐E on endothelial permeability using a fluorescent dextran assay to determine whether VEGF‐E is less likely than VEGF‐A to cause increased permeability in vitro and edema in vivo. Studies to further characterize the effects of VEGFE‐MSC CM on EC in vitro are underway. Future studies will examine the ability of VEGFE‐MSC to repair the injured lung in COPD.

Grant Funding Source: Supported by Oklahoma Center for Adult Stem Cell Research
Systemic LPS attenuates the acute hypoxic ventilatory response: implications for a uniquely sensitive window of neonatal development (1177.4)
Authors: Rourke Kyle , Mayer Catherine , MacFarlane Peter
Published: June 2021

Sustained hypoxia exposure during a finite window of neonatal development (~10‐15 days of postnatal (P) age) attenuates the acute hypoxic (HVR) and hypercapnic (HCVR) ventilatory response in rats. In the current study, we investigated whether the HVR and HCVR are also modified by LPS exposure during a similar period of development. Rat pups received an intraperitoneal injection of either saline or 0.1mg/kg of LPS at either P5, P10, or P20; the acute HVR and HCVR were measured using whole‐body plethysmography ~2.5hrs later. LPS attenuated the HVR (but not the HCVR) in P10 rats (1.8 ml/g/min) compared to saline (2.4 ml/g/min) treated rats, whereas younger (P5) or older (P10) rats were unaffected. LPS was lethal in some rats at some time within 24 hours after LPS; P10 pups appeared particularly vulnerable with a higher rate of mortality (27% of pups) than P5 (9% of pups) or P20 (0% of pups) treated rats. These data reveal the presence of a developmental window (~P10) that may be at a heightened level of vulnerability to inflammatory challenges.
Impaired arousal associated with prenatal alcohol exposure in rat pups is related to an increase in brainstem GABA (1177.5)
Authors: Sirieix Chrystelle , Wrocklage James , Schneider Robert , Darnall Robert
Published: June 2021

Prenatal alcohol exposure (PAE) increases the risk for The Sudden Infant Death Syndrome (SIDS). Many SIDS infants are exposed to repeated hypoxia before death. Arousal in response to hypoxia is impaired after PAE and is modulated by medullary raphe GABAergic mechanisms. We therefore hypothesized that arousal impairment after PAE would be associated with increases in medullary GABA. Pregnant dams received an ethanol liquid diet (ETOH), an iso‐caloric pair fed diet (PF) or a standard chow diet (CHOW). Artificial cerebrospinal fluid (ACSF) or nipecotic acid (NIP), a GABA reuptake inhibitor, was microinjected (50 nL) into the raphe magnus (RMg) of P15 and P21 pups from each diet group. After recovery, pups were exposed to 4 episodes of hypoxia and the time to arousal (latency) was determined behaviorally. Brainstem GABA concentration was measured (HPLC) in P5, P15 and P21 pups from each diet group. At P21, ACSF injected pups in the ETOH group had longer arousal latencies than those in the PF and CHOW pups combined (CON group) (P<0.001). ETOH pups also had higher brainstem GABA concentrations (P=0.013). Moreover, CON pups injected with NIP had arousal latencies similar to those in ETOH pups after ACSF injection and NIP injected ETOH pups had no further increases in arousal latency. Our results show that the effects of PAE on arousal are associated increases in brainstem GABA and that RMg GABA reuptake mechanisms may be involved.

Grant Funding Source: Supported by NIH PO1 HD36379 and R21 AA020279
Arousal from sleep produced by optogenetic stimulation of C1 neurons is reduced following sleep deprivation or hypoxia, but restored with systemic adenosine 1A antagonism (1178.11)
Authors: Burke Peter , Guyenet Patrice
Published: June 2021

Arousal from sleep is the major defense mechanism against asphyxia and is sensitive to environmental stressors. Here we determined whether sleep deprivation or hypoxia reduces the probability of arousal from sleep and cardiorespiratory activation by optogenetic stimulation of C1 neurons in freely‐behaving rats. Channelrhodopsin‐2 was selectively introduced into C1 neurons of Tyrosine Hydroxylase Cre‐driver rats with a Cre‐dependant viral vector. Sleep stages were identified from EEG and neck EMG recordings. Breathing was measured using whole body plethysmography and blood pressure by telemetry. During non‐rapid eye movement sleep, unilateral photostimulation of C1 neurons produced arousal in 86 ± 5% of trials and sighs in 65 ± 7% of trials (n=5). After sleep restriction (6am ‐ 12am), incidence of arousal and sighs were significantly reduced to 37 ± 3% and 28 ± 2% (n=4), respectively, though cardiorespiratory responses remained unchanged. A hypoxic insult (12% O2 for 30min) also reduced arousals and sigh in subsequent trials to 28 ± 4% and 18 ± 6%, respectively (n=5), but caused no change in cardiorespiratory activation. The impaired arousal and sigh incidence in both stressor models were fully restored with an adenosine 1A receptor blocker (DPCPX; 0.2 mg/kg ip), suggesting that an increase in adenosine inhibitory tone selectively depresses the wake‐promoting responses to C1 neuronal activation.

Grant Funding Source: Supported by NIH (HL28785, HL74011)
Coordination of swallow and breathing: in vivo and computational model simulations (1178.12)
Authors: Pitts Teresa , Gestreau Christian , Morris Kendall , Lindsey Bruce , Davenport Paul , Bolser Donald
Published: June 2021

Coordination of swallow and breathing is vital for airway protection. However, the central mechanisms for the coordination of these behaviors are not known. We speculated that coupled oscillators could be used to explain swallow‐breathing interactions. In vivo recordings in anesthetized cats (n=6) demonstrate that swallow exhibits respiratory phase preference with 83% during expiration (E), 7% during inspiration (I), and 10% phase transition. A swallow oscillator circuit with two, mutually inhibitory, neuron populations, (A & B) was added to the extant medullary breathing network. The swallow A group excites downstream motoneurons and the swallow B group controls inter‐swallow duration. To loosely couple the oscillators excitatory connections were added: 1) I populations to swallow B, controlling swallow occurrence during E phase; 2) E‐augmenting population to swallow B, increasing the probability of swallow occurrence near the end of E. Additionally, to reduce expiratory activity during swallow, an excitatory connection was added between the swallow A population and an E decrementing population, which in turn suppressed E‐Aug bulbospinal activity. Simulations exhibited repetitive breathing, and swallow occurred during the appropriate breathing phase. We conclude significant features of the swallow‐breathing relationship can be modeled with loosely coupled oscillators. NIH HL89104, HL103415, HL89071, HL109025, HL 111215(K99/R00)

Grant Funding Source: NIH HL89104, HL103415, HL89071, HL109025, HL 111215 (K99/R00)
Effects of hypothyroidism in female hamsters on D1 receptor modulated breathing and expression of D1 receptors (1178.2)
Authors: Schlenker Evelyn , Del Rio Rodrigo , Schultz Harold
Published: June 2021

The D1 receptor antagonist, SCH23390 (SCH), decreased ventilation (VE) in euthyroid (E) male hamsters exposed to air and hypoxia, but increased VE in hypothyroid (H) hamsters exposed to air, but not during hypoxic exposures. In H relative to E males, D1 receptor expression was decreased in the carotid body (CB), striatum, and paraventricular nucleus of the hypothalamus (PVN), but not in the nucleus tractus solitarius (NTS). For the present study we hypothesized that in H female hamsters D1 receptor expression in the CB, PVN, NTS and striatum and modulation of breathing would be dissimilar to that observed in H males. H and E female hamsters received SCH or saline. Physiological parameters were evaluated in awake animals exposed to air, 5 minutes of hypoxia (10% O2 in N2) and then air. SCH decreased body temperatures in both groups, had no effect on metabolism, but VE was increased in the H hamsters relative to E hamsters’ responses during air and hypoxic exposures due to increased tidal volumes. SCH relative to saline decreased breathing frequency, but increased tidal volume in the E hamsters during baseline and post‐hypoxic air exposures. In both groups SCH decreased hypoxic responsiveness. Expression of D1 receptors was similar in both groups in all regions. Thus, the ventilatory responses in H females were similar to SCH relative to H males (but not in E males), expression of D1 receptors was dissimilar in 3 regions.

Grant Funding Source: Internal Sanford School of Medicine Grant
Attenuation of pulmonary chemoreflex following acute cervical spinal cord injury (1178.4)
Authors: Tsai I‐Lun , Lee Kun‐Ze
Published: June 2021

Pulmonary C‐fibers are the main chemosensitive afferents in the lung. Activation of pulmonary C‐fibers can evoke pulmonary chemoreflex characterized by apnea, hypotension and bradycardia which is a critical defense reflex to modulate cardiorespiratory responses. The present study was designed to investigate whether pulmonary chemoreflex is altered following acute cervical spinal injury. Adult Sprague Dawley rats received high cervical unilateral hemisection (C2Hx) or C2 laminectomy (sham) were used. Intra‐jugular capsaicin induced pulmonary reflex was evaluated by measuring respiratory airflow in spontaneously breathing rats and phrenic nerve activity in mechanically‐ventilated rats. Capsaicin treatment evoked a cessation of respiratory airflow and phrenic bursting in sham but not C2Hx animals. In order to clarify whether attenuation of pulmonary chemoreflex in C2Hx animals is merely induced by capsaicin or generally applied to other stimuli that can excite pulmonary C‐fibers, another pulmonary C‐fiber stimulant (phenylbiguanide, PBG) was used to evoke pulmonary chemoreflex in spontaneously breathing rats. We observed that PBG‐induced pulmonary chemoreflex was also blunted in C2Hx animals. These results suggested that pulmonary chemoreflex is attenuated after acute C2Hx and the blunted reflex may unfavorably impact the ability to prevent inhaling respiratory irritants.

Grant Funding Source: Supported by NSC 102‐2320‐B‐110‐004‐MY3, NHRI‐EX102‐10223NC
Paradoxes in cell‐based transcriptomic and systematic research data in aging and obesity studies and the attempt of bridging (1180.1)
Authors: Tan Ningzhi , Zhou Yanjiao , Lu Jihong , Gao Ling , Yao Yisang , Dong Yunzi , Bartlett Mark , Zhu Jia‐Shi
Published: June 2021

Transcriptomic studies unmask important molecular information at cellular levels, which may meet paradoxes in interpretation in whole body settings when hyper‐/atrophy or hyper‐/hypoplasia occurs (FASEB J 2007, 21:A1352). In an obesity study, microarray assay revealed an unaltered expression of adiponectin gene in obese adipocytes, paradoxically to the reduced plasma adiponectin (‐27%, p<0.01). After normalized with total DNA and tissue weight, we observed reduced adiponectin mRNA at the tissue level (‐26%, p<0.01), consistent with the plasma changes under the physiological control. In an aging study, Chuk (NF‐κB activator) was down‐regulated (‐20%, P<0.01), inhibitor Ikbkb gene up‐regulated (+20%, p=0.02) & NF‐κB gene (Rela) unchanged at a hepatocyte level in the enlarged, inflammatory liver, but these genes were all up‐regulated at the tissue level (+40% → +100%; p<0.02). But NF‐κB activity was not changed in the whole liver. Gstk1 & Gstk7 genes were unchanged with aging per cell, but up‐regulated at the level of whole liver (p<0.01). In contrast, GSH‐Px activity was reduced in the aged liver (p<0.01). Our studies indicate that bridging the cell‐based trancriptomic data to systems physiology levels is necessary for understanding the whole body physiological and pathological regulations, including feedback compensatory regulations and decompensation, of different biological pathways.
Mechanosignaling with ischemia‐reperfusion: restart of flow triggers a signaling cascade (1180.11)
Authors: Abe Shigeru , Tao Jian‐Qin , Wang Hui , Hong Nankang , Hood Elizabeth , Feinstein Sheldon , Cantu Edward , Christie Jason , Fisher Aron , Chatterjee Shampa
Published: June 2021

We concluded earlier that endothelial mechanotransduction with ischemia in the lung activates a signaling cascade comprised of KATP channel closure that leads to NOX2 activation and reactive oxygen species (ROS) production. We studied the mechanosignaling with reperfusion. Isolated perfused mouse lungs and pulmonary endothelial cells showed a 2 fold increase in ROS production with ischemia that increased by 3‐3.5 fold upon reperfusion. This response is not due to hypoxia or reoxygenation since alveolar pO2 is unchanged in these ventilated lungs. ROS production was not observed in lungs and cells from NOX2 null, KATP channel null mice or lungs pre‐treated with KATP agonist (cromakalim) or a NOX2 activation inhibitor (MJ33). Wild type mice (WT) showed ~1.8 fold increase in oxidative lung damage (as assessed by 8‐isoprostanes, TBARS, alveolar permeability) with pulmonary ischemia and a ~2.5 fold increase with reperfusion; no significant change was observed in NOX2 null mice. Calcium influx, observed with ischemia in WT cells increased further with reperfusion; this was not observed in KATP and NOX2 null cells. Reperfusion thus amplifies the effects of the mechanosignaling cascade that is associated with ischemia. Prevention of the cascade with inhibitors such as MJ33 or cromakalim has potential application in lung transplantation which requires reperfusion of a previously ischemic lung.

Grant Funding Source: NIHRO1HL‐075587
Pinhão‐manso (Jatropha curcas) demonstrates potent antibacterial properties in a rat model of third degree burns (1180.18)
Authors: Paulillo Luis , de Sousa Lessa Gladssinay , Isaacson Janalee , Mo ChengLin , Brotto Leticia , Abreu Eduardo , Brotto Marco , Gomes Mourão e Lima Januario
Published: June 2021

Pinhão‐manso (Jatropha curcas L.; JC) is multipurpose perennial plant native from Brazil that belongs to the Euphorbiaceae family. This plant has mainly been considered for its potential as an alternative source of fuel. We recently demonstrated that JC has very potent protective activity against the toxic effects of ethanol in both tenocytes and muscle cells. Here we employed a rat model of 3rd degree burn to compare the effects of JC with silver sulfadiazine (SS). Microbiological analyses were conducted 1, 7, and 21 days after localized burn was induced. We found that at Day 1, all experimental groups presented with the same type of bacteria in their burns. During the course of these observations it make evident that there was significant reduction of bacterial infection in the group treated with JC as compared with the group treated with SS. The morphological appearance of the burns in JC treated group was also improved as compared to JC. Our new results suggest that JC has anti‐bacterial actions that could be further explored for medicinal use.

Grant Funding Source: Supported by Conselho Nacional de Pesquisa (Brazil), CAPES (Brazil) and the Thompson Endowment Fund
Manipulation of the differentiation program of induced pluripotent stem cells (1180.19)
Authors: DiVincenzo Lola , Yin Liya , Logan Suzanna , Snyder Sarah , Kolz Christopher , Chilian William
Published: June 2021

Since the discovery that genetic reprogramming of somatic cells produced pluripotent cells (termed induced pluripotent stem cells [iPSCs]), there were myriad reports using these cells in regenerative medicine. Because of their pluripotency, iPSCs have potential to regenerate any type of tissue; but the “flip side” of this characteristic is that pluripotency confers potential to be tumorigenic and/or form undesired cell types. Thus for iPS cell‐derived regenerative therapies, strategies must be employed to control differentiation. To address this issue, we hypothesized that iPS cell fate is determined by the “secretome,” in that products released by one cell type will induce the differentiation program of iPSCs into that particular cell type. To test this we obtained conditioned media from adult rat endothelial cells (EC‐CM) and compared the expression of endothelial markers (VE‐Cadherin [VE‐C], CD31, eNOS) in native rat endothelial cells (EC), iPSCs (derived from rat fibroblasts), and iPSCs treated with the EC‐CM for 5‐7 days. Expression of CD31 was highest in endothelial cells, lowest in the iPSCs, and increased in the EC‐CM treated iPSCs (compared to iPSCs). This treatment effect waned with increased passage of the treated cells. Expression of VE‐C paralleled CD31, in that native ECs had the highest expression and EC‐CM‐treated iPSCs had higher expression than untreated iPSCs. Expression of eNOS was greatest in EC, and lower in iPSCs regardless of treatment. In conclusion, the products of the endothelial secretome influence the differentiation of iPS cells by inducing expression of certain markers consistent with an endothelial phenotype; however, we cannot yet state that these cells have become fully differentiated endothelial cells as they do not express eNOS. Nonetheless, the use of the secretome may provide queues for the controlled differentiation of iPS cells into specific cell types.
Real‐time monitoring of oxygenation in cultured organotypic brain slices (1180.20)
Authors: Dmitriev Ruslan , Borisov Sergey , Kondrashina Alina , Pakan Janelle , Papkovsky Dmitri
Published: June 2021

Cultured brain slices are important models in studies of brain development, regeneration of neural tissue upon (patho)physiological conditions and development of novel tissue remodeling systems. Efficient control of temporal and spatial oxygenation in such models is indispensable. We have developed novel phosphorescent O2 probe MM2X, which allows efficient in‐depth intracellular staining of neural tissue (~50‐100 μm, 2‐3 h) and imaging in different modalities (one‐, two‐photon, intensity, PLIM), over the range of 0‐200 μM of O2. We found minimal probe toxicity upon staining and imaging (analysis of membrane integrity, polarization of mitochondria and endocytosis), while its spectral properties allow multiplexing with conventional fluorescent markers such as GFP. We investigated staining efficacy of brain slices of different age (E12‐P21) and found a modest decrease in adult tissue. In proof‐of‐concept study, we analysed brain tissue oxygenation and correlated it with the distribution of BLBP‐GFP‐positive cells and glial progenitor cells. After culturing at 1‐3DIV, brain slices displayed high spatial heterogeneity (50‐150 μM) and no correlation between O2 and BLBP distribution. This study demonstrates how O2 imaging can be combined with the study of processes of neurogenesis in complex 3D models of developing tissue.

Grant Funding Source: Supported by the Science Foundation Ireland.
Regulation of epithelial sodium channel activity in A6 cells via the peptide hormone, prolactin (1181.9)
Authors: Greenlee Megan , Mitzelfelt Jeremiah , Duke Billie Jeanne , Eaton Douglas
Published: June 2021

Prolactin (PRL) is a peptide hormone well‐known for its role in lactation. PRL also has osmoregulatory actions via alteration of ion and water transport, actions characterized mostly in aquatic life. In 2003, researchers reported that PRL stimulates epithelial sodium channel (ENaC) activity in adult frog skin. Since that time, PRL‐mediated regulation of ENaC has been largely ignored. Given that ENaC present in the kidney plays an essential part in blood pressure control, we hypothesized that PRL may control blood volume and blood pressure via regulation of ENaC activity. To investigate this hypothesis, we used a well‐known frog kidney cell model (A6‐2F3) possessing amiloride‐sensitive transepithelial current and highly selective sodium channels with a conductance of ~5pS. Amiloride‐sensitive (ENaC‐mediated) current dramatically increased within 30 minutes after application of 1µg/mL PRL to the basal side of the cells ‐ an effect sustained even at 24 hours after treatment. We confirmed this finding using cell attached patch clamp. We found an almost 6 fold increase in ENaC NPo in PRL‐treated versus vehicle‐treated cells. The mechanism, although still not clear, at least partially involves JAK2 signaling since AG‐490, a JAK2 inhibitor, reduced the effect of PRL on amiloride‐sensitive current in A6 cells. Inhibitors of PI3K and SRC had no effect on PRL’s induction of amiloride‐sensitive current. In conclusion, we observed a significant induction of ENaC activity in frog kidney cells exposed to PRL ‐ an effect partially mediated through JAK signaling pathways. Future studies will finalize the signaling mechanisms involved in PRL’s activation of ENaC and the role of these pathways in blood pressure control.

Grant Funding Source: Supported by NIH K12GM000680 and T32HL076118
Are the upper limits of cerebral autoregulation at gross and microcirculatory levels different? Does nitric oxide play a role? (1183.1)
Authors: Thompson Emma , Coney Andrew , Marshall Janice
Published: June 2021

In Alfaxan‐anaesthetised, male Wistar rats, we infused phenylephrine (0.1‐200µg/kg/min i.v.) to raise arterial pressure (ABP). Dual‐line least‐squares analysis of gross cerebral blood flow (CBF), recorded from the common carotid with external carotid ligated, vs ABP, gave an autoregulatory upper limit (UL) of 168±3mmHg (n=14). By contrast, after neuronal NO synthase (nNOS) inhibition (ЅMTC; 0.56mg/kg i.v, or 7‐NI, 25mg/kg i.p. n=24), or non‐selective NOS inhibition (L‐NAME, 10mg/kg, n=9), the UL was either not reached, even when ABP reached 171±2 or 186±6* (vs Control; P<0.05) respectively or was increased to 173±2 (n=11) and 187±2mmHg* (n=4) respectively.

When laser speckle imaging was used to follow tissue red cell flux (RCF) and diameters of arterial and venous vessels in the cerebral cortex, gross CBF UL was 168±5mmHg, but cortical tissue UL was higher; 170±5mmHg† (vs gross CBF UL, n=6). Further, in 4 rats after L‐NAME, the UL was not reached (either gross CBF or cortical tissue RCF) even when ABP was increased to 190±2mmHg*.

We suggest that autoregulatory constriction of proximal cerebral arterial vessels when ABP rises is protective of cerebral tissue circulation, as indicated by the higher cortical tissue UL. We propose that NO synthesised mainly by nNOS induces tonic dilatation of cerebral circulation, such that its absence allows greater scope for autoregulatory vasoconstriction, leading to a raised UL.

Grant Funding Source: Supported by the British Heart Foundation
The effects of transcranial Doppler probe placement on cerebral autoregulation measurements (1184.1)
Authors: de Jong Daan , Lagro Joep , Meel ‐ van den Abeelen Aisha , Slump Kees , Meulenbroek Olga , Claassen Jurgen
Published: June 2021

Problem description: In measuring cerebral autoregulation (CA), transcranial Doppler (TCD) is currently the standard measurement technique for cerebral blood flow velocity (FV). However, this technique has a great operator dependency, affecting the reproducibility of the signals. This study assessed the effect of probe placement on TCD signal and on CA outcome measures.

Methods: In 15 healthy young volunteers, blood pressure and FV were continuously measured. Changes in probe placement were applied systematically to assess the influence of probe position on absolute TCD measures (systolic, mean and diastolic FV), the pulsatility index (PI), and CA transfer function measures (gain, phase, and coherence).

Results: Ranges of FV obtained after placement changes were 74‐133 % of the initially measured FV. With increases in measured FV , most subjects showed small decreases in the PI (up to ‐0.15 with p = 0.017). Transfer function analysis outcome measures varied, but did not show association with placement.

Conclusion: This study indicates that the effects of placement of the TCD probes on CA measures are, if present, negligible. However, probe placement does have a strong influence on measured FV. This influence can be reduced by using a strict placement protocol, improving placement quality.
Nonstationary multivariate modeling of cerebral autoregulation during resting state and hypercapnia (1184.10)
Authors: Kostoglou Kyriaki , Poulin Marc , Mitsis Georgios
Published: June 2021

We examined the time‐varying characteristics of cerebral autoregulation and hemodynamics during resting state and hypercapnia by using recursively estimated multivariate (two‐input) models which quantify the dynamic effects of mean arterial blood pressure (ABP) and end‐tidal CO2 tension (PETCO2) on middle cerebral artery blood flow velocity (CBFV). Experimental measurements of spontaneous variations of these signals were obtained from thirteen healthy subjects under normal, free‐breathing conditions. Beat‐to‐beat values of ABP and CBFV, as well as breath‐to‐breath values of PETCO2 were also obtained in 8 female subjects during baseline and sustained euoxic hypercapnia. The multiple‐input, single‐output linear models used to describe the relationship between ABP, PETCO2 and CBFV were based on the Laguerre expansion technique. In order to account for the different dynamics associated with each input, the model parameters were updated using a recursive least squares scheme with constant and adaptive multiple forgetting factors. The results reveal the presence of nonstationarities that are more pronounced in the very low frequency range. By comparing one‐input (MABP) and two‐input (MABP and PETCO2) models, our results point out that the incorporation of PETCO2 as an additional input yields less time‐varying estimates of dynamic pressure autoregulation obtained from single‐input (MABP‐CBFV) models, suggesting the important role of PETCO2 and the possible shortcomings of assessing dynamic autoregulation using such models.
Interindividual relationships between blood pressure and cerebral blood flow variability (1184.11)
Authors: Witter Trevor , MacRae Braid , O'Donnell Terry , Berry Max , Tzeng Y.C.
Published: June 2021

The relationships between blood pressure variability (BPV) and cerebral blood flow variability (CFV) are poorly understood. This study sought to characterize the interindividual characteristics between spontaneous BPV and CFV across individuals with intact cerebrovascular control. We analyzed blood pressure and flow velocity data from 105 healthy subjects. Spontaneously occurring fluctuations in mean arterial blood pressure (MAP) and middle cerebral artery flow velocity (MCAvmean; Transcranial Doppler ultrasound) were characterized using power spectral and transfer function analysis in the very low‐ (0.02‐0.07 Hz), low‐ (0.07‐0.20 Hz), and high‐frequency (0.20‐0.40 Hz) ranges. Across our study sample, MAP was a positive predictor of MCAvmean to varying degrees in all three frequency ranges (Very Low Frequency (R2= 0.088, P< 0.01) Low Frequency (R2= 0.47, P< 0.01), High Frequency (R2 = 0.55, P< 0.01)). The increased predictive power of MAP for MCAvmean in higher frequencies demonstrates that BPV is a determinant of CFV. However, factors other than BPV are influential, especially in the very low frequencies. These results demonstrate that intermittent blood pressure, a clinical measurement predominantly representative of very low frequency BPV content, is an inadequate surrogate of cerebral perfusion variability.
Evaluating the repeatability of measuring CBFV and estimating ARI at the MCA vs. ICA (1184.2)
Authors: Nogueira Ricardo , Saeed Nazia , Panerai Ronney , Robinson Thompson , Bor‐Seng‐Shu Edson
Published: June 2021

Assessment of dynamic cerebral autoregulation (CA) at the internal carotid artery (ICA), as an alternative to blood flow velocity (BFV) measurements in the middle cerebral artery (MCA), has been recently demonstrated.This alternative is particularly important in cases where insonation of the MCA is not possible. However, the reliability and reproducibility of this approach using multiple operators have not been previously reported.

Following confirmation of a plaque free carotid artery, BFV was measured at the ICA and MCA, synchronously with blood pressure, heart‐rate and end‐tidal‐CO2. The Autoregulation Index (ARI) was estimated using Tieck’s model. 11 healthy volunteers (10 female:, 48±11 years) were recruited. There were no significant differences in BFV MCA, BFV ICA or MCA ARI, ICA ARI between the left and right hemispheres or between operators. ARI estimates were not significantly different between MCA and ICA (operator 1: 6±2 and 5±2, operator 2: 5±2 and 5±2,respectively) or between operators within each location (MCA P=0.75, ICA P=0.98). Good reproducibility over repeated measurements between operators was evidenced by low coefficient of variation (%) between operators for BFV (MCA: 15%, ICA: 14%) and ARI (MCA: 24%, ICA: 27%). Therefore this study confirms that the ICA is a suitable location for CBFV and CA estimates, showing significant promise to evaluate the ICA ARI in patients where the temporal window is not available.
The effect of different body positions on the assessment of dynamic cerebral autoregulation (1184.4)
Authors: Jara José , Chacon Max
Published: June 2021

The Effect of Different Body Positions on the Assessment of Dynamic Cerebral AutoregulationJL Jara1, Max Chacón1: 1Departamento de Ingeniería Informática, Universidad de Santiago de Chile, Avda. Ecuador 3659, 9170124, Santiago, Chile.

Dynamic Cerebral Autoregulation (dCA) has been assessed for changes in arterial blood pressure induced by a change in the position of the subject, as in the sit‐to‐stand technique (Sorond et al. 2009). However, the most common method to evaluate dCA has been the thigh‐cuff maneuver proposed by Tiecks et al. (1995). But this technique has been performed with the subject lying in supine or in Fowler's position, and there are no studies of its use with other body positions.

In this work, the classic index of autoregulation (ARI) and a recently‐proposed index, named model‐free ARI (mfARI), are utilized to assess dCA in 18 healthy subjects when they are laying supine, seated and standing. Three thigh‐cuff maneuvers are performed in each position. Subject means by position were compared with one‐way repeated‐measures ANOVA. Separated analysis were conducted for each index and cerebral hemisphere.

Significant differences were only observed with the classic ARI on the left hemisphere (p<0.01), which might be attributed to the great variability that this index exhibits in different conditions rather than to a real physiological hemispheric difference.
Influence of dynamic cerebral autoregulation on presyncope in endurance athletes (1184.5)
Authors: Paquette Myriam , Le Blanc Olivier , Gaudreau Alexandra , Moreau Pascale‐Renée , Clément Andrée‐Anne , Thibault Guy , Brassard Patrice
Published: June 2021

The impact of dynamic cerebral autoregulation (dCA) on presyncope symptoms (PS) is controversial in endurance athletes. We examined the influence of dCA during transient hypotension on PS in 11 male athletes using 1) the thigh‐cuff method (rate of regulation [RoR]), and 2) the relative reduction in cerebrovascular conductance index (CVCi) during transient hypotension induced by squat‐stand maneuvers at different frequencies (0.025, 0.05 and 0.1 Hz). Prevalence of PS was assessed using a 20‐min glyceryl trinitrate‐provoked head‐up tilt. Correlations were assessed between dCA metrics. Subjects were then divided into two groups according to presence (n=6; PS group) or absence (n=5; non‐PS group) of PS. There were no correlations between dCA metrics and time to PS. dCA metrics were unrelated, except CVCi response to hypotension at 0.05 Hz and 0.1 Hz (B=0.846, p=0.008). ROR was comparable between PS and non‐PS groups. The CVCi response to hypotension was attenuated in the PS group vs. non‐PS group (0.05 Hz: 1.51 ± 0.31 vs. 2.47 ± 0.27 %/%; 0.1 Hz: 1.35 ± 0.36 vs. 2.32 ± 0.41 %/%, all p<0.05). These results suggest that although CVCi was better adapted to coping with transient hypotension in athletes with PS vs. those without PS, this was unrelated to PS. Thus, metrics of dCA assessing the cerebral blood flow response to hypotension cannot be used to predict PS risk in endurance athletes.

Grant Funding Source: Supported by a research contract from the Ministère de l'Éductation, du Loisir et du Sport (Québec)
Are there differences in cerebral autoregulation between small increases or decreases of blood pressure? (1184.7)
Authors: Simpson David , Birch Anthony , Panerai Ronney
Published: June 2021

Cerebral blood flow velocity (CBFV) in the middle cerebral and arterial blood pressure (ABP) were recorded while a pseudorandom sequence of lower body negative pressure (LBNP) steps was applied for approximately 5 minutes in 31 healthy adult volunteers (53 recodings). The LBNP provoked ABP changes of 4.5 ± 4.1 mmHg (mean ± standard deviation). Cerebral autoregulation (CA) was quantified from phase at 0.1 Hz, calculated over the whole recording (Pwhole) and then only over segments with increasing or decreasing LBNP (Plbnp2, Plbnp1), respectively. Phase was also estimated over all signal segments with only increasing (or only decreasing) ABP (Pabp1, Pabp2) ‐ whether spontaneous or provoked. The results showed small, but significant differences between the estimates (Friedman, p<0.0001), with the largest mean values for Pabp1 and Pabp2. There was no significant difference (Wilcoxon) between Pabp1 and Pabp2, or between Plbnp1 and Plbnp2, but all were significantly different to Pwhole. There was no significant difference in dispersion from the mean, between phase estimates from different signal segments. The results thus indicate that the response to small increases or decreases in LBNP and ABP are similar. The data also provides further evidence that some signal segments within a recording are more suitable for CA analysis than others. Selection of only the segments with increasing or decreasing LBNP was not beneficial.

Grant Funding Source: Supported by EPSRC (UK)
Improvement of hemopoieticstem cell register of donors in Kazakhstan (1185.4)
Authors: Omarova A. , Turgambayeva A. , Omarkulov B. , Alihanova K. , Magzumova R. , Tulemisova A. , Zhunusova D.
Published: June 2021

Study of histocompatibility or HLA‐ typing, has not lost its significance in transplantation,so long as success of transplantation correlates with the number of coincidence of HLA‐ antigens in donor and in recipient to date.

We analyzed 505 potential donors HSC registered in the database of RD HSC on sex, nationality, as well as on prevalence of HLA antigens on locus ‐A, B, C, DRB1, DQB1. SSP technique typing was carried out on low resolution and SBT method was carried outon high resolution.

Typing carried out on low and high resolutionsof locucA, B, C, DRB1 averaged 9‐10%, and by locuc DQB1typing on high resolution was over 39%. Frequency of locus among potential HSC donors situation is as follows: 17 antigen species were found on this locus A. Meanwhile, the most frequent following antigens were found: A2 ‐ 25.4%, A24 ‐ 16.4%,and A1 ‐ 11.9%.

31 antigen species were foundon locusB; the most frequent were antigen B35‐11.4%, B44‐7.9%,and B15‐7.8%.

12 antigen species were found on locus C, the most frequentwere C7 ‐20.6%, 15.4% C6,and C1‐ 14,5%.

The most spread antigens were DRB1 7‐14,3%, DRB1 4‐13,5%,and DRB1‐15‐ 12.1%, respectively, among 13 identified genes among potential HSC donors.

DQB locus is presented by 6 types: DQB103, 02, 06, 05, 04, 16,the most spread antigen wasDQB1‐ 03, which covers more than one third of antigens presented in a given locus.