bluuebunny/crossref_metadata_2025 huggingface dataset:
Nrf2 mutation eliminates vasoprotective effects of low dose angiotensin II infusion in Sprague‐Dawley rats fed high salt diet (664.12)
Authors: Priestley Jessica , Weinberg Brian , Geurts Aron , Lombard Julian
Published: June 2021

Nuclear factor erythroid‐derived 2‐like 2 (Nrf2) is a master transcriptional regulator of antioxidant and cytoprotective genes. In the Sprague‐Dawley (S‐D) rat, suppression of angiotensin II (ANG II) by high salt (4.0% NaCl) diet leads to endothelial dysfunction and oxidant stress, which can be prevented by low‐dose ANG II infusion to restore normal plasma ANG II levels. To understand the role of Nrf2 in maintaining normal vascular function, the MCW genomics group developed a colony of S‐D Nrf2 mutant rats using transcriptional activator‐like effector nuclease (TALEN) technology targeting the first exon of the Nrf2 gene. This resulted in a frame shift mutation of 43 base pairs, removing the initiation codon from the resultant mRNA. We hypothesized that this mutation would prevent the restoration of vasodilation phenotypes in salt‐fed rats by ANG II infusion. In isolated, cannulated middle cerebral arteries from salt‐fed rats, diameter change (ΔD) in response to 10 µM acetylcholine was restored in WT animals receiving ANG II (ΔD=9.67 ± 0.33 µm), but not in WT rats receiving saline infusion, or mutant animals receiving either ANG II or saline (ΔD=0.33 ± 0.67 µm, ΔD=2.00 ± 2.47 µm, or ΔD=1.75 ± 3.09 µm, respectively; p < 0.001 for each comparison). These data support the hypothesis that physiological levels of ANG II help preserve vascular function by preserving redox balance in an Nrf2‐dependent manner.

Grant Funding Source: NIH HL065289
Orally‐delivered, tetrahydrobiopterin‐containing nanoparticles targeting gastrointestinal lymphatics modulate nitric oxide synthesis by aortic endothelial cells (666.4)
Authors: Meininger Cynthia , Stees Melinda , Kelly Katherine , Heaps Cristine , Wu Guoyao , Labhasetwar Vinod
Published: June 2021

A deficiency of tetrahydrobiopterin (BH4), a critical cofactor for endothelial cell (EC) nitric oxide (NO) synthase, may be a common basis for vascular dysfunction in many diseases. Our goal was to modulate BH4 levels in ECs to increase NO bioavailability and improve vascular function in diabetes. We previously showed that BH4 encapsulated in poly(D,L‐lactide‐co‐glycolide) nanoparticles (NPs) and injected i.v. significantly improved vascular function in diabetic rats. In the current study, we synthesized solid lipid NPs (SLNPs) containing BH4. Our objective was to deliver BH4 to the systemic circulation via uptake and transit of SLNPs by the gastrointestinal lymphatics. SLNPs were administered via oral gavage once per day for 2 days (SLNPs) and endothelium‐dependent vessel relaxation assessed 24 hours later by measuring the response to increasing concentrations of acetylcholine. BH4 levels were determined using a reversed phase high‐performance liquid chromatography method. BH4‐loaded SLNPs taken up by cultured ECs increased cellular BH4 levels significantly, compared to vehicle/control NPs. Importantly, BH4‐loaded SLNPs increased vasodilatory responses of aortic rings taken from gavaged diabetic rats (maximal relaxation 75% vs. 55% in rats receiving control NPs). These data support the feasibility of oral NPs to deliver therapeutic agents to ECs in the general circulation via the lymphatic system.

Grant Funding Source: Supported by AHA 11GRNT7930004 and NIH HL093689
Pannexin 1‐dependent ATP release from venous endothelium promotes acute vascular inflammation (669.8)
Authors: Lohman Alexander , Isakson Brant
Published: June 2021

Tumor necrosis factor α (TNFα) activates endothelial cells (EC) promoting EC‐leukocyte interactions in the venous circulation which is mediated, in part, by purinergic signaling. However, it is currently not known if ECs release ATP to TNFα. Employing in vitro ATP release assays, we report that TNFα significantly increased ATP release from venous ECs (Human Umbilical and Saphenous) which was not observed in arterial ECs (Human Coronary and Aortic). ATP release was inhibited by WP9QY, a peptide antagonist of the TNF receptor 1 (TNFR1), indicating receptor activation in the initiation of this event. Pharmacological inhibition of Pannexin 1 (Panx1) channels in venous ECs with CBX or 10panx1 and siRNA knockdown of Panx1 significantly inhibited ATP release, whereas inhibition of connexin hemichannels, CALHM1 channels and exocytosis had no effect. This indicates that Panx1 channels are the conduit for ATP liberation. Additionally, we show that Src family kinases mediate Panx1 channel activation, as the Src inhibitor PP2 significantly reduced TNFα‐induced ATP release. Functionally, both Panx1 inhibition and degradation of extracellular ATP attenuated leukocyte‐venous EC interactions to TNFα stimulation. Utilizing ex vivo cannulated mesenteric venules, luminal perfusion of TNFα significantly increased ATP release into the perfusate, supporting a role for TNFα‐mediated ATP release in intact venules. Finally, we developed an EC‐specific, inducible Panx1 knockout mouse (VEcadCreERT2+/Panx1fl/fl) to determine the role of Panx1 in vascular inflammation in vivo. In conclusion, TNFα activates Panx1 channels in venous ECs inducing ATP release to promote leukocyte‐EC interactions.

Grant Funding Source: Supported by NIH, AHA
Vasodilators of active hyperaemia effect one another’s action: potassium attenuates the vasodilation produced by nitric oxide and adenosine (677.15)
Authors: Lamb Iain , Murrant Coral
Published: June 2021

Potassium (K+), nitric oxide (NO) and adenosine (ADO) are present contemporaneously during active hyperaemia, thus, we sought to test if interactions occur between these vasodilators. Using intravital microscopy of the hamster cremaster in situ, we assessed arteriolar diameter response to the presence of dilators added in succession or simultaneously. In succession experiments a dose response (10‐8–10‐4M) of NO donor, S‐nitroso‐N‐acetyl penicillamine (SNAP) was preformed and repeated after 5‐minute pre‐exposure and continuous presence of 10mM KCl. Separately, a dose response of KCl (10 and 20mM) was preformed and repeated after 5‐minute pre‐exposure and continuous presence of 10‐7M SNAP. A similar protocol was used using the remaining combinations of KCl, ADO and SNAP. In simultaneous experiments 10‐7M SNAP and ADO were applied independently, then each were simultaneously applied with 10mM KCl. In succession experiments KCl attenuated the dilatory response of both ADO and SNAP at all concentrations (average decreases of 43.2±9.7% and 39.8±11.7%, respectively). In simultaneous experiments KCl reduced the dilatory response of SNAP and ADO (by 69.1±3.7% and 75.1±7.1%, respectively), KCl‐induced attenuation occurred after 5 and 7 minutes for SNAP and ADO, respectively. Therefore, K+ reduces dilatory action of ADO and NO implying dilators released during active hyperaemia interact.

Grant Funding Source: Supported by NSERC Canada
Protein kinase C, VEGF and ADAM metalloproteases regulate proteolytic cleavage of Flt1 to control the abundance of a functionally active ectodomain (679.3)
Authors: Raikwar Nandita , Liu Kang , Thomas Christie
Published: June 2021

Flt1 or VEGFR1 (vascular endothelial growth factor receptor‐1/fms‐related tyrosine kinase 1) is a tyrosine kinase receptor that regulates angiogenesis by sequestering VEGF from its signaling receptor Flk1 or VEGFR2. We have previously described the proteolytic release of an ectodomain of Flt1 that can also function as an angiogenesis inhibitor (Exp Cell Res 2013). We demonstrate that the metalloprotease inhibitor GM6001 reduces the abundance of sFlt1 in conditioned media in vascular endothelial cells consistent with the notion that sFlt1 arises in part via metalloprotease activity. Flt1 cleavage is increased by overexpression of ADAM10 and ADAM 17 while knockdown of either ADAM10 or ADAM17 reduced cleavage in HEK cells confirming that these metalloproteases regulate Flt1 ectodomain release. Interestingly, PMA stimulates Flt1 ectodomain cleavage while VEGF inhibits Flt1 ectodomain cleavage and neither requires the C‐terminal tyrosine kinase domains. Proteolytic cleavage of Flt1 can be regulated independently of ligand binding and intracellular signaling. NIDDK
Alpha1 adrenergic receptors and cerebral artery angiogenesis (679.4)
Authors: Goyal Ravi , Goyal Dipali , Wolfe Craig , Longo Lawrence
Published: June 2021

The largest neural component of the cerebral vasculature is adrenergic in nature. This serves an important role in regulating angiogenesis and maintaining adequate oxygenation to the brain tissue. Importantly, radio‐ligand binding and molecular cloning in several species have demonstrated that the α1‐adrenergic (α1‐AR) family has three structurally distinct subtypes (α1A‐, α1B‐, α1D‐) which are widely expressed in tissues including cerebral arteries. We tested the hypothesis that cerebral artery angiogenic responses are mediated by one of the three α1‐AR subtypes. In an attempt to identify the specific α1‐AR subtype, we examined their role and downstream signaling pathways in angiogenic responses in sheep cerebral arteries. In three dimensional organ culture experiments, we observed that the α1B‐AR subtype (but not α1A‐ or α1D‐AR subtypes) plays a critical role in angiogenesis responses. Also, these responses are mediated by canonical ERK‐MAPK signaling pathways. We conclude that the development of specific α1B‐AR agonists may prove to be effective angiogenic therapy in stroke as well as cerebrovascular degenerative diseases. Moreover, such therapy would be free from the side effects produced by activation of α1A‐ or α1D‐AR.
Sex differences in the fetal programming of hypertension: role of adrenal phenylethanolamine N‐methyltransferase gene expression (680.21)
Authors: Khurana Sandhya , Grandbois Julie , Nguyen Phong , Peltsch Heather , Lo Suet , Venkataraman Krishnan , Tai T.C.
Published: June 2021

Prenatal exposure to high levels of glucocorticoids (GCs) predisposes to hypertension in later life. GCs regulate catecholamine biosynthesis and are critical for blood pressure homeostasis. This study examined whether prenatal exposure to elevated GCs influences the development of adult hypertension via alterations in phenylethanolamine N‐methyl transferase (PNMT), a gene that is linked to hypertension. Pregnant Wistar‐Kyoto dams were injected with 100 μg/kg/day of the synthetic GC dexamethasone (DEX) in their third trimester. Blood pressure and weights of the offspring were measured from week 3‐18, at which point the animals were sacrificed. Total RNA and protein were extracted from the adrenals, and the expression of PNMT and its regulatory transcription factors analyzed by qPCR and Western blot. Results reveal that in utero exposure to DEX promotes the development of hypertension in the offspring. A more pronounced increase in blood pressure was seen in males than females. The mRNA transcripts for PNMT, and its transcriptional regulators Egr‐1, Sp1, GR and AP‐2, were elevated in the adrenals of these animals compared to saline controls, with differences in both sexes. Preliminary Western data also shows upregulation of PNMT and the transcription factor Egr‐1. These results suggest that prenatal GC exposure increases adrenal PNMT gene expression via altered transcriptional regulatory mechanisms.

Grant Funding Source: Supported by CIHR
Dietary NaCl alters several sympathoexcitatory and sympathoinhibitory reflexes: dependence on the ventral lamina terminalis (683.4)
Authors: Simmonds Sarah , Stocker Sean
Published: June 2021

Elevated dietary NaCl intake enhances both sympathoexcitatory and sympathoinhibitory responses evoked from the rostral ventrolateral medulla. These enhanced responses are prevented by prior lesion of the LT. The purpose of the present study was to test whether increased dietary NaCl intake enhanced a number of sympathetic reflexes dependent on neurotransmission in the rostral ventrolateral medulla and whether this was prevented by LT lesion. Male Sprague‐Dawley rats were fed 0.1% (low) or 4.0% (high) NaCl for 14‐18 days. Electrical stimulation of sciatic afferent nerves (1ms, 1‐20Hz, 500 uA) produced greater increases in renal SNA (5 Hz: 125±9% vs 187±23%, n=6/group) and mean ABP (5Hz: 14±2mmHg vs 28±2mmHg, n=6/group) of rats ingesting high versus low NaCl, respectively. LT lesion prevented the enhanced responses of renal SNA (low: 129±13% vs high: 144±13%, n=3/group) and mean ABP (low: 17±2mmHg vs high: 14±5mmHg, n=5/group). Stimulation of the aortic depressor nerve (1ms, 1‐20 Hz, 500 uA) produced greater decreases in renal SNA (low: ‐53±10% vs high: ‐74±7%, n=6/group) and mean ABP (low: ‐16±3mmHg vs high: ‐32±3mmHg, n=6/group) of rats fed low versus high NaCl diets. Again, LT lesion prevented the enhanced responses of renal SNA (low: ‐58±10% vs high: ‐59±6%, n=3/group) and mean ABP (low: ‐18±5mmHg vs high: ‐15±3mmHg, n=3/group). Similar findings were observed for additional reflexes including activation of vagal afferents and intracerebroventricular infusion of 1M NaCl. These findings suggest dietary salt activates LT neurons to alter the responsiveness of hindbrain neurons and enhance sympathetic reflexes.

Grant Funding Source: Supported by NIH R01HL113270 and AHA Established Investigator Award
Chronic isometric strength training reduces anxiety‐like behavior in Wistar rats (684.15)
Authors: Conceicao Rodrigo , Laureano‐Melo Roberto , Almeida Claudio , Pinto Guilherme , Cortes Wellington
Published: June 2021

The objective of this study was to assess the anxiety‐like behavior in rats subjected to isometric strength training. For this purpose, the animals were randomized into 2 groups (n=10): control group and trained, the trained rats undergone a 8 week of isometric strength training. At the end of the training period, the rats underwent a behavioral assessment in an open field test, elevated plus maze test and marble burying test. Then, the animals were euthanatized and blood was collected for determination of serum corticosterone by radioimmunoassay method. Statistical analysis was performed by t student test, considering the significant differences between means at p <0.05. According to the results, in the elevated plus maze, it was demonstrated that trained group had higher time spent in the open arms (302%, p = 0.007) and greater percentage of entries into the open arms (288%, p = 0.007). Moreover the serum levels of corticosterone were significantly lower in the trained group (41%, p = 0.005). Together, these results suggest that to isometric strength training improves anxiety regulation by engaging inhibitory mechanisms related to stress responses.

Grant Funding Source: FAPERJ/CNPq
IL‐1β microinjections into the nucleus tractus solitarius in rodents alter cardiorespiratory patterning consistent with endotoxemia (686.19)
Authors: Hsieh Yee‐Hsee , Nethery David , Jacono Frank , Dick Thomas
Published: June 2021

Endotoxemia increases heart (HR) and respiratory (fR) rates and disrupts cardio‐respiratory coupling (CRC). We previously identified expression of proinflammatory cytokines in the nTS during endotoxemia which was associated with increases in HR and fR, as well as uncoupling of cardio‐respiratory patterns. We hypothesized that microinjection of the proinflammatory cytokine IL‐1β into the nTS would increase HR, fR, and alter cardio‐ventilatory coupling (CVC), defined as the significant tendency for a heartbeat to occur at a preferred latency prior to the next breath. To test this hypothesis, we injected IL‐1β (0.5 ng, n=8) bilaterally into the nTS of anesthetized (isoflurane) spontaneously breathing adult male Harlan Sprague‐Dawley rats (n=12) and recorded diaphragmatic electromyogram (EMG) and electrocardiogram (ECG). 2h after focal injections of IL‐1β, fR increased 32% (from 42.9 ± 2.2 to 57.0±15 brths/min; p<0.05). CVC was analyzed with custom software and χ2 from multi‐ordered histograms was calculated. 2h post IL1‐β injection, the average χ2 value reduced from 40.76 ± 5.9 to 15.91 ± 3.6 (p<0.001) indicating loss of CVC. We conclude that exogenous IL‐1β injections into the nTS (reduced model of brainstem inflammation) reproduces some of the cardiorespiratory changes observed in experimental endotoxemia, and may contribute to altered cardio‐respiratory coupling.

Grant Funding Source: T32 HL007913, NS069220, VA I01BX000873
Impaired PVN neuronal activity in response to acute sodium challenge drives persistent elevations in MAP in conscious rats lacking CNS Gαi2 proteins (686.6)
Authors: Carmichael Casey , Wainford Richard
Published: June 2021

Aim: We have reported CNS Gαi2 proteins mediate the sympathoinhibitory responses to chronic high salt‐intake. These studies assessed the role of CNS Gαi2 proteins in the activity of PVN neurons in response to acute sodium challenge.

Methods: 24‐h ICV Gαi2 or SCR oligodeoxynucleotide (ODN; 25μg/5μl)‐pretreated conscious Sprague‐Dawley rats were monitored for changes in MAP in response to IV bolus NaCl (3M; 0.14 ml/100g). Separate groups of rats were sacrificed at time of the peak MAP change (40 min post‐IV NaCl) and Fos IHC was performed (N=6/gp).

Results: No difference was observed in sodium‐evoked peak change in MAP between treatment groups (IV 3M NaCl ΔMAP [mmHg] SCR: 13±4 vs. Gαi2: 10±3), however MAP returned to control levels by 100 min in SCR but not Gαi2 treated rats (MAP 100min post‐IV NaCl [mmHg] SCR: 134±2 vs. Gαi2: 146±3, P<0.05). In SCR, but not Gαi2, treated rats, an IV NaCl bolus produced significant reduction in Fos immunoreactivity ([PVN Fos positive cells] SCR control: 95±9 vs. post NaCl: 5±1, p<0.05, Gαi2 control 90±13: vs. post NaCl: 66±9).

Conclusion: These data reveal a novel role of CNS Gαi2 proteins in the regulation of the neuronal response within the hypothalamic PVN to an acute sodium challenge that significantly elevates MAP. The sodium‐induced decrease in Fos‐positive PVN cells in SCR ODN rats likely reflects inhibition of neuronal activity facilitating the return of MAP to basal levels. In Gαi2 pretreated rats, failure to inhibit PVN neuronal activity represents a neural mechanism by which impairment of Gαi2 signal‐transduction pathways may contribute to the pathophysiology of salt‐sensitive hypertension.

Grant Funding Source: Supported by RO1 HL107330, K02 HL112718, & T32 GM008541
Cyclic stretch increases the expression of fibronectin through cPLA2, STAT3, and Akt phosphorylation in renal tubular epithelial cells (690.18)
Authors: Sridhara Rashmi , Alexander Larry
Published: June 2021

Extracellular matrix accumulation contributes to the progression of chronic renal disease. Recent in vivo data indicate that unilateral ureteral obstruction (UUO), a model for chronic obstructive nephropathy, induces profibrotic renal tubular cell injury via STAT3 activation. A characterization of obstructive nephropathy and UUO is an increase in intrarenal pressure resulting in tubular mechanical stretch. However, in the kidney, the influence of mechanical strain on renal tubular epithelial (RTE) cells remains unclear. It is possible that tubular mechanical strain may attribute to increased STAT3 activation leading to accumulation of the extracellular matrix protein, fibronectin, in RTE cells. Human proximal tubular HK‐2 cells were either or not subjected to cyclic mechanical stretch on a uniform biaxial strain device. Fibronectin expression increased significantly in response to cyclic stretch, which was attenuated by inhibition of cPLA2, but not sPLA2 or iPLA2. In addition, treatment of cells with arachidonic acid resulted in an increased expression of fibronectin. Cyclic stretch also stimulated phosphorylation of STAT3 and Akt, and inhibition of STAT3, Akt, or its upstream activator phosphatidyl inositol 3‐kinase (PI3‐K), attenuated stretch‐induced fibronectin accumulation. Expression of dominant negative Akt or transfection of small interfering RNA targeting STAT3 significantly blocked stretch‐induced increase in fibronectin, indicating a role for these kinases in fibronectin accumulation. Taken together, these results provide the first evidence for a role of cPLA2, STAT3, and Akt in stretch‐induced fibronectin expression in RTE cells.
Coenzyme Q supplementation or over‐expression of the yeast Coq8 putative kinase stabilizes multi‐subunit Coq polypeptide complexes in yeast coq null mutants (693.17)
Authors: He Cuiwen , Xie Letian , Allan Christopher , Clarke Catherine
Published: June 2021

Coenzyme Q biosynthesis in yeast requires a multi‐subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over‐expression of the putative Coq8 kinase in certain coq null mutants restores steady‐state levels of the sensitive Coq polypeptides and promotes the synthesis of late‐stage Q‐intermediates. Here we show that over‐expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria on two‐dimensional blue‐native gels. The Coq4 polypeptide persists at high molecular mass with over‐expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady‐state levels of Coq4, Coq7, Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late‐stage Q‐intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over‐expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q‐biosynthetic complex, termed the CoQ‐synthome.

Grant Funding Source: NIH GM45952, Ruth L. Kirschstein National Research Service Award GM007185, and USPHS National Resear
Short distance transportation stress in the adult domestic chicken as modulated by pretreatment with ascorbic acid and α‐tocopherol (693.18)
Authors: Azeez Odunayo , Oyagbemi Ademola , Oyewale Johson
Published: June 2021

The effects of short distance transportation stress and pretreatment with non‐enzymic antioxidants ‐ ascorbic acid and α‐tocopherol on the haematology, erythrocyte osmotic fragility (EOF), Total plasma protein (TP), H2O2 generation, Malondialdehyde (MDA) and glutathione (GSH) levels were investigated using the domestic chicken.

Thirty‐two, adult female domestic chickens of the Nera black strain were used. The birds were divided into four groups A‐D of 8 birds each. Groups A and B did not receive any medication. Group C received ascorbic acid (650mg/kg diet) while D, α‐tocopherol (270mg/kg diet) in feed for 2 weeks. Birds in groups B, C and D were transported through a distance of 200km. Blood samples were collected within 30 minutes after the journey for analysis. Haematocrit (PCV), red blood cell counts (RBC), haemoglobin, erythrocyte indices (MCV, MCH and MCHC) and EOF, TP, H2O2, LP and GSH levels were evaluated after transportation.

Group B had lower EOF than the control, while those pretreated with Vitamins C and E were higher. Haemoglobin and MCHC values were higher in Group B while other haematological parameters were similar. There were no significant differences in the total protein, H2O2, MDA and GSH levels in Group B compared with the control. Group C however, had higher H2O2 and MDA levels but lower TP and GSH than the control. Whereas, Group D had higher H2O2 and GSH levels but lower protein than the control.

This study demonstrated that nucleated erythrocytes of the domestic chicken respond to acute stress by increase in osmotic resistance and membrane stability in hypotonic solution, this was antagonized by pretreatment with Vitamins C and E. Furthermore, short distance transportation may not increase oxidative stress and lipid peroxidation in the chicken. Prolonged use of the antioxidants especially ascorbic acid may however lead to oxidative stress
The antioxidative detection of Prdx6 to cell membrane lipid peroxidation of lung type II epithelial cells by DPPP (693.3)
Authors: Wang Yan , Manevich Yefim , Fisher Aron
Published: June 2021

Peroxiredoxin 6 (Prdx6) is a peroxidase antioxidant enzyme, we observed the protection of Prdx6 to cell membrane lipid peroxidation of lung alveolar epithelial typeIIcells (AEC II) by using a novel fluorescent probe Diphenyl‐1‐pyrenylphosphine (DPPP). Hydrogen peroxide (H2O2) can diffuse across cell membrane, so the dose of 500 µM H2O2 was used to AEC II exposure with labeled DPPP at 400nmol/m for 1 to2 h, the AECIIwere isolated from wild type (WT) and Prdx6 transgenic overexpressor (Tg) mice and observed under fluorescence microscopy and PTI spectrofluorometer. We found that AECIIwith H2O2 exposure induced time‐related fluorescence signal increasing in cell membrane under fluorescence microscopy, and the AECIIfrom Prdx6 Tg mice showed about 36% less cell membrane lipid peroxidation compared to WT group; under PTI spectrofluorometer study, the decreased cell membrane phospholipid peroxidation in Tg Prdx6 AECIIconsistently presented about 60% decreasing remarkably in 2h‐ H2O2 exposure and about 30% in 1h‐ H2O2 exposure compared to WT group. Thus, transgenic overexpression of Prdx6 in AECII defends from H2O2‐induced cell membrane lipid peroxidation.

Grant Funding Source: Heart and Lung Society
The protective role of peroxidase 6 against peroxidative stress in pulmonary microvascular endothelial cells includes its ability to repair peroxidized cell membranes (693.4)
Authors: Li Haitao , Feinstein Sheldon , Fisher Aron
Published: June 2021

Peroxiredoxin 6 (Prdx6) is known to play a critical role in antioxidant defense of lung and other organs. Our aim in this research was to investigate whether this protective function of Prdx6 includes an ability to repair peroxidized cell membranes. Mouse pulmonary microvascular endothelial cells (PMVECs) were treated with tert‐butyl hydroperoxide (tBOOH) and lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances (TBARS). PMVECs from Pxdx6 null mice showed much higher levels of lipid peroxidation upon tBOOH (50‐300 μM) exposure compared with wild‐type (WT).To determine whether Prdx6 is necessary for repair, PMVECs were treated with tBOOH (300 μM) for 24 h and then evaluated at 0,2,4 and 6 h after removal of the oxidant. TBARS in WT PMVECs returned to the levels of unexposed control level in 2 h. On the contrary, TBARS in PMVECs from Pxdx6 null mice were unchanged from their elevated levels during 6 h observation after cessation of tBOOH exposure. These results indicate that Prdx6 plays a critical role in protecting PMVECs against peroxidative stress both by preventing lipid peroxidation during the oxidant exposure and through its ability to repair peroxidized cell membrane during the subsequent recovery period following removal of the oxidative stress.

Grant Funding Source: Supported by 5T32HL007748‐20, RO1‐HL 102011, and PO1‐HL19737
Differential effect of apoptosis‐associated tyrosine kinase on K‐Cl cotransporter activity in C6 glioblastoma cells (694.2)
Authors: Gagnon Kenneth
Published: June 2021

Expression of the potassium‐chloride (K‐Cl) cotransporter is linked to the growth and spread of gynecological cancers. Immunofluorescent localization of three K‐Cl cotransporter isoforms (KCC1, KCC3, and KCC4) to migrating extensions of rat glioblastoma (C6) cells suggests their potential role in the enhanced motility of these tumors. RT‐PCR amplification of mRNA from C6 cells validated the expression of kinases and phosphatases known to control cation‐chloride cotransporter activity. Xenopus laevis oocytes co‐injected with KCC3 and apoptosis‐associated tyrosine kinase (AATYK1) cRNA showed isosmotic K+ influx similar to hyposmotically‐activated K+ influx, whereas no change in isosmotic K+ influx was observed with co‐injection of KCC1 and AATYK1 cRNA. This differential effect of AATYK1 on two KCC isoforms prompted testing of the potential of AATYK1 as a novel target for affecting glioblastoma motility. Genistein, a general tyrosine kinase inhibitor, failed to reduce the hyposmotically‐stimulated K+ influx of C6 cells or inhibit their motility in a scratch wound assay indicating that the catalytic activity of the tyrosine kinase is not involved. Molecular identification of both serine/threonine kinase and phosphatase binding sites on AATYK1 suggests that it may scaffold other K‐Cl cotransporter regulatory proteins and thus represent a novel target for anti‐cancer therapeutics.

Grant Funding Source: Supported by a Special Projects Grant from the University of Saskatchewan College of Medicine
Characteristics of collective cell migration in human keratinocytes at subatmospheric pressure (694.5)
Authors: Hsu Chih‐Chin , Chow Shu‐Er
Published: June 2021

A negative pressure incubator incorporating the electric cell‐substrate impedance sensing (ECIS) technique has been built to quantify monolayer keratinocytes movement in serum free media at ambient pressure (AP) and at the subatmospheric pressure of 635 mmHg (SP). Monolayer cell motion at the two pressures was continuously recorded by ECIS in the frequency range of 22.5‐64 kHz. Cells in both pressures had similar viability. Greater migration distance was found in cells at SP. We underwent fluorescent staining and protein analysis for E‐cadherin, phosphorylated ‐catenin (p‐catenin), and acetylated ‐tubulin in cells at the both pressures serially at 0, 3, 6, 9, and 12 hours after wounding the monolayer cells. A significant decrease of cell‐substratum conductance (AP: SP= ~5 cm0.5: ~3 cm0.5) and increase of membrane capacitance (AP: SP= ~4F/cm2: ~8F/cm2) were observed early in the monolayer cell movement at SP. Increased cell‐substratum distance and membrane ruffling observed in scanning electron microscopic (EM) images may be respectively responsible for these measurements. Long and stable microtubules may also contribute to the cell deformation. In cells treated with SP, a progressive drop of intercellular resistance from 1.2 to 0.8 cm2 and a significant change were observed 6 hours after wound closure. The gradual loss of cell junctions associated with decreased adherent junction proteins was identified in serial fluorescent stains and transmission EM images in cells at SP. Characteristics of collective cell migration at SP include early release of cell‐substratum adhesion, quick formation of membrane ruffling, and an ensued decrease of cell junction protein expressions.

Grant Funding Source: Chang Gung Medical Research Programs
Life and death in the living embryo: live imaging of germ cell migration and programmed cell death in Drosophila melanogaster (694.7)
Authors: Elliott Emily , Harischandra Hiruni , Pohl Danielle , Coffman Clark
Published: June 2021

Primordial germ cells (PGCs) in Drosophila melanogaster undergo a choreographed process of migration and programmed cell death during embryogenesis. PGCs generate the future gametes and thus are critical for the continuation of the species. PCGs are the first cells generated in the embryo, and remain sequestered from the somatic cells until after gastrulation. They then migrate through the external epithelial layer into the mesoderm, and diverge as they migrate away from the midgut. Finally, PGCs coalesce with somatic gonadal precursor cells, forming the gonads. Programmed cell death is a crucial part of this process, preventing the persistence of ectopic germ cells. Though a few of the signaling molecules that regulate PGC migration and programmed cell death are known, the molecular mechanisms are not well understood. The scattershot (sctt) mutation of the receptor tre1 leads to disrupted germ cell migration, and appears to have a role in germ cell death as germ cells survive outside of the gonads. Since PGC migration is dynamic and occurs quickly, we use live imaging to observe migration and germ cell death in real time. Using membrane‐localized fluorophores expressed under a germ‐cell‐specific promoter, we will track the migration and death of wild type and tre1sctt germ cells, to further understand how the tre1‐sctt mutation leads to the failure of proper gonad formation and the persistence of ectopic germ cells.
P2X4 receptors mediate atheroprotective gene expression in an in vitro model of steady flow in vascular endothelial cells (696.6)
Authors: Sathanoori Ramasri , Olde Björn , Rosi Federica , Müller Christa , Erlinge David
Published: June 2021

Vascular endothelial cells are in direct contact with blood flow and are exposed to fluid shear stress. The role of P2X4 receptors in shear stress‐mediated mechanotransduction is well documented. Studies have shown endothelial cells to release ATP in response to steady flow that in turn modulates cellular functions via P2 receptors in the vasculature. However, the molecular mechanisms governing ATP‐mediated P2 receptor signaling downstream of steady flow in endothelial cells remains largely unknown. Although steady flow‐induced KLF2 expression implicated in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the aim of this study.

Human umbilical vein endothelial cells (HUVECs) were either exposed to steady flow at 12 dynes/cm2 or to exogenous administration of a non‐hydrolysable analog of ATP (ATPγS) in the presence of the P2X4 antagonist (PSB‐12253) and P2X4 siRNA. We validated the in vitro orbital shaker model for steady flow showing cell alignment in the direction of flow and upregulation of KLF2 and NOS3. Further, addition of ATPγS to static cultures led to an increase in KLF2 mRNA as early as 3h. Exposing HUVECs to apyrase and PSB‐12253 reduced KLF2 and NOS3 expression under steady flow. Furthermore, the knockdown of P2X4 confirmed its role in regulating KLF2. These results suggest that ATP is involved in regulating KLF2 in part via the P2X4 receptor.
miR‐23b regulates CDK‐activating kinase complex through cyclin H repression to modulate endothelial transcription and growth under flow (696.7)
Authors: Wang Kuei‐Chun , Li Yi‐Shuan , Chien Shu
Published: June 2021

The flow regulation of microRNAs (miRNAs) in endothelial cells (ECs) plays a significant role in vascular homeostasis and diseases. We used a combination of an in vitro cell perfusion system and in vivo studies on experimental animals to elucidate the molecular mechanism by which the pulsatile shear flow (PS)‐induced miR‐23b exerts anti‐proliferative effects on ECs. Our results demonstrated that PS induces the transcription factor KLF2 to promote miR‐23b biosynthesis; the increase in miR‐23b then represses cyclin H (CCNH) to impair the activity and integrity of CDK‐activating kinase complex (CAK). The inhibitory effect of miR‐23b on CAK further suppresses cell cycle progression via reducing the basal transcription and phosphorylation of CDK2/4. While PS regulates the miR‐23b/CAK pathway to exert anti‐proliferative effects on ECs, oscillatory shear flow (OS) has little effect on the miR‐23b/CAK pathway and does not cause EC growth arrest. Such flow pattern‐dependent phenomena are validated with an in vivo model on rat carotid artery. Local delivery of miR‐23b precursors to rat carotid artery significantly reduces the EC proliferative phenotype induced by flow disturbance. Our findings unveil a novel mechanism by which hemodynamic forces modulate endothelial homeostasis through miR‐23b and demonstrate the potential of developing miR‐23b‐based therapeutics for cardiovascular diseases.

Grant Funding Source: Supported by NIH HL106579 and HL108375
Integration of background genome variation to dissect complex cardiovascular phenotypes (697.5)
Authors: Dwinell Melinda , Lazar Jozef , Shimoyama Mary
Published: June 2021

The genetic underpinnings of cardiovascular (CV) disease are far more complex than originally imagined. Human genome wide association studies (GWAS) have identified candidate genes, but to date have predicted only a small fraction of the genetic basis. Some of the “missing heritability” is likely due to the interaction of multiple genes and alleles contributing to the disease. Phenotypic and genotypic characterization of two complete comsomic rat panels provides the foundation to analyze the influence of genetic background on CV traits. Phenotyping of PhysGen consomic strains, developed by introgressing Brown Norway (BN) chromosomes onto the Salt‐Sensitive (SS) or Fawn Hooded Hypertensive (FHH) backgrounds, identified consomic strains in introgressed BN chromosomes alter phenotypes on only one genomic background or under only one environmental condition. Introgression of BN chromosome 1 differentially regulates left ventricle infarct size following ischemia on SS and FHH backgrounds. HIF pathway genes on chromosome 1 are nearly identical between SS and FHH, although 12 genes on background chromosomes contain non‐synonymous SNPs (2 in SS only, 6 in FHH only, and 4 in both compared to BN), potentially altering the HIF pathway and CV phenotypes in response to chronic hypoxia. Additional in silico analysis, using gene, QTL and phenotype annotations, in conjunction with strain‐specific sequence variation, enables virtual variant profiles for complex phenotypes or disease pathways to be created for each consomic strain.

Grant Funding Source: Supported by R01HL094271 and R01HL064541
Polimeric nanoparticles contain a new NO donor (RuBPY) that induces rat aorta relaxation (700.5)
Authors: Araujo Luciana , Da Silva Roberto , Lopez Renata , Bendhack Lusiane
Published: June 2021

This work aimed to prepare the formulation containing free RuBPY and loaded in PNP and to verify if it induces relaxation in isolated rat aorta. We have analyzed the maximum effect (ME) and potency (pD2). P values 蠄0.05 were considered significant. Vascular reactivity experiments were performed on denuded phenylephrine‐contracted aorta and simultaneous amperometric measurements of NO were performed in time course experiments. It was evaluated the effect of the NO scavengers oxyhemoglobin and hydroxocobalamin on the relaxation induced by the formulation. Formulation containing RuBPY induced relaxation with similar ME (103.5±3.3%) as free RuBPY (100.2±0.7%) and similar pD2 values for formulation (7.15+0.33, n=5) and free RuBPY (7.76+0.20, n=5). Time‐course for free and in formulation RuBPY induced relaxation, and NO released was detected in the solution only by free RuBPY. The NO scavengers reduced the relaxing effect for both with greater inhibitory effect for the formulation. In conclusion, RuBPY formulation is a potential vasodilator and the formulation protects the complex RuBPY avoiding the NO leaking to the solution. Supported by FAPESP and CNPq.
Acute ingestion of dietary nitrate increases muscle blood flow via local vasodilation during exercise in young adults (705.9)
Authors: Richards Jennifer , Racine Matthew , Hearon Christopher , Patel Rakesh , Dinenno Frank
Published: June 2021

Dietary nitrate (NO3‐) supplementation with beetroot juice (BR) can reduce the O2 cost of submaximal exercise and increase exercise tolerance. NO3‐ is converted to nitrite and can be reduced to the vasodilator nitric oxide amid a low O2 environment and BR is observed to increase hindlimb blood flow in rats during treadmill exercise. We tested the hypothesis that BR increases forearm blood flow (FBF) via local vasodilation during handgrip exercise in young adults (n=7; 24±2 years). FBF (Doppler ultrasound) and blood pressure (finometer) were measured at rest and during graded handgrip exercise at 5, 15, and 25% maximal voluntary contraction (MVC) lasting 4 minutes each. During 25% MVC exercise, systemic hypoxia (80%SaO2) began and exercise continued for 3 additional minutes. Subjects then ingested concentrated BR (12.6 mmol nitrate) and repeated the exercise bout 2h post consumption. Compared to control, BR significantly increased FBF at 15% (183±29 vs. 165±21 ml/min), 25% (331±40 vs.293±43 ml/min), and 25% + hypoxia (393 ±59 vs. 357±49 ml/min; all P<0.05), and this was due to increases in vascular conductance. Further, the increase in FBF from control was greatest at 25% MVC in normoxia and hypoxia (Δ~35‐40 ml/min) compared with 15% (Δ~18 ml/min) and 5% (Δ~5 ml/min). These findings may have important implications for aging and diseased populations that have impaired muscle perfusion and exercise intolerance.

Grant Funding Source: Supported by HL095573
Activation of lymphocyte autophagy/apoptosis mirror reflects hemodynamic inefficiency and functional aerobic impairment in patients with heart failure (706.19)
Authors: Wang Jong‐Shyan
Published: June 2021

This study investigated whether lymphocytopenia results from activated lymphocyte autophagy/apoptosis, which reflects hemodynamic inefficiency and functional aerobic impairment in patients with heart failure (HF). One hundred twenty‐seven HF patients were divided into the HF with non‐, mild‐, and severe‐lymphocytopenia groups. Lymphocyte autophagy/apoptosis and ventilatory/hemodynamic efficiencies were analyzed in these HF patients and 35 normal counterparts. The results showed that HF patients with severe lymphocytopenia revealed (i) more G protein‐coupled receptor kinase‐2 level, (ii) lesser mTOR level with higher LAMP‐2 expression and acridine orange staining, (iii) lower mitochondrial transmembrane potential with higher caspase‐3 activation and phosphatidylserine exposure, and (iv) greater extents of epinephrine induced apoptosis in lymphocytes, and higher plasma norepinephrine/epinephrine, myeloperoxidase, and interleukin‐6 concentrations than HF patients without lymphocytopenia and normal counterparts did. Therefore, we conclude that increased sympathetic activation and oxidative stress/pro‐inflammatory status may cause lymphocytopenia by activating programmed lymphocyte death in the HF patients. Moreover, a low lymphocyte count correlates with reduced hemodynamics and functional aerobic capacity.
Effects of aerobic interval and moderate continuous training on erythrocyte rheological property modulated by hypoxic exercise in sedentary men (706.9)
Authors: Huang Yu‐Chieh , Wang Jong‐Shyan
Published: June 2021

This study investigated how aerobic interval (AIT) and moderate continuous training (MCT) influence hypoxic exercise‐mediated erythrocyte aggregability and deformability under various shear conditions in sedentary men. Twenty‐four sedentary males were randomized to perform AIT 3‐minute intervals at 40% and 80% VO2 peak, n=12) or MCT (sustained 60% VO2 peak, n=12) for 30 minutes/day, 5 days/week for 6 weeks. Erythrocyte aggregability and deformability under various shear conditions were determined by a slit‐flow ektacytometer. The results showed that, before exercise training, the 60%VO2max exercise under 12%O2 decreased maximal elongation index (EImax), as well as, increased aggregation index (AI) and shortened aggregation half‐time (t1/2) in erythrocytes under shear flow. However, both AIT and MCT diminished erythrocyte EImax and AI, as well as, lengthened erythrocyte t1/2 during the 12%O2 exercise. Accordingly, we conclude that either AIT or MCT effectively improves hemorheological function by decreasing aggregability and increased deformability of erythrocytes during hypoxic exercise in sedentary men.
Influence of 5‐HT1A agonist administration associated with E2 replacement on neuroendocrine in ovariectomized rats (707.11)
Authors: Fonseca Fabricia , Mecawi André , Araujo Iracema , Monteiro Lívia , Pereira Gislaine , Rodrigues José , Reis Luís
Published: June 2021

We intend to examine the effect of repeated use of an agonist of 5‐HT1A receptor in sodium appetitive response in ovariectomized (OVX) rats and investigate the effect of estrogen therapy. Female Wistar rats (n=6‐14) were ovariectomized and formed groups as follow: OVX and OVX + E2 (estradiol cypionate, 80µg/Kg, SC, 14 days) separated in groups of 8‐OH‐DPAT (5‐HT1A receptor agonist, 250µg/Kg, IP, 7 days) or vehicle (0.9% NaCl) in baseline and sodium depletion (furosemide 20mg/kg, SC) protocols. The animals were submitted to following analyses: (i) plasma Na+ (ii) radioimmunoassay to measure oxytocin (OT), vasopressin (AVP), angiotensin II (ANG II) plasma levels. The E2 increased Na+ (P<0.01) plasma levels in OVX‐E2‐8‐OH‐DPAT compared to the OVX‐E2‐OH‐DPAT group in sodium depletion condition. Plasma OT increased (P<0.001) in OVX‐E2‐8‐OH‐DPAT group compared to OVX‐E2 group in baseline condition. OT plasma levels also significantly increased in OVX‐E2 group compared to OVX rats (P<0.01). AVP plasma concentration increased in OVX‐E2‐DPAT group in relation to OVX‐E2 (P<0.001) group following volume depletion. Plasma concentration of ANG II decreased in OVX group compared to OVX‐E2 (P<0.01) group in basal condition. Plasma concentration of ANG II decreased in OVX group compared to OVX‐E2 (P<0.01) group in basal condition. Thus, these results suggest that repeated administration of 5‐HT1A agonist associated with E2 replacement differentially trigger mechanisms for neuroendocrine in electrolyte homeostasis.

Grant Funding Source: FAPERJ, CNPq and CAPES
A module implemented in Excel® for predicting fluid requirements in complex thermal environments (707.7)
Authors: Gonzalez Richard , Kenefick Robert , Charkoudian Nisha , Cheuvront Samuel , Muza Stephen
Published: June 2021

Military and public health reports have expressed a need for improved sweating rate prediction models (msw,pred) that calculate hourly and daily water needs. Using both meta‐analysis and experimental studies (150 volunteers and >1000 cases), we developed an energy balance module within the Microsoft Excel® spreadsheet package. The module incorporates previous legacy heat balance data and predicts msw,pred . The core model inputs environmental parameters, metabolic (Pandolf equation), clothing coefficients, and calculates net effective radiation fluxes, sensible and latent heat fluxes, and outputs evaporative heat loss to estimate fluid requirements. The altitude elevation (<3000 m) can be specified in the model. An output file estimating hourly and daily water requirements and the energy fluxes is generated. The main advantages of the module are: (1) it requires only a laptop/smartphone computation running standard Microsoft Windows®, enabling it to be used at a desktop or in the field; and (2), it can be adapted quickly to different environmental scenarios and meteorological data formats. Model calculations of msw,pred compared with measured data showed a predictive accuracy ranging from 95% for the best to 75% for the worst prediction in complex thermal environments with solar loads. The module is a useful tool allowing guidance in military, public health, occupational and sports medicine settings

Grant Funding Source: Supported by:Knowledge Preservation Program, USAMRMC