bluuebunny/crossref_metadata_2025 huggingface dataset:
B‐methyl‐cyclodextrin (B‐methyl‐CD) blocks constitutive NKCC2 endocytosis, increases surface NKCC2 and stimulates net NaCl absorption in Thick Ascending Limbs.
Authors: Ortiz Pablo A. , Ares Gustavo R
Published: June 2021

The Na/K/2Cl cotransporter (NKCC2) mediates NaCl absorption by the epithelial cells of the thick ascending limb (TAL). Trafficking into (exocytosis) and out (endocytosis) of the apical membrane regulates surface NKCC2 levels. It is not known whether NKCC2 undergoes constitutive endocytosis and whether this regulates NaCl absorption by the TAL. We hypothesized that constitutive endocytosis regulates steady‐state surface NKCC2 and its activity in TALs. We measured surface NKCC2 and the rate of NKCC2 endocytosis by biotinylation and Western blot. To block endocytosis we used B‐methyl‐CD, a compound that depletes membrane cholesterol. In control TALs, we observed constitutive NKCC2 endocytosis over 30 min that averaged 7.0 ± 0.8 a.u/min. However, in TALs incubated with B‐methyl‐CD (5 mM) NKCC2 endocytosis was blocked, averaging only 0.9 ± 0.3 a.u/min (n = 6, p<0.01 vs control). Acute (20 min) blockade of endocytosis with B‐methyl‐CD increased steady‐state surface NKCC2 by 60 ± 16% (p<0.05). The total pool of NKCC2 was not changed by B‐methyl‐CD. Finally, we tested whether the increase in surface NKCC2 caused by inhibiting endocytosis stimulates Cl absorption. Incubating isolated perfused TALs with B‐methyl‐CD for 20 min enhanced net Cl absorption by 46 ± 15 % (n = 5, p<0.05). We concluded that constitutive NKCC2 endocytosis maintains steady‐state surface NKCC2 and regulates NaCl absorption.
Pro‐inflammatory / Pro‐fibrotic effects of Interleukin‐17A on human proximal tubule epithelium
Authors: Dudas Paul L , Sague Sarah L , Elloso M. Merle , Farrell Francis X
Published: June 2021

Chronic allograft nephropathy is manifested by inflammation and tubulointerstitial fibrosis. IL‐17A is a T‐cell‐derived cytokine upregulated in allograft nephropathy, therefore, the present study sought to characterize the actions of IL‐17A on human proximal tubule epithelium.

First, pro‐fibrotic gene expression was examined by real‐time PCR in human proximal tubule cells following treatment with IL‐17A. e‐cadherin was significantly decreased while CTGF, CD44 and TGFβR1 were increased. These changes were reduced by anti‐IL‐17A antibody treatment. Next, IL‐17A‐stimulated soluble mediator secretion was examined. IL‐17A induced the secretion of fractalkine, G‐CSF, GM‐CSF, VEGF, IL‐6 and IL‐8. Finally, ZO‐1 expression was examined by immunocytochemistry as an indicator of epithelial integrity. Treatment with IL‐17A caused a substantial downregulation of ZO‐1 that was inhibited by anti‐IL‐17A antibody treatment.

These data indicate IL‐17A can induce the upregulation of genes associated with ECM remodeling and cell‐cell interaction, and stimulate secretion of inflammatory mediators and immune cell chemoattractants. IL‐17A decreased ZO‐1 protein expression indicating it may negatively impact epithelial barrier function. Thus, IL‐17A may play an important role in the progression of chronic renal injury through the above‐described mechanisms. Centocor is a Johnson & Johnson company.
Calpain10: A new marker of kidney aging and dysfunction
Authors: Covington Marisa , Schnellmann Rick G
Published: June 2021

Aging is associated with abnormalities in kidney function, but the exact mechanisms or causes are unknown. We examined calpain 10 protein expression in aging kidneys from rats, mice, and humans. Calpain 10 protein expression decreased in the kidney of aging Fischer 344 rats, and this decrease was attenuated with caloric restriction. However, there was no change in calpain 10 protein expression in the liver nor in calpain 1 or 2 protein expression in the kidney or liver in normal and caloric restricted Fischer 344 rats. A decrease in calpain 10 protein expression was also seen in aging mice and human kidneys in the absence of a change in calpain 1 or 2. We next examined the functional consequences of calpain 10 protein loss. A single intravenous dose of a siRNA directed against calpain 10 decreases calpain 10 protein in rat kidney cortex. Calpain 10 siRNA had no effect on kidney cortex calpain 1, demonstrating calpain isoform specificity, and calpain 10 siRNA had no effect on liver calpain 10 levels, demonstrating renal specificity. Importantly, serum creatinine levels increased at days 7 and 10 in rats treated with calpain 10 siRNA, suggesting loss of calpain 10 results in decreased renal function. In summary, we have shown calpain 10 protein levels are decreased in aging rat, mice, and human kidney tissues and loss of calpain 10 in vivo results in decreased kidney function.
The Mediation of Aldosterone‐Induced Cortical Collecting Duct Cell Growth Through The Activation of Protein Kinase D1
Authors: Thomas Warren , McEneaney Victoria , Dooley Ruth , Harvey Brian J.
Published: June 2021

Aldosterone elicits transcriptional and rapid signaling responses in target tissues such as the distal nephron. Aldosterone treatment of the M1 cortical collecting duct cell line (M1‐CCD) led to the stimulation of cell growth. This effect was protein kinase D1 (PKD1), PKCδ and ERK1/2‐dependent. Aldosterone induced the rapid activation of ERK1/2 with peaks of activation at 2 min and at 10 to 30 min after hormone treatment, followed by sustained ERK1/2 activation lasting beyond 120 min. M1‐CCD cells suppressed in PKD1 expression exhibited only the early, transient phase in ERK1/2 activation. Co‐immunoprecipitation experiments showed that aldosterone promoted the physical association of PKD1 with ERK1/2 within 2 min of treatment. The MR antagonist RU28318 inhibited the early and late phases of aldosterone‐induced ERK1/2 activation, and also the aldosterone‐induced cell growth. Aldosterone promoted the sub‐cellular redistribution of ERK1/2 to the nuclei of M1‐CCD cells after 2 min treatment and to sites, proximal to the nuclei after 30 min. The sub‐cellular redistribution of ERK1/2 was inhibited in PKD1 knock‐down cells. The stabilization of ERK1/2 activation by PKD1 and ERK1/2 cellular redistribution are a pre‐requisite for aldosterone‐induced cell growth. PKD1 activation by aldosterone may be an important factor in both renal differentiation and proliferative disease.
Application of lymphocytes as model for proteolytic activation of ENaC
Authors: Friis Ulla G , Bistrup Claus , Jensen Boye L
Published: June 2021

We have recently identified plasmin as an activator of epithelial Na+ channels (ENaC) in urine from humans and rats with nephrotic syndrome (1). Lymphocytes express ENaC similarly to ENaC in renal principal cells. We therefore wanted to study if human lymphocytes could be used as a model for proteolytic activation of ENaC. Human lymphocytes were isolated from peripheral blood samples from healthy young males. When lymphocytes were flushed with urine from control rats, it did not significantly increase sodium currents, while when flushed with urine collected on day 4 after puromycin injection resulted in a 4.24 ± 0.97 fold increase in the sodium current compared to controls (control ‐77 ± 11 pA vs. nephrotic urine ‐240 ± 17 pA, n=4, P <0.005). The stimulatory effect was blocked by pre‐treatment of nephrotic urine with aprotinin (700 μg/mL), which is an inhibitor of serine proteases. The identity of the sodium current was further established by blockade of the current with 2 μmol/L amiloride, which is specific for ENaC inhibition when used in this concentration. These data thus confirm the applicability of lymphocytes in the study of ENaC.

This study was supported by grants from the Danish Research Agency and the Novo Nordisk Foundation.

(1) Svenningsen S et al. JASN: in press.
Irbesartan (Irb) Treatment Ameliorates Renal Damage in High Protein Fed FHH Rats: Role of L‐Arginine (arg) Pathways
Authors: Chen Gin‐Fu , Wagner Laszlo J , Moningka Natasha , Baylis Chris
Published: June 2021

The FHH rat is a model of spontaneous glomerulosclerosis, resulting from five renal failure genes (Rf 1‐5), exacerbated by high protein intake. We studied the effects of AT‐1 blockade, via Irb, on arg synthesis and utilization. Arg is synthesized by renal argininosuccinate synthase/lyase (ASS/ASL) and then either consumed within the kidney by arginase II or NOS, or released into the circulation. Arg competes for NOS with ADMA, which is degraded by dimethylarginine dimethylaminohydrolase (DDAH) 1/2. Male FHH rats (6wks old) were put on a 40% casein diet, with (HP+Irb; n=10) or without (HP; n=10) Irb treatment, for 13 wks. Data are summarized in Table (* p<0.05, ** p<0.01, *** p<0.001). HP+Irb rats had reduced proteinuria (UpV mg/24h/100gBW), blood pressure (BP, mmHg), kidney weight (KW, g), glomerulosclerosis index (GSI), and creatinine clearance (CCr, ml/min/100gBW). HP+Irb rats had reduced expression of eNOS (70%) and DDAH2 (65%), higher DDAH1 (120%) and nNOS (>500%) vs. HP. There was no change in the expression of ASS and ASL and Irb reduced arginase II (35%), which allow more arg for NO synthesis. Increased arginase II, and decreased nNOS and DDAH1 expression in HP rats are potential mechanisms for renal injury. These data suggest that Irb may improve renal outcome both by increasing arg supply to the kidney by lowering arginase, and by increasing nNOS expression.
Microglia/motor neuron interactions in response to ATP and BDNF
Authors: Watters Jyoti J , Mitchell Gordon S , Richards Victoria E
Published: June 2021

Microglia are CNS resident immune cells that remove cellular debris during normal brain development and synaptic remodeling. Their ability to sense and respond to neuronal signals is critical for maintaining a healthy CNS. Microglial activities are also important in disease, including motor neuron diseases such as ALS as well as spinal injury. However, interactions between microglia and motor neurons are not well understood. Two regulators of microglial and motor neuron function are ATP and BDNF; BDNF plays a central role in neuroplasticity, and ATP potently regulates microglial immune functions. We hypothesized that cell/cell interactions between microglia and motor neurons modulate their respective responses to BDNF and ATP. To test this hypothesis, a transwell system was used wherein motor neuron‐like NSC‐34 cells and N9 microglia were cultured alone or together in transwells without physical contact. BDNF and ATP synergistically promoted ERK activation within 2 hrs in microglia (not motor neurons) in the transwells, but not when cultured alone. ATP also increased TrkB receptor expression in both cell types 24 hrs following exposure, but only when cultured in transwells. Although the soluble molecule(s) mediating this communication are not yet clear, cell/cell interactions appear to be important for amplifying cellular responses to ATP and BDNF.

Supported by NIH NS040933 and T32 HL007654 (VER).
Effect of combined nitric oxide and prostaglandin inhibition on hypoxic exercise hyperemia
Authors: Crecelius Anne R , Voyles Wyatt F , Dumke Breanna R , Davis Julia A , Kirby Brett S , Dinenno Frank A
Published: June 2021

We tested the hypothesis that endothelium‐derived nitric oxide (NO) and prostaglandins (PGs) contribute to the augmented muscle blood flow in hypoxic exercise relative to normoxia. In 8 healthy adults, we measured forearm blood flow (FBF; Doppler ultrasound) during 5 minutes of rhythmic handgrip exercise at 20% maximal voluntary contraction in normoxia (NormEx) and isocapnic hypoxia (HypEx; O2 saturation ~85%). All trials were performed after α‐ and β‐adrenergic blockade (via brachial artery catheter) to eliminate sympathoadrenal influences on vascular tone. FBF responses were determined under control conditions and during combined NO synthase (L‐NMMA) and cyclooxygenase inhibition (ketorolac). Prior to inhibition, steady‐state FBF was significantly augmented during HypEx compared to NormEx (348±28 vs 306±20 ml/min; P<0.05). After inhibition, FBF during NormEx was similar to that observed prior to blockade (290±18; P = 0.11). In contrast, FBF during HypEx was significantly reduced after inhibition (315±24; P<0.05 vs pre), and this was no longer greater than the hyperemic response observed during control NormEx (P = 0.12). We conclude that under sympathoadrenal blockade, NO and PGs contribute modestly to NormEx hyperemia, whereas these locally‐derived substances appear to play a significant role in the augmented hyperemic response observed during HypEx.

Supported by HL‐087952
Facilitatory Interactions between 5‐HT and Thromboxane A2 in Stimulation of Ischemically Sensitive Cardiac Sympathetic Afferents
Authors: Fu Liang‐Wu , Longhurst John
Published: June 2021

We have demonstrated that the endogenously produced mediators serotonin (5‐HT) and thromboxane A2 (TxA2) contribute to activation of cardiac afferents during ischemia. In the present study we hypothesized facilitatory interactions between these two metabolites in stimulation of cardiac afferents during ischemia. Nerve activity of single cardiac afferent units was recorded from the left sympathetic chain or rami communicantes (T2 ‐ T5) of anesthetized cats. Twenty ischemically sensitive afferents (CV= 0.28 ‐ 3.62 m/s, 4 Ad‐ and 16 C‐fibers) were identified. We observed that a repeat injection of 5‐HT (40 μg/kg) into left atrium (LA), 4 min after LA 2.5 μg of U46619, a TxA2 mimetic, induced a significantly larger cardiac afferent response than the first 5‐HT response (0.46±0.08 to 1.06±0.11 vs.0.45±0.13 to 1.90±0.18 imp/s, first vs. second injection, n=3). In a control group, repeated 5‐HT (40 μg/kg, LA) evoked consistent responses in three other ischemically sensitive afferents. Furthermore, LA administration of 5‐HT (40 μg/kg) and U46619 (2.5 μg) caused a larger response than simple addition of the individual responses (n=5). Additionally, the responses of three cardiac afferents to repeat U46619 (2.5 μg, LA) were enhanced compared with the U46619 response before 5‐HT, while the responses of three other cardiac afferents to repeated U46619 stimulation were consistent. Finally, blockade of the 5‐HT3 receptors with tropisetron (200 μg/kg, iv) attenuated the responses of three cardiac afferent to U46619 (2.5 μg, LA) by 52%. These preliminary data suggest that 5‐HT and TxA2 each facilitate the responses of ischemically sensitive cardiac afferent endings to the actions of other metabolite (Supported by NIH HL66217).
Cholinergic protection against apoptosis in primary cardiac cells
Authors: Li Yi‐Fan , LaCroix Carly , Bergstrom Tyler , Freeling Jessica
Published: June 2021

The autonomic imbalance, characterized as the elevated sympathetic activity and the suppressed parasympathetic activity, is commonly present in a variety of cardiovascular diseases. However, the functional role of the parasympathetic suppression in cardiac pathology has not been well addressed. Our recent in vivo studies revealed that treatments with cholinergic reagents protected against cardiac dysfunction, while deficiency of M2 receptors increased cardiac susceptibility to stress. In this study, we further observed the cholinergic protection in primary cardiac cells. Pretreatments with cholinergic reagents reduced cell death induced by H2O2 and serum withdrawal. Furthermore, cholinergic treatments attenuated activity of caspase3/7, a central apoptotic signaling pathway. This cholinergic protection against cell death was associated with an enhanced ERK activation. These results suggest that the cholinergic direct effect against cell death may be one of the mechanisms of parasympathetic cardiac protection in cardiac diseases.

This study was supported by American Heart Association Grant No. 0835256N, NIH COBRE grant No. 5 P20 RR017662, and INBRE Grant No. 2 P20 RR016479
Effect of AT1 and AT2 receptor antagonism of the subfornical organ (SFO) on baroreflex regulation in normotensive rats
Authors: Maliszewska‐Scislo Maria , Rossi Noreen F.
Published: June 2021

Our earlier studies have shown the subfornical organ (SFO) is involved in the baroreflex control of renal sympathetic nerve activity (RSNA) and heart rate (HR). Specifically, lesioning the SFO leads to an increase in the upper plateau and range of the baroreflex in normotensive rats, but the mediator of this response is not known. The present study was carried to test the hypothesis that activation of angiotensin receptors type 1 (AT1) and/or type 2 (AT2) within the SFO by endogenous angiotensin II (Ang II) contributes to the modulation of the baroreflex. Rats were instrumented with vascular catheters, renal nerve electrode and a cannula directed to the SFO. Baroreflex was tested in conscious rats with graded iv infusions of phenylephrine and nitroprusside before and after microinjection of 20 nmol losartan or 10 nmol PD123319 or both together. None of the treatments changed resting parameters significantly. Losartan alone did not alter the baroreflex response of either HR or RSNA. The combination of AT1 and AT2 blockade increased the upper plateau of the HR baroreflex from 486±12 to 507±10 bpm (P = 0.05) and the upper plateau of the RSNA baroreflex by 24% (P < 0.03), thereby significantly increasing the range of both responses. Thus, combined AT1/AT2 blockade results in a similar pattern of responses as lesioning the SFO and these receptors may be partially responsible for the modulation of baroreflex by the SFO.
Impaired Baroreflex Control of Renal Sympathetic Nerve Activity (RSNA) and Elevated Basal RSNA in Type 1 Diabetic Mice (OVE26)
Authors: Gu He , Zhang Zhihua , Epstein Paul N. , Li Lihua , Harden Scott . W , Wurster Robert D. , Cheng Zixi (Jack)
Published: June 2021

To investigate the effects of chronic diabetes on baroreflex control of renal sympathetic nerve activity (RSNA) and basal RSNA, OVE26 diabetic (transgenic mouse line which develops hyperglycemia within the first three weeks after birth) and FVB control mice 5‐6 months old were studied. Under anesthesia, RSNA in response to sodium nitroprusside (SNP) ‐ and phenylephrine (PE)‐induced mean arterial pressure changes (ΔMAP) were measured. Baroreflex‐induced inhibition of RSNA during PE infusion was characterized using the sigmoid logistic function curve. Baroreflex‐induced excitation of RSNA during SNP infusion was characterized by the RSNA vs. ΔMAP relationship. MAP responses to the left aortic depressor nerve (ADN) stimulation were evaluated. Basal RSNA was estimated as the resting RSNA divided by the maximal RSNA measured during SNP‐infusion. Compared to FVB control, we found in OVE26 mice that (1) RSNA in response to MAP increase during PE infusion was dramatically reduced, as characterized by the maximal gain of the RSNA sigmoid logistic function curve (FVB: ‐20.0 ± 5.1; OVE26: ‐7.6 ± 0.8 %/mmHg, p<0.05); (2) RSNA in response to MAP decrease during SNP infusion was also attenuated (p<0.05); (3) MAP responses to ADN stimulation were reduced (p<0.05); (4) the basal RSNA was elevated (P<0.05). We concluded that chronic diabetes impairs baroreflex control of RSNA and elevates basal RSNA in OVE26 diabetic mice. The use of the transgenic OVE26 diabetic mouse model may underlie a foundation for the further understanding of diabetes‐induced autonomic neuropathy. Supported by NIH HL79636.
Increased urothelial ATP release from a subset of OAB patients
Authors: Contreras‐Sanz Alberto , Janska S , Alexander SLH , Lunawat R , Bishara S , Khasriya R , Taylor KMG , Malone‐Lee J , Wildman SSP
Published: June 2021

Overactive bladder (OAB) is characterised by frequency, urgency and urge incontinence in the absence of infection. We have identified previously unrecognised pyuria in patients diagnosed as having OAB; associated with more severe symptoms.1 Antibiotic treatment of these patients is successful if maintained for long periods. Others have shown that bacteria can thrive in urothelium, resisting host immunity and antibiotics. It is also documented that inflammation, when caused by infection, is associated with increased ATP release from cells, and extracellular ATP signalling is involved in bladder sensation.

We hypothesise that in OAB, with pyuria, there is enhanced ATP release from urothelium caused by intracellular bacteria, which ultimately results in increased sensory nerve excitation and heightened symptoms of OAB. Here we begin to investigate our hypothesis.

Human bladder urothelium biopsies were obtained using cystoscopy. ATP release from urothelium was measured using a luciferin luciferase assay.

Basal and stretch‐evoked (using a hypotonic solution) ATP release was significantly greater from the urothelium of pyuric OAB patients than from non‐pyuric OAB or asymptomatic patients.

In summary, this data supports our hypothesis.

1. Malone‐Lee et al, Urinary white cells and symptoms of the Overactive bladder. Urodyn. Supplement for International Continence Society (Rotterdam, 2007) Abstract 42.
Effects of postural changes on arterial to venous blood flow in the dependent limbs of conscious cats
Authors: Misra Sunil P , Yavorcik Kevin J , Erwin Monica , Cotter Lucy A , Reighard Derek A , Wilson Timothy D , Yates Bill J
Published: June 2021

It is routinely assumed that the force of gravity can cause blood pooling in the dependent limbs, as venous return from the body region is reduced. However, the effects of postural alterations on blood flow to and from the limbs have not been directly compared or quantified. In the present study, blood flow in the femoral artery and vein of conscious cats were measured using transit‐time ultrasound technology as the animals were rotated head‐up by amplitudes up to 60°. 60° head‐up rotations produced significant (ANOVA, p<0.05) decreases in venous blood flow beginning ≈3 sec following tilt onset, which became maximal at ≈5 sec, with an alteration of 55‐65% from pre‐tilt values. As the animals were maintained head‐up, venous blood flow began to increase, and was 18‐29% lower than pre‐tilt values by 40 sec following rotation initiation. This recovery of venous flow was accompanied by a 17‐34% decrease in arterial blood flow that peaked ≈9 sec after tilt onset. These data confirm that venous return from the dependent limb is greatly attenuated during postural alterations, and the magnitude may be larger than expected. However, the largest perturbation in venous return is transient, and this combined with accompanying decreases in arterial blood flow limits peripheral pooling.
Particulate matter exposure‐induced decreased heart rate variability is associated with a reduced excitability of cardiac vagal neurons in mice
Authors: Pham Hai , Pinkerton Kent E , Bonham Ann C , Chen Chao‐Yin
Published: June 2021

Exposure to PM2.5 increases the total daily cardiovascular mortality by an estimated 3‐4% for every 25 μg/m3 increase in PM2.5. Impaired cardiac autonomic function, indexed by reduced heart rate variability (HRV), is one underlying cause. We previously showed that 3 d (6 hr/d) of PM2.5 exposure in the form of iron‐soot reduced HRV in mice. Here we test the hypothesis that exposure to airborne PM impairs vagal regulation of HRV by decreasing the excitability of the cardiac vagal neurons in the nucleus ambiguus (NA) in C57BL/6 mice. We measured 24 hr HRV in time domains from ECG telemetry recordings obtained in conscious freely moving mice. In parallel studies we determined the intrinsic properties of anatomically identified NA cardiac vagal neurons, retrogradely labeled with a fluorescent dye (DiI) applied to the SA node. PM2.5 exposure in the form of soot decreased the short term HRV (rMSSD). With the addition of iron, all HRV parameters were significantly reduced. The reduced HRV is more prominent during the dark cycle of the day. Blocking the vagal influence with Methylatropine significantly reduced all HRV parameters suggesting that HRV is under vagal regulation in mice. PM2.5 exposure also decreased the spiking responses of the identified cardiac vagal neurons to depolarizing current injections (P<0.05). The data suggest PM‐induced neuroplasticity of cardiac vagal neurons maybe one mechanism contributing to the cardiovascular consequences associated with PM exposure seen in humans. (Support: Support: 1R01ES012957‐01).
NOX2 deficiency prevents angiotensin II‐induced hypertrophy and inward remodeling in cerebral arterioles
Authors: Chan Siu Lung , Baumbach Gary
Published: June 2021

We examined the role of NAD(P)H oxidase subunit NOX2 on structural alterations in cerebral arterioles induced by angiotensin II (Ang II). NOX2‐deficient (NOX2‐/‐) and wild‐type (WT) mice were infused with a pressor or a sub‐pressor dose of Ang II (1000 or 200 ng/kg/day) or saline via osmotic minipumps for 4 weeks. Systolic arterial pressure (SAP) was measured by a tail‐cuff method. Pressure and diameter of cerebral arterioles were measured via an open cranial window in anesthetized mice. In WT and NOX2‐/‐ mice, the pressor, but not the sub‐pressor, dose of Ang II increased SAP (135±5 vs 106±3 mmHg). Superoxide levels in cerebral arterioles (hydroethidine staining) were increased by both doses of Ang II in WT (staining density: 41±5 and 32±2 vs 18±5 %), but not in NOX2‐/‐ mice. The sub‐pressor, as well as the pressor, dose of Ang II significantly reduced external diameter of maximally dilated cerebral arterioles in WT mice (51±3 and 52±3 vs 58±2 μm at 30 mmHg), but not in NOX2‐/‐ mice. Cross‐sectional area (CSA) of the arteriolar wall was significantly increased in WT mice (497±58 vs 388±50 μm2) by the pressor, but not the sub‐pressor, dose of Ang II. In contrast, the pressor dose of Ang II did not increase CSA in NOX2‐/‐ mice. These findings suggest that generation of superoxide may play an important role in Ang II‐induced hypertrophy and inward remodeling in cerebral arterioles.
Antioxidant treatment with tempol prevents obesity induced remodeling of middle cerebral arteries in Sprague‐Dawley rats
Authors: Pires Paulo W , McClain Jonathon L , Dorrance Anne M
Published: June 2021

Obesity is a risk factor for stroke, especially when associated with diabetes. Cerebral vessel remodeling may contribute to this increased risk of cerebrovascular accidents. We hypothesized that a diet rich in fat and carbohydrates would contribute to middle cerebral artery (MCA) remodeling, and that oxidative stress would play a key role in the process. Three week old male Sprague‐Dawley rats were fed a high‐fat (HF) diet containing 36% fat and 36% carbohydrate (n=21), or HF plus the super oxide dismutase mimetic, tempol (n=9) or regular chow (n= 18) for 18 weeks. MCA's were mounted in a pressure myograph under calcium‐free conditions to assess vascular structure. All data are shown as mean±SEM. Body weight was higher in the groups that received HF diet (442±6, 484±19, 374±9 grams, HF, HF+tempol and control); the same was true for visceral fat weight (16.2±0.9, 22.1±3.1, 5.7±0.5 grams, HF, HF+tempol, control). Blood glucose was increased in the HF group (197±10, 143±5 mg/dl, HF vs control) and treatment with tempol reversed this effect (115±10 mg/dl). Wall/lumen ratio was higher in the HF rats and reduced by tempol treatment (0.15±0.02, 0.08±0.01, 0.08±0.01, HF, HF+tempol, control, at 80mmHg p<0.05), the same was true for wall thickness and wall area. These results suggest that a HF diet increases MCA remodeling and blood glucose, and tempol treatment reverses these processes. ADA‐7‐07‐RA28, AHA‐0840122N
Phospholipase Cγ1 is Required for Pressure‐Induced Vasoconstriction of Cerebral Arteries
Authors: Herrera Carlos Eduardo , Crnich Rachael , Earley Scott
Published: June 2021

Phospholipase C (PLC) hydrolyzes the phosphodiester bond of the membrane phospholipid phosphotidylinositol (4,5)‐bisphosphate (PtdIns(4,5)P2) to generate the second messengers diacylglycerol (DAG) and inositol 1,4,5‐trisphosphate (IP3). A number of PLC isoforms are present in vascular smooth muscle cells, including members of the PLCβ, PLCg, and PLCd families. Pharmacological inhibition of PLC activity blocks both pressure‐ and agonist‐induced vasoconstriction. To test the hypothesis that specific PLC isoforms influence discreet vasoconstrictor pathways, we used small interfering RNA (siRNA) to silence PLCγ1 expression in intact cerebral resistance arteries. Expression of PLCγ1 in intact cerebral arteries treated with siRNA was reduced by approximately 70% compared to controls. Arteries treated with PLCγ1 siRNA failed to constrict in response to increases in intraluminal pressure. In contrast, vasoconstriction in response to the purinergic receptor agonist uridine triphoshphate (UTP) did not differ between PLCγ1 siRNA‐treated arteries and controls. These finding demonstrate that PLCγ1 is required to regulate myogenic responsiveness, whereas other PLC isoforms regulate agonist‐induced vasoconstriction. AHA0535226N.
Diverse impairment of vasodilator pathways in brain following selective interference with PPAR‐gamma in vascular muscle
Authors: Modrick Mary L. , Chrissobolis Sophocles , Halabi Carmen M. , Sigmund Curt D. , Faraci Frank M.
Published: June 2021

Peroxisome proliferator‐activated receptor‐gamma (PPARg) may play an important protective role in the vasculature. We examined the hypothesis that selective interference with PPARg in vascular muscle would alter regulation of vasomotor tone. We studied basilar arteries from mice expressing a dominant negative mutation in human PPARg (P467L) under the control of the smooth muscle myosin heavy chain promoter (S‐P467L). We examined responses to stimuli that produce vasodilation via nitric oxide and cGMP (acetylcholine, ACh; nitroprusside, NP), cAMP (forskolin), and potassium channels [cromakalim and potassium ion (K+)]. In non‐transgenic controls, all agonists produced dilation of the basilar artery in vitro. In S‐P467L mice, responses to ACh, NP, forskolin, and activators of K+ channels were reduced by 40‐70% compared to controls (P<0.05). For example, vasodilation to K+ (10 mM) was 67±6 and 22±4% in control (n=9) and S‐P467L (n=6) mice, respectively (P<0.01). Arteries from both groups responded similarly to the calcium channel agonist Bay K8644 and the calcium channel antagonist nifedipine. These data provide the first evidence that cell‐specific interference with the function of PPARg in vascular muscle produces marked, but selective, impairment of diverse vasodilator pathways. Thus, normal PPARg function in vascular muscle plays an essential role in regulation of cerebral vascular tone.
Differential role of α1‐adrenergic receptor subtypes in Ca2+‐dependent and Ca2+‐independent contraction in developing cerebral arteries
Authors: Mittal Ashwani , Goyal Ravi , Wedhas Nia , Shi Lijun , Longo Lawrence D.
Published: June 2021

Objective

Cerebral arteries in the fetus differ functionally from adult. We tested the hypothesis that developmental differences are a consequence of differential activities of α1‐adrenergic receptor (α1‐AR) subtypes.

Methods

In middle cerebral arteries from fetal and adult sheep, we measured responses of Ins(1,4,5)P3, vascular tension, and [Ca2+]i to the α1‐AR agonist phenylephrine (PHE, 10−5 M), in presence of blockers.

Results

In fetal cerebral arteries, expression levels of each α1‐AR subtype was only ~20% that of adult. In fetus, each subtype specific blocker, 5‐MU (α1A), CEC (α1B) and BMY‐7378 (α1D) inhibited to a degree greater degree PHE‐induced tension and [Ca2+]i, compared to adult. Although in arteries of both age groups, 5‐MU and CEC reduced Ins(1,4,5)P3 responses to PHE ~35%, BMY‐7378 showed no significant decrease in Ins(1,4,5)P3 responses. Also in fetal arteries, the PHE‐induced increase in activated ERKs was blocked by CEC and BMY‐7378, but not by 5‐MU or WB‐4101.

Conclusions

In both age groups α1A‐ and α1B‐AR appear to mediate α1‐adrenergic‐Ins(1,4,5)P3 responses. In the fetus in contrast, α1D‐AR appear to activate the MAPK/ERK1/2 cascade by non‐Ins(1,4,5)P3‐dependent mechanisms. In the immature organism, relative deficiency of specific α1‐AR subtypes, and their differing signaling pathways, may be critical factors associated with dysregulation of cerebral blood flow. (USPHS HD 03708)
Estrogen receptor‐mediated protection of cerebral endothelial cell viability and mitochondrial function during ischemic conditions
Authors: Guo Jiabin , Weiss John H. , Krause Diana N. , Duckles Sue P.
Published: June 2021

We recently demonstrated 17ß‐estradiol (E) improves mitochondrial efficiency and reduces free radicals in brain blood vessels and endothelial cells. We hypothesized that during ischemic insult, E would protect brain endothelial mitochondrial function and enhance cell viability. To test this, oxygen‐glucose deprivation (OGD)/reperfusion was applied to immortalized mouse cerebral endothelial cells (bEnd.3) in culture. OGD‐induced cell death was prevented by long‐term (24, 48 h), but not short‐term (<24 h) pretreatment with E (10 nM). E also suppressed OGD‐induced translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei. E inhibited lactate dehydrogenase (LDH) leakage during OGD and reperfusion and also abrogated OGD‐induced ATP depletion. The latter effects were blocked by the estrogen receptor (ER) antagonist, ICI‐182,780 and mimicked by an ERalpha‐selective agonst but not an ERbeta agonist. We found previously ERalpha stimulates mitochondrial protein expression and suppresses mitochondrial superoxide in brain endothelial cells. Together these findings suggest estrogen receptor‐mediated protection of mitochondrial function helps preserve cerebral endothelial cells during ischemic insult. [Supported by NIH Grant R01 HL‐50775 and Chinese Government Scholarship 20073020].
Bi‐directional neurovascular communication in the brain
Authors: Filosa Jessica A , Blanco Victor M , Higashimori Haruki
Published: June 2021

We previously showed that changes in the resting tone of intracortical arterioles results in opposite‐type vascular responses (dilations and constrictions). Such findings also suggest that in addition to the well‐studied flow of information characteristic of functional hyperemia, namely neuron‐to‐astrocyte‐to‐arteriole, stimulation in a reverse manner may uncover yet another important function associated to brain information processing and/or neuroprotection. Here we show that three different modes of stimulation (depolarization, endothelin‐1 and mechanical) which induced pial arteriolar constriction subsequently increased intracellular calcium in cortical astrocytes (P<0.0001, n=5). A potential mechanism leading to this event is the activation of mechanosensitive TRPV4 channels expressed in astrocytic endfeet processes. Attesting to the presence of these channels, bath applied 4 alpha‐phorbol 12,13‐didecanoate significantly (P<0.0001) increased calcium oscillations in astrocytes (0.008±0.002 to 0.026±0.004 Hz, n=4) with prominent changes occurring in perivascular processes. We propose that stretching of astrocytic endfeet membranes, following vessel diameter changes, unveils an important mode of communication between the vasculature and the brain. Ongoing studies aim to address whether neuronal and glial activity is indeed regulated by changes in blood flow.
Decreases in Cerebral Blood Flow When Upright Are Related to Vestibular Function Regardless of Age
Authors: Serrador Jorge M , Lipsitz Lewis , Black F. Owen , Wood Scott J
Published: June 2021

Introduction

Decreases in cerebral blood flow occur when moving from the supine to upright posture, yet the cause remains unclear. We hypothesized that impaired vestibular function would produce greater drops in cerebral blood flow in the upright posture.

Methods

155 subjects were screened for otolith function and based on ocular counter‐roll (OCR), 21 were classified as either young low (YL), young high (YH), old low (OL) or old high (OH). Mean OCR ‐ YL & OL ‐ 0.08±0.05; YH & OH 0.22±0.05. Age for young 33±12, old 69±6 years. Subjects underwent two 30‐min tilt tests on separate days. Cerebral flow velocity (CFV), by TCD, blood pressure (Finapres) and end‐tidal CO2 were measured continuously.

Results

Reduced otolith function was associated with significantly greater drops in CFV at all ages (YL: ‐20±4%; OL: ‐22±3%) as compared to normal function (YH: ‐8±2%; OH: ‐13±2%), despite the fact that blood pressure changes were similar in all groups. The reduced OCR groups also demonstrated greater postural hypocapnia (YL: ‐3±1mmHg; OL: ‐4±1mmHg) vs (YH: ‐1±1mmHg; OH: ‐2±1mmHg), however this hypocapnia could only explain ~9‐12% of the 20‐22% decrease in CFV..

Discussion

These data demonstrate that otherwise healthy subjects of all ages with impaired vestibular function demonstrate greater decreases in cerebral blood flow when upright. Thus, vestibular function may play an important role in cerebral orthostatic response.
Deficiency of telomerase activity affects the blood brain barrier disruption and neurological outcome in a model of middle cerebral artery occlusion
Authors: Zhang Bei , Chen Lei , Swartz Karin R , Bruemmer Dennis , Hennig Bernhard , Toborek Michal
Published: June 2021

Epidemiology and genetic studies indicate that patients with telomere length shorter than average are at higher risk of dying from heart disease or stroke. Telomeres are located at the ends of eukaryotic chromosomes whose length is progressively reduced in most somatic cells during aging. The enzyme telomerase can compensate for telomere loss during cell replication. The present study is aimed to investigate the contribution of telomerase to stroke and the blood‐brain barrier (BBB) dysfunction. Telomerase reverse transcriptase knock‐out (TERT‐/‐) and wild‐type mice (males, 4‐month‐old) were subjected to permanent 24 h middle cerebral artery occlusion (MCAO). Neurological testing revealed that TERT‐/‐ mice showed enhanced deficits as compared to wild‐type animals. These changes appeared to be associated with greater infarct volume in TERT‐/‐ mice. Expression of tight junction proteins, such as ZO‐1 and ZO‐2, was decreased in ischemic hemispheres both in TERT‐/‐ and wild type mice. These results suggest that TERT deficiency can predispose to the development of stroke and diminish the recovery processes in an experimental model of this disease.
Neutrophil activation during reperfusion after ischemic stroke in mice
Authors: Morrison Helena Willington , Davis‐Gorman Grace , Frye Jennifer , McDonagh Paul , Funk Janet , Ritter Leslie
Published: June 2021

Experimental evidence in non‐murine models suggests that activated neutrophils play a pivotal role in cerebral reperfusion injury. Neutrophils release toxic mediators as they initially adhere to the microcirculation and migrate out of the circulation into to brain tissue. In this study we investigated whether systemic neutrophils are activated in mice during the first minutes and 24 hours after reperfusion following ischemic stroke. The filament method of middle cerebral artery occlusion (60 min) and reperfusion (MCAOR) was performed in male C57Bl/6J mice (22‐25g). Blood was drawn from the aorta after 15 minutes (MCAOR 15min, n=6) or 24 hours of reperfusion (MCAOR 24hr, n=2) and compared to blood obtained from a sham surgery (Sham, n=4). Neutrophil CD11b expression was measured in whole blood using flow cytometry methods. Compared to the shams, there was a 70% increase in neutrophil CD11b expression following MCAOR at 15 min (p = 0.06) and a 108% increase following MCAOR at 24 hours (p = 0.03). There was also an 83% and 94% increase of LPS‐stimulated neutrophil CD11b expression at 15 min (p = 0.08) and 24 hours (p = 0.13) of reperfusion. These results indicate that in a mouse model of MCAOR, systemic neutrophils 1) are activated and primed during the first minutes and after 24 hours of reperfusion, and 2) can be easily monitored to assess the effects of new treatments for reperfusion injury after stroke.

Supported by NIH NRSA 1 F31 NR010658‐01 (Morrison) NIH HL 58859 (McDonagh) University of Arizona Emmons (Ritter)
Hypoxic Exercise Training Promotes Cytotoxicity of Natural Killer Cells to Nasopharyngeal Carcinoma Cells
Authors: Weng Tzu‐Pin
Published: June 2021

Nasopharyngeal carcinoma (NPC) is common cancer in South China. Natural killer cells (NKs) incur apoptosis of carcinoma by employing the perforin/granzyme‐mediated cytotoxicity. This study investigates how hypoxic exercise training affects the cytotoxicity of NK to NPC and further explores its underlying mechanisms. Twelve sedentary males were trained on a bicycle ergometer at about 60%VO2max under 15% O2 air condition for 20 minutes per day, 5 days per week for 4 weeks. Our results demonstrated that before the training, strenuous, acute exercise enhanced NK‐induced phosphotidyl serine exposure of NPC, which were accompanied by increases in NK count and cellular perforin/granzyme levels. Moreover, 4 weeks of hypoxic exercise training induced higher NK count and cellular perforin/granzyme levels as well as the percentage of NK cytotoxicity to NPC at rest and following exercise compared to pre‐training state. Therefore, we conclude that hypoxic exercise training promotes anti‐NPC cytotoxicity mediated by NK though increasing NK count and cellular perforin/granzyme levels.
Atorvastatin improves function of vascular endothelium by decreasing oxidative stress
Authors: Wang Jian , Ru Xiaochen , Qian Lingbo , Tu Yeqiang , Xia Qiang
Published: June 2021

Vascular oxidative injury is involved in numerous cardiovascular diseases, particularly in hypertension and atherosclerosis. Statins ‐‐ a family of lipid lowering drugs ‐‐ appear to display non‐lipid modifying abilities. The aim of this study was to investigate the effect of atorvastatin on vascular tone and its vasoprotection against oxidative stress injury. The tension of the isolated rat thoracic aortic rings was measured using organ bath technique and superoxide arterial injury was induced by pyrogallol (which produces superoxide anions) incubation. We found that atorvastatin (10−7 M ‐ 10−4 M) caused a concentration‐dependent relaxation in endothelium‐intact aortic rings precontracted by phenylephrine (PE, 10−6 M), which was abolished by L‐NAME (10−4 M), a nitric oxide synthase inhibitor. However, atorvastatin did not relax endothelium‐denuded aortic rings. Exposure to pyrogallol (3 ?10−4 M) for 15 min inhibited the acetylcholine (ACh, 10−9 M ‐ 10−5 M)‐induced relaxation in aortic rings precontracted by PE. This superoxide anion‐induced impairment was attenuated by pre‐incubation of atorvastatin (10−5 M) for 30 min. Atorvastatin administration (2, 10, 50 mg·kg−1·d−1, respectively) by gavage for 14 days did not alter the body weight of the rats, but showed improvement of ACh‐induced relaxation in aortic rings exposure to pyrogallol. These results indicate that atorvastatin has an endothelium‐dependent vasodilating effect on rat aorta, mediated by nitric oxide. Both acute and chronic administration of atorvastatin protect aorta from oxidative stress injury.
The effects of a phytogenic feed additive versus and antibiotic feed additive on oxidative stress in broiler chicks
Authors: Settle Tabatha Lynn , Moritz Joseph , Leonard Stephen , Klandorf Hillar , Falkenstein Elizabeth , Caro Maria
Published: June 2021

Phytogenic feed additives are plant‐derived products used in poultry feeding to improve overall performance of broilers while reducing oxidative stress. In the first study 588 one day‐old Cobb 500 chicks of mixed sex were fed one of four diets and housed on either dirty or clean litter for 3wks. Blood samples were obtained from chicks at 18 days of age for measurement of leukocyte oxidative activity. Results of the study showed that chicks in the treatment groups fed either the phytogenic additive or the antibiotic had lower levels of oxidative stress (P< 0.02). In a second study electron spin resonance (ESR) spin trapping was used to measure feed additive of hydroxyl or superoxide radicals. Fenton chemistry was utilized in generation of hydroxyl radicals and a xanthine/xanthine oxidase reaction generated superoxide radicals. Radicals were also measured after stimulation of RAW 264.7 mouse peritoneal monocytes. Treatments were based on different concentrations and age of the antibiotic or phytogenic feed additive. Results from both studies showed that both the antibiotic and the phytogenic feed additive scavenged hydroxyl and superoxide radicals although the antibiotic was determined to be more efficient.
Cannabidiol attenuates cisplatin‐induced nephrotoxicity by decreasing oxidative/nitrosative stress, inflammation and cell death
Authors: Pacher Pal , Mukhopadhyay Partha , Pan Hao , Rajesh Mohanraj , Patel Vivek , Mukhopadhyay Bani , Becker Lauren , Bátkai Sandor , Gao Bin , Haskó György
Published: June 2021

The platinum compound cisplatin is one of the most potent chemotherapy agents available to treat various malignancies. Nephrotoxicity is a common complication of cisplatin chemotherapy, which involves increased oxidative and nitrosative stress, limiting its clinical use. In this study we have investigated the effects of a nonpsychoactive cannabinoid cannabidiol, which was reported to exert antioxidant effects and has recently been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in patients, in a mouse model of cisplatin‐induced nephropathy. Cisplatin induced increased expression of superoxide generating enzymes RENOX and NOX1,, enhanced ROS generation, nitrotyrosine formation, apoptosis , PARP activity, and inflammation in the kidneys of mice, associated with marked histopathological damage and impaired renal function. Pretreatment of mice with cannabidiol markedly attenuated the cisplatin‐induced oxidative/nitrosative stress, inflammation and cell death in the kidney, and improved renal function. Thus, our results suggest that cannabidiol may represent a promising new protective strategy against cisplatin‐induced nephrotoxicity.
Small Interfering RNA (siRNA) Suppresses the Fas Response Amplification Loop in Sulfur Mustard (SM)‐Exposed Normal Human Bronchial/Tracheal Epithelial (NHBE) Cells
Authors: Keyser Brian , Andres Devon , Benton Betty , Ray Radharaman
Published: June 2021

Our previous studies using NHBE cells and caspase type‐specific peptide inhibitors demonstrated that SM induces apoptosis via a dominant caspase‐8 (Fas response) mediated pathway (Ray et al., Drug & Chem. Toxicol. 37:137‐48, 2008). Here, we validate this observation by using a more specific siRNA technology to suppress the Fas receptor as well as caspases‐9 and ‐6. Caspase activation was measured by using a luminescent caspase substrate hydrolysis assay. The effectiveness of the siRNA constructs was determined at both protein (western blot) and functional (caspase activation) levels. We found that down regulation of caspase‐9 by siRNA was able to significantly (~60%) reduce caspase‐3 activation by CH11, monoclonal antibody that activates Fas (caspase‐8 pathway); similar results were seen in SM‐exposed NHBE cells. These results provide further support to an amplification loop between caspases‐8, ‐9, ‐6, and ‐3 in apoptosis due to SM (300 μM) exposure in NHBE cells. These findings may be useful in developing siRNA based therapeutics against SM injury. Disclaimer: The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army or the Department of Defense. This research was supported by the Defense Threat Reduction Agency ‐ Joint Science and Technology Office, Medical S&T Division.