bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of prenatal nicotine exposure on in vitro central respiratory‐related cholinergic neurotransmission
Authors: Pilarski Jason Quinn , Fregosi Ralph Frank
Published: June 2021

Prenatal nicotine exposure (PNE) is correlated with breathing abnormalities in both humans and other animals. Despite evidence this relationship results from alterations in nicotinic acetylcholine receptors (nAChRs), which mediate vital brain stem respiratory‐related cholinergic neurotransmission, a complete mechanistic understanding is lacking. In the present study, we hypothesize that PNE alters medullary nAChRs, which then leads to a blunted respiratory motor output following acute nAChR excitation. To test this hypothesis, we used an in vitro “split‐bath” brain stem spinal cord preparation and recorded C4 ventral root (C4VR) nerve bursts from 0–4 day old rats. Rats were exposed to either nicotine (6 mg/kg/day) or saline (control) in utero. Results show that following PNE peak C4VR burst frequency in response to bath application of nicotine was decreased 57% at [0.25 ìM] nicotine and 45% at [0.5 ìM] compared to controls. Our results also indicate that á4â2 nAChRs play a dominant role in the nicotine‐induced increase in C4VR burst frequency, since its antagonism completely blocked nicotine's effect on C4VR burst frequency in both PNE and control animals, while á7 antagonism had no effect in either group. These data suggests that the reduction in nicotine‐induced C4VR burst frequency in PNE animals is due to an anatomical and/or functional decrease in excitatory neurotransmission mediated by á4â2 nAChRs.
Activation of 5HT2A receptors alters temporal and spectral characteristics of phrenic nerve discharge in arterially‐perfused adult rat
Authors: Bale Tejus A , Warren Kelly A. , Solomon Irene C.
Published: June 2021

Previous studies in the in vitro transverse medullary slice have demonstrated that activation of serotonergic 5HT2A receptors increases inspiratory burst amplitude and frequency as well as the incidence of augmented (sigh) bursts. The effects of 5HT2A receptor activation in a more intact (and mature) respiratory neuroaxis, and its influence on the spectral dynamics underlying inspiratory motor discharge remains to be identified. Therefore, we examined the effects of 5HT2A receptor activation using DOI (1 μM) on phrenic nerve discharge in 7 arterially‐perfused adult rat preparations. Perfusion with DOI increased phrenic burst frequency from 14.5±2.9 to 31.7±1.9 bpm (P=0.003), which was mediated by a decrease in both TI (from 865+77 to 708+30 ms; P=0.09) and TE (from 4.4+0.9 to 1.3+0.1 s; P=0.015). Phrenic burst amplitude was also decreased during perfusion with DOI. Spectral activity was also modified during perfusion with DOI, such that the onset of both medium (MFO) and high (HFO) frequency oscillations was shifted to and earlier time point in the phrenic burst; power of the MFO peak was also decreased. These findings demonstrate a modulatory role for serotonin acting on the 5HT2A receptor in establishing burst timing and spectral dynamics of phrenic nerve discharge. Supported by NS045321
Lowered set‐point for hypothalamic temperature regulation alters hippocampal activity and may enhance neuroprotection in hibernating hamsters
Authors: Goo Marisa S , Nguyen Yen H , Becker Jeffrey J , Hamilton Jock S , Horowitz John M
Published: June 2021

The hypothalamus regulates temperature in hibernators at all stages of the hibernation cycle. Thus, in addition to direct neural signaling, it may modify neural activity in other CNS regions via its regulation of low brain temperatures. We hypothesized that as temperature falls and hamsters enter hibernation, hippocampal NMDAR channels are arrested, minimizing potential excitotoxic cell damage. To test this hypothesis, we measured in hippocampal slices the generation of long‐term potentiation (LTP), which requires Ca2+ influx through NMDAR channels to activate the process; and dispersion and amplitude of population spikes (PS), because synchronized activity is required to gate NMDARs. Although LTP following tetanus was readily generated (n=10) above 18°C, it could not be established below 18°C (n = 4). In slices from 10 euthermic hamsters, PS amplitude decreased from 1.6 ± 0.1 at 25°C to 0.9 ± 0.1 mV at 15°C; and PS half‐max duration increased from 1.7 ± 0.1 to 4.3 ± 0.4 msec (mean ± SEM, p<.001). In 7 slices from hibernating hamsters, similar changes were seen (PS amplitude fell from 2.1 ± 0.2 to 0.8 ± 0.2 at 15°C, and PS half‐max duration rose from 1.8 ± 0.1 to 5.6 ± 0.6, p<.001). These data are consistent with our hypothesis that cellular pathways activated by Ca2+influx through NMDARs are silenced in hibernating hamsters and may contribute to neuroprotection during hibernation. (Supported in part by a PUF grant to M.G.)
Sex Differences in Resting Hemodynamics and Vascular Distensibility Following Resistance Exercise Training in Hypertensive Individuals
Authors: Collier Scott Robert , Frechette Vincent , Sandberg Kathryn , Smulyan Harold , Tobin Melissa Marie , Fernhall Bo
Published: June 2021

Hypertension is associated with increased arterial stiffness, which leads to ventricular remodeling and increased risk for cardiovascular disease. While exercise can reduce blood pressure (BP) and attenuate the risk of hypertension, few studies have compared the hemodynamic and vascular effects of exercise in both men (M) and women (W). Moreover, while the BP‐lowering effects of aerobic exercise (AE) are well known, the antihypertensive effects of resistance exercise (RE) training (T) are less understood. We studied the effects of 4 weeks of AE and RE on resting hemodynamics (systolic and diastolic BP (SBP & DBP)), and arterial distensibility (pulse wave velocity (PWV)), in 34 pre‐to stage‐1 age‐matched essential hypertensives (21M and 13W, 48.2±7.0 yrs). Both AE and RE significantly decreased SBP (mm Hg) [(pre vs post T): M‐AE, 136±3.5 vs 131±2.9 & M‐RE, 132±2.9 vs 128±2.4; F‐AE, 147±5.5 vs 143±6.4 & F‐RE, 148±7.3 vs 142±8.5] and DBP (mm Hg) [(pre vs post T): M‐AE, 80±1.9 vs 76±1.9 & M‐RE, 78±2 vs 76±1.6; F‐AE, 81±2.5 vs 78±2.6 & F‐RE, 78±3.2 vs 68±3.4] in both sexes. In contrast, RE significantly increased PWV (m/s) in men [(pre vs post T): 11.4±0.86 vs 13.3±0.95] but had no effect in women [(pre vs post T): 10±1.6 vs 10.9±1.7]. These data suggest that both sexes would benefit from the BP‐lowering effects of RE but that hypertensive women have the added advantage compared to men, that RE does not increase arterial stiffness.
Fatty acid induced NF‐κB activation and insulin resistance in skeletal muscle are chain length dependent
Authors: Hommelberg Pascal , Langen Ramon C.J. , Plat Jogchum , M.W.J. Schols Annemie , Mensink Ronald P.
Published: June 2021

Palmitate‐induced insulin resistance has been associated with inflammatory signaling in cultured skeletal muscle cells. The aim of this study was to evaluate if other saturated fatty acids (SFA) also exert these effects on skeletal muscle and whether these relate to chain length. In differentiated L6 GLUT4‐cMyc myotubes, we observed a decrease in insulin stimulated GLUT4‐cMyc translocation to the cell membrane after incubation with the long chain SFAs palmitic (C16:0) and stearic (C18:0) acid. However, incubation with equimolar amounts of the medium chain SFAs caprylic (C8:0) and lauric (C12:0) acid did not affect insulin sensitivity in these cells. In addition, in C2C12 myotubes with a genomically integrated Nuclear Factor kappa B (NF‐κB) reporter gene, C16:0 and C18:0 increased NF‐κB transcriptional activity, while the SFA with shorter chain lengths (C8:0 and C12:0) were not able to elevate NF‐κB transcriptional activity.

In conclusion, we have shown that SFA‐induced insulin resistance only occurs in response to long‐chain but not medium‐chain fatty acids and is associated with NF‐κB activation. The exact mechanisms underlying these differential effects of SFA on insulin resistance and NF‐κB activation remain to be investigated, but this finding supports the notion that inflammatory signaling in skeletal muscle may be regarded as a target for improving or restoring insulin sensitivity.
Effects of acute isometric knee extension exercise at 40% MVC on muscle mechanical properties and Ca2+ pump function
Authors: Stewart Riley David , Sayer Ryan A , Hopf Andrew , Vigna Chris , Bombardier Eric , Tupling A. Russell
Published: June 2021

To investigate the effects of intermittent isometric knee extension exercise on muscle mechanical properties and sarcoplasmic reticulum (SR) Ca2+‐ATPase activity, 8 healthy males performed 30 minutes of single‐legged knee extension exercise at 40% maximal voluntary contraction (MVC) with a 50% duty cycle (5 sec. contraction, 5 sec relaxation). Quadriceps isometric contractile properties were measured during voluntary and evoked contractions before (PRE) and after (POST) exercise and on recovery day 1 (R1), 2 (R2), 3 (R3) and 6 (R6). Exercise caused a reduction (p<0.05) in the maximal twitch force POST when compared with PRE (141 ± 15 vs. 74 ± 8, N) that had recovered by R1 (135 ± 11, N). MVC force was also depressed (p<0.05) POST when compared to PRE (557 ± 58 vs. 313 ± 16), but unlike evoked force, did not fully recover until R6 (p<0.05).Measurements of maximal Ca2+‐ATPase activity (uM/mg protein/min) were made in homogenates that were prepared from muscle biopsy samples taken before and after exercise and throughout recovery. Compared with PRE, maximal Ca2+‐ATPase activity was not different at any time point. In conclusion, muscle fatigue caused by intermittent isometric knee extension exercise at 40% MVC is not associated with intrinsic alterations in SR Ca2+‐ATPase function in humans. Supported by NSERC
Temperature and Doublet Potentiation: Effects of Varied Interspike Interval
Authors: Barnes William Stephen
Published: June 2021

The addition of two closely spaced pulses (doublets) at the beginning of alow‐frequency train of stimuli has been shown to increase peak isometric tension (PT) and tension‐time integral (TTI). This study examined the effects of cooling on potentiation associated with paired pulses having varied (10–100 ms) interspike intervals (ISIs). Sartorii muscles (60–70 mg) from male R. pipiens were dissected free and mounted vertically in water‐jacketed chambers containing oxygenated (100%) Ringer solution (pH 7.2). Muscles were exposed serially to single (SES) and paired (i.e., doublet; DES) electrical stimulation. DES was adjusted to produce ISIs ranging from 10–100 ms. PT (g/g) was measured via field stimulation with supramaximal square wave pulses (1 ms). At 20°C, PT in response to DES (ISI = 100 ms; PTD) was elevated when compared to SES (PTS): 105.7 ± 21.5 v. 79.5 ± 10.0, respectively. Decreasing ISI produced further increases in PTD, with maximum PTD occurring at an ISI of 10 ms: 178.0 ± 34.0 v. 79.5 ± 10.0, respectively. At 10°C, PTS at was increased when compared to 20°C: 120.7 ± 8.1 (10°C) v. 79.5 ± 10.0, respectively. Furthermore, PTD (10°C; ISI = 100 ms) was also elevated when compared to SES (10°C); 196.7 ± 22.5 v. 120.7 ± 8.1, respectively. Decreasing ISI produced additional increases in PTD. However, maximum PTD was developed when paired pulses were actually than 10 ms (i.e., ISI = 40 ms). ISIs < 40 ms failed to produce further increases in PTD. Calculated values for TTI (g·s) were similarly affected. These results suggest that: 1) the potentiating effect of paired stimulation is temperature‐sensitive, and 2) the efficacy of paired stimulation in vivo, as a potential neural control strategy to augment force production, may be mediated by environmental factors.
Hepatocyte Growth Factor Signaling in Mediating Human Muscle Satellite Cell Activation and Proliferation Following Eccentric Exercise
Authors: McKay Bryon Robert , O'Reilly Ciara E. , Kawka Stefany , Phillips Stuart M. , Tarnopolsky Mark A. , Parise Gianni
Published: June 2021

Eight subjects (20.6 ± 2.1 yr; 81.4 ± 9.8 kg) performed 300 isolateral eccentric contractions (knee extensions at 180 °/s) to evaluate the skeletal muscle satellite cell (SC) response and the potential of hepatocyte growth factor (HGF) signaling in mediating SC activation and proliferation following acute damaging exercise. Muscle biopsies were obtained from the vastus lateralis of the non‐exercised leg prior to exercise (PRE) and the exercised leg at 4 (T4), 24 (T24), 72 (T72), and 120 h (T120) post‐exercise. The number of SC (N‐CAM+) increased (p < 0.001) by T4 and remained elevated at T120 (p = 0.002). Serum HGF increased at T4 (p < 0.05) with a trend for increased muscle HGF and HGF activator (HGFA) protein expression at T24 with active HGF detected in all PRE samples. HGFA inhibitors (HAI‐1 and HAI‐2) protein content increased at T72 and remained elevated at T120 (p < 0.05). Myogenic regulatory factors (MyoD and Myf5) mRNA expression increased at T4 and T24 (p < 0.05) respectively, with increased myogenin mRNA expression at T24 (p < 0.05). Furthermore, plasma interleukin‐6 concentration increased ∼200% (p < 0.05) by T4, illustrating a roll of both local and systemic signaling in the activation and proliferation of SC; with HGF signaling playing an important regulatory role in the SC response to exercise‐induced muscle damage.

Supported by NSERC
Mechanisms involved in cAMP mediated inhibition of the Ubiquitin‐Proteasome system
Authors: Lira Eduardo Carvalho , Gonçalves Dawit Albieiro , Zanon Neusa M. , Baviera Amanda M. , Bedard Nathalie , Wing Simon S. , Kettelhut Ísis do Carmo , Navegantes Luiz Carlos
Published: June 2021

Aim: This study was undertaken to investigate the role of beta‐2 adrenoceptors and cAMP in regulating the Ubiquitin‐proteasome system (UPS) in skeletal muscle from normal rats.

Methods and results: The activity of UPS and the Akt/FoxO signaling pathway were measured in skeletal muscle from rats treated with clenbuterol (3 mg/kg wt; sc), a selective beta‐2 adrenergic agonist, for 3 days. In extensor digital longus (EDL) muscle, clenbuterol increased by 30% muscle weight, reduced by 45% the UPS activity and increased by 30% the phosphorylation of Akt and FoxO3. The addition of isobutylmethylxanthine (IBMX; 10−3M), a cAMP phosphodiesterase inhibitor, to the incubation medium increased cAMP levels (4‐fold) and decreased by 50% the UPS activity in isolated soleus and EDL muscles from normal rats. IBMX in vitro also reduced the levels of ubiquitin‐protein conjugated and the mRNA levels of the atrogin‐1/MAFbx (50%) and the E2‐14KDa ubiquitin conjugating enzyme (30%) transcripts in muscles from normal rats. Ubiquitin and MuRF1 mRNA were not altered by IBMX in vitro.

Conclusions: These data suggest that stimulation of beta‐2 adrenoceptors, through the activation of cAMP and Akt signaling pathways, inhibit ubiquitin‐proteasome proteolysis by increasing FoxO3 phosphorylation and suppressing atrogin‐1 mRNA expression in skeletal muscle from normal rats.

Financial support: CNPq, CAPES, FAPESP
A NOVEL REGULATORY ROLE FOR THE LACTATE ION ON EXCITABILITY AND FORCE
Authors: de Paoli Frank Vincenzo , Jørgensen Rasmus , Pedersen Thomas Holm , Ørtenblad Niels , Nielsen Ole Bækgaard
Published: June 2021

We examined the effect of lactate (lac) per se on excitability and tetanic force development. Rat soleus muscles were incubated in KR‐buffer (95% O2) containing 20 mM Na‐methanosulfonate (MS). Lac was added as Na‐lac and an equimolar of MS was omitted to maintain osmolarity and constant Na+ and Cl− concentrations.

Elevation of [K+]o to 11 mM reduced M‐wave area and force in muscles stimulated via the nerve to 16 and 17% of control values at 4 mM [K+]o, respectively. Subsequent addition of 20 mM lac recovered M‐wave area and force to 53 and 70% of control values at 4 mM [K+]o, respectively. In similar experiments with field stimulation, the force recovered from 29 to 88% of control values at 4 mM [K+]o. 20 mM lac reduced pHi from 7.19 to 7.06 within 40 sec, followed by a recovery of pHi to 7.21 over the next 45 min. We have previously shown that a reduction in pHi can lead to force recovery in K+ depressed muscles, it was of interest to estimate to what extend the reduction in pHi after addition of lac was responsible for the recovery of force. The reduction in pHi induced by lac was mimicked by increasing CO2 to 7%, which reduced pHi from 7.19 to 7.04. This pHi reduction, however, only produced a negligible and insignificant recovery of force in muscles depressed by elevated [K+]o. Conclusion: In K+ depressed muscles, a physiological increase in intracellular lac restores excitability and force.

Danish Medical Research Council: j.nr. 271‐05‐0304
Pervanadate‐induced shedding of the intercellular adhesion molecule (ICAM)‐1 ectodomain is mediated by membrane type‐1 matrix metalloproteinase (MT1‐MMP)
Authors: Essick Eric E. , Sithu Srinivas D. , Dean William , D'souza Stanley E.
Published: June 2021

Intercellular adhesion molecule(ICAM)‐1 is a member of the immunoglobin(Ig)‐like family of endothelial cell (EC) adhesion molecules responsible for adhesive interactions with circulating leukocytes, necessary for subsequent transmigration to damaged tissues. In several vascular diseases, the ectodomain of ICAM‐1 becomes cleaved by a zinc‐dependent endopeptidase, releasing a soluble form of the protein (sICAM‐1), a common marker for inflammatory diseases. Since several reactive oxygen species (ROS) generated during conditions of oxidative stress are known to induce endopeptidase‐mediated cleavage of transmembrane proteins, we sought to explore the cleavage and enzymatic effects that the ROS pervanadate, a protein tyrosine phosphatase inhibitor, has on ICAM‐1 cleavage. Our data indicates that pervanadate treatment of ECs and human embryonic kidney (HEK) 293 cells expressing ICAM‐1 results in shedding of its ectodomain. Furthermore, by employing specific tissue inhibitors of metalloproteinases (TIMPs) and small interfering(si) RNA against endopeptidase activity, we found that this cleavage is mediated by membrane type‐1 matrix metalloproteinase (MT1‐MMP). Confocal microscopy provides evidence that ICAM‐1 and MT1‐MMP colocalize at the cellular surface following pervanadate treatment, further implicating the involvement of MT1‐MMP in this mode of ICAM‐1 shedding.
The expression of cofilin‐1 via PKC pathway induced by hyperglycemia in endothelial cells
Authors: Liu Yongming , Wang Yinfang , Wang Fengjie , Deng Juan , Wu Dehong , Su Hui , Zhang Yemin , Wang Nian , Zhang Deling , Ouyang Jingping
Published: June 2021

Cofilin is an important cytoskeletal associated protein which regulated the actin fiber. Our aim is to research the associated relation between the expression of cofilin‐1 and the regulative effect of protein kinase C(PKC) in endothelial cells induced by hyperglycemia.

Methods: Endothelial cell line (ECV304) was routinely cultured in low glucose DMEM medium as control (5mmol/L), and then were treated with high glucose (25mmol/L) and different factors for 24h. Activity of protein kinase C (PKC) within the cells was detected by PepTag non‐radioactive detection assay (Promega). The expression of cofilin‐1 and actin was detected by Western blot, the distribution of actin fiber was labeled by fluorescent probe Alexa‐488 and detected by Confocal (Leica).

Results: The activity of PKC within cells obviously increased (p<0.01), and the expression of cofilin‐1 significantly strengthened after stimulation of hyperglycemia (p<0.01). The cells were pretreated with GF109203X (PKC inhibitor), the activity of PKC which increased formerly by hyperglycemia was apparently inhibited(p<0.01), and expression of Cofilin‐1 was also inhibited(p<0.05). But GF109203X had no apparent effect on the expression of cofilin‐1 and activity of PKC within low‐glucose cultured cells.

Conclusion: The high‐glucose can improve the expression of cofilin‐1 through PKC signaling pathway. It may be an important pathogenesis for diabetic vascular complications.

The research is granted by“ChenGuang Youth Scientific Project of Wuhan”(20015005045).
Role of Connexin40 in Coronary Endothelial Cell Dysfunction in Type 1 Diabetic Mice
Authors: Makino Ayako , Platoshyn Oleksandr , Suarez Jorge , Yuan Jason X.‐J. , Dillmann Wolfgang H.
Published: June 2021

Gap junction intercellular communication (GJIC) in the endothelium plays an important role in regulating vascular tone and in revascularization, although little is known about the contribution of connexins (Cxs, component of GJIC) to vascular complications in the diabetic heart. Coronary endothelial cells (ECs) isolated from diabetic mice exhibit lower protein levels of Cx37 and Cx40 (but not Cx43) and less GJIC than control. Vasodilatation induced by endothelium‐dependent hyperpolarization was significantly reduced in diabetic coronary artery (CA). The Cx40‐specific inhibitory peptide, 40GAP27, attenuated endothelium‐dependent relaxation in diabetic CA at the concentration that does not affect the relaxation in control CA, suggesting that the total amount of Cx40 is lower in diabetic CA than in control CA. In diabetic mice, coronary capillary density was significantly decreased in vivo. In vitro, GJIC inhibitor attenuated EC capillary network formation. High glucose treatment caused a decrease in Cx40 protein expression in ECs and impaired EC capillary network formation. Furthermore, we found that the decrease in Cx40 might be due to the lowered protein expression of Sp1, a transcriptional factor that regulates Cx40 expression. These data indicate that downregulation of Cx40 protein expression and resultant inhibition of GJIC contribute to coronary vascular complication in diabetes.
Role of CYP2C generated metabolites in adenosine‐mediated relaxation using A2A AR−/− mice
Authors: Nayeem Mohammed A. , Poloyac Samuel M. , Falck John R. , Zeldin Darryl C. , Ledent Catherine , Mustafa S. Jamal
Published: June 2021

We hypothesize that A2A‐adenosine receptor (A2A AR)‐mediated relaxation is caused by CYP2C generated metabolites whereas, lack of A2A AR promotes contraction via CYP4A in mouse aorta. We measured EETs and DHETs in A2A AR+/+ /A2A AR−/− aorta. DHETs were significantly higher in A2A AR+/+ vs. A2A AR−/− aorta (14,15‐DHET: 1.66±0.25 vs. 0.83±0.17; 11,12‐DHET: 1.39±0.20 vs. 0.84±0.12; 8,9‐DHET: 0.89±0.15 vs. 0.35±0.08 pmol/mg/min p<0.05). No significant differences were observed in EET levels in A2A AR+/+ vs. A2A AR−/− aorta. CYP2C29 was higher in A2A AR+/+ aorta (p<0.05) while CYP4A was higher in A2A AR−/− aorta (p<0.05). CYP2C inhibitor, 1‐ABT (5μM) changed the response to NECA (10‐6M) from relaxation (+33.99±4.70%) to contraction (25.70±4.75%, p<0.05) in A2A AR+/+. Similar results were obtained in A2A AR+/+ to NECA at 10‐6 M with selective CYP2C inhibitor MS‐PPOH (10μM, 22.74±5.11%, p<0.05). 1‐ABT also changed the response of CGS 21680 (10‐6M) from relaxation (+28.44±4.36%) to contraction (18.60±4.66%, p<0.05) in A2A AR+/+. In contrast, no effect was seen in A2A AR−/− with 1‐ABT and MS‐PPOH. MS‐PPOH also caused contraction to CGS 21680 at 10‐6M (−18.54±6.06%, p<0.05) in A2A AR+/+, while 1‐ABT and MS‐PPOH were without an effect in A2A AR−/−. The data show that CYP2C generated metabolites play an important role in A2A AR‐mediated relaxation, whereas, in A2A AR−/−, CYP4A induces contraction. Supported by HL027339, GM31278, NIEHS, NS052315, S10RR023461, S10RR023461 & NS052315.
Post‐transcriptional regulation of inducible nitric oxide synthase (iNOS) by Protein Interacting with Never in Mitosis Gene A‐1 (PIN1) in murine aortic endothelial cells
Authors: LIU TONGZHENG , Likhotvorik Rostislav , Huang Yongcheng , Keshvara Lakhu , Hoyt Dale G
Published: June 2021

PIN1, a peptidyl‐proline isomerase, increases the level or activity of several transcription factors that can induce iNOS. Thus it was hypothesized that depletion of PIN1 would reduce induction of iNOS by lipopolysaccharide (LPS) and interferon‐gamma (IFN). Murine aortic endothelial cells (EC) were stably transfected with a vector producing a PIN1 short hairpin RNA (shRNA) or an inactive control sequence. PIN1 shRNA reduced PIN1 levels >85% compared to control. Unexpectedly, suppression of PIN 1 enhanced NO production in response to LPS/IFN, and iNOS protein was induced 4.7‐fold more. The increased iNOS was cytotoxic to EC lacking PIN1, but not to control EC, in a crystal violet staining assay. Furthermore, cytotoxicity was prevented by a nitric oxide synthase inhibitor, L‐NAME. LPS/IFN increased iNOS/β‐actin mRNA ratio as expected, but PIN1 knockdown did not affect iNOS mRNA levels measured by RT‐PCR. Instead, PIN1 depletion stabilized iNOS protein in cycloheximide‐treated EC such that the time to a 50% decrease was extended from 8 h to more than 24 h. The loss of iNOS was antagonized by the calpain inhibitor, MDL 28170, but not by the selective proteasome inhibitor, epoxomicin. These results suggest that PIN1 normally restrains the induction of iNOS by facilitating its calpain‐mediated degradation in EC. Modulation of PIN1 may significantly affect iNOS‐dependent vascular activity and inflammatory diseases.
Regulation of calpain and calpastatin by Protein Interacting with Never in Mitosis Gene A‐1 (PIN1) in murine aortic endothelial cells
Authors: Liu Tongzheng , Likhotvorik Rostislav , Huang Yongcheng , Keshvara Lakhu , Hoyt Dale G
Published: June 2021

The peptidyl‐proline isomerase, PIN1, regulates protein turnover and function. We found that PIN1 normally restrains the induction of iNOS by facilitating calpain‐mediated degradation in murine aortic endothelial cells (EC), suggesting that PIN1 facilitates calpain activity in EC. To test this theory, EC were stably transfected with a vector producing a PIN1 short hairpin RNA (shRNA) or an inactive control sequence. PIN1 shRNA reduced PIN1 levels >85% compared to control. Calpain activity in cell extracts was measured with a fluorescently quenched substrate, (DABCYL)‐TPLK~SPPPSPR‐(EDANS). Incubation of control EC with 10 μg LPS and 20 ng IFN for 24 h increased the initial velocity of cleavage by >5.8 fold. Activity in EC lacking PIN1 was only 45% of the control cells, with or without LPS/IFN treatment. However, there was no reduction of Calpain‐1 or ‐2 protein levels by western blotting. Instead, calpastatin, an endogenous protein inhibitor of Calpain‐1 and ‐2, was increased >2.5‐fold in PIN knockdown cells. PIN1 depletion did not affect calpastatin mRNA levels, suggesting a post‐transcriptional effect. The results suggest that PIN1 normally enhances calpain activity by post‐transcriptional suppression of calpastatin. Alterations in PIN1 expression or function may significantly affect a broad range of calpain‐dependent processes, particularly in EC.
Effect of ozone on nitric oxide synthases gene expression in endothelial cells
Authors: Castillo Carmen , Villanueva Cleva , Medina‐Santillan Roberto , Floriano Esau , Mera Elvia , Castillo‐Henkel Carlos
Published: June 2021

The goal was to explore the effects of ozone (O3) on gene expression of nitric oxide synthases (NOSs) in endothelial cells (EC) isolated from rat aorta. EC grew until confluence and then they were exposed (1h) to 1ppm of O3 or to 0.05 ppm of O3 (to assess the effect of separating the cells from the culture environment) under sterile conditions. After O3 exposure EC were incubated within normal culture environment during 3, 6 or 9 hs. mRNA was determined by means of real time RT‐PCR. O3 at 0.05 ppm significantly decreased the expression of NOSs after 6 h (p<0.05, compared to basal expression) with a partial recovery after 9 h. O3 exposure at 1ppm also decreased NOSs expression but it happened earlier (3 h, p<0.001 compared to basal), was more significant at 6 h (p<0.001) and greater than the effect produced by 0.05 ppm of O3 (p<0.01), and it had also a partial recovery after 9 h. It is concluded that O3 inhibits NOSs gene expression in isolated EC, which is different from the effect on the whole animal where O3 decreases the vascular endothelial and neuronal NOS while increases the inducible isoform, probably because of the systemic induction of inflammatory mediators by O3
Hypochlorous acid stimulates heme oxygenase‐1 gene expression in vascular endothelium
Authors: Wei Yong , Liu Xiao‐ming , Peyton Kelly J. , Wang Hong , Durante William
Published: June 2021

Hypochlorous acid (HOCl) is the major product of the enzyme myeloperoxidase which is active in human atherosclerotic lesions and often closely associated with endothelial cells. HOCl is a potent antimicrobial oxidant that plays an important role in host defense; however, it may also contribute to tissue injury. In the present study, we examined whether HOCl regulates the expression of the cytoprotective protein heme oxygenase‐1 (HO‐1) in human umbilical vein endothelial cells (HUVEC). Treatment of HUVEC with HOCl (3–300μM) stimulated a concentration‐ and time‐dependent increase in HO‐1 protein. A significant rise in HO‐1 protein was first detected after 4 hours and levels progressively increased over 24 hours of HOCl exposure. HOCl‐mediated increases in HO‐1 protein were preceded by significant elevations in HO‐1 mRNA that peaked 4 to 8 hours after HOCl treatment. The induction of HO‐1 by HOCl was dependent on de novo RNA synthesis since it was blocked by the transcriptional inhibitor, actinomycin D (2ìg/ml). In addition, HO‐1 expression was inhibited by the antioxidant N‐acetyl‐L‐cysteine (10mM). In conclusion, this study demonstrates that HOCl stimulates HO‐1 gene expression in vascular endothelium via a transcriptional‐ and redox‐sensitive pathway. The ability of HOCl to induce HO‐1 may represent a critical adaptive response to preserve cell viability at sites of atherosclerosis. Supported by NIH.
Rat thoracic vena cava ETB receptors re‐sensitize faster than venous ETA receptors
Authors: Thakali Keshari , Gariepy Cheryl E. , Galligan James J. , Fink Gregory D. , Watts Stephanie W.
Published: June 2021

We previously demonstrated that aorta completely and vena cava partially desensitize to ET‐1. We hypothesized that vena cava from male ETB receptor deficient rats (sl) would completely desensitize to ET‐1, like aorta from male wild‐type (WT) rats because they both lack functional ETB receptors. Initial maximal contraction to ET‐1 (100 nM) was not different between vena cava from sl and WT rats [sl: 409±65% norepinephrine (10 μM) contraction; WT: 499±68%]. After washing out the first ET‐1 challenge, the second maximal contraction to ET‐1 (100 nM) was similarly reduced compared to the first between vena cava from sl and WT rats (sl: 99±37%; WT: 97±7%). Vena cava from male Sprague Dawley rats were contracted with the ETB receptor agonist sarafotoxin 6c (S6c, 100 nM, 15 minutes), the S6c was washed out for 15 and 30 minutes, and then challenged again with S6c (100 nM). After 15 minutes of washing, vena cava did not contract to the second S6c challenge. After 30 minutes of washing, the contraction to the second S6c challenge was the same as the initial S6c‐induced contraction [initial: 145±20% norepinephrine (10 μM) contraction; 30 min: 139±38%]. Our data demonstrate that the venous ETB receptor does desensitize, and is not involved in remaining venous ET‐1‐induced contraction after desensitization. Our data also suggest that the mechanism of ET receptor desensitization differs between arteries and veins.
Nongenomic effects of T3 on NO production in vascular smooth muscle cell (VSMC)
Authors: Sepulveda Maria Alicia Carrillo , de Chaves Maria Luiza Morais Barreto
Published: June 2021

Thyroid hormone (T3) has a broad effect on the vascular system. It is known as a vasodilator that acts directly on VSMC causing a rapid relaxation by a nongenomic action. However, the molecular basis of vascular relaxation and signal transduction in VSMC promoted by T3 is unclear. The purpose of this study was to investigate the rapid effect of T3 on nitric oxide (NO) production, potent vasodilator gaseous. Considering that AKT signaling can be involved in the NO production, we also evaluated whether the T3 activates the AKT. Methods: Rat aortas VSMC culture, passage 3th, were treated with 0,1uM T3 for 5, 10, 20, 30 and 60 min. Production of NO was assessed by NO‐specific fluorescent dye DAF‐2 and phosphorylated AKT levels by Western Blot. Statistical analysis was performed by Student T test and one‐way ANOVA. p<0.05 was considered as statistically significant. Results:Treatment of VSMC for 30min with T3 stimulated NO production (p<0,001) in 6 fold of control, which was accompanied of increase on AKT phosphorylation levels (p<0,01) in residue serine 473. This activation AKT was noted since 10min (p<0,001) and sustained until 30min. Conclusion: These results demonstrated that T3 is able to stimulate the NO production by nongenomic and endothelium‐independent mechanisms in VSMCs. Moreover, these findings suggest, in part, that AKT signaling activation could be mediating this rapid vascular effect of T3 in the NO production.
A new and simple method for detecting non‐invasive vascular endothelial function in mice
Authors: Furuta Tomoaki , Matsushita Shonosuke , Sugimori Haruhiko , Sato Fujio , Sakakibara Yuzuru
Published: June 2021

Purpose: The importance of non‐invasive measuring of vascular endothelial function is widely recognized. However, it has not been established in mice as far as we know. From this background, we tried to establish a new method to measure a vascular endothelial function through nitric oxide (NO) mediated vasodilation.

Method: Mouse hindlimb (n = 6) was compressed for a short time (30 seconds) and released immediately. The arterial blood flow was measured continuously using laser Doppler flow meter at arcus plantaris.

Result: Reactive hyperemia (127+/−10% from baseline) at reperfusion was reproducibly‐observed. This reaction was mostly inhibited by L‐NAME (5mg/kg i.p.) (107+/−5%, p<0.005 vs. without L‐NAME). And it was completely‐recovered by supplying exogenous NO (122+/−16%, p<0.05 vs. after L‐NAME). Therefore, this method was considered to reflect NO‐mediated endothelial function. On the other hand, this was not inhibited by non‐selective cyclooxygenase inhibitor indomethacin (IDM; 20 mg/kg i.p.) (baseline: 120+/−11 vs. after IDM: 128+/−10%, p:NS).

Conclusion: From these results, we confirm that this method is a new and simple technique measuring NO‐mediated endothelial function non‐invasively in mice.
Combined epigenomic and genomic approach to hypertension
Authors: Michalkiewicz Mieczyslaw , Michalkiewicz Teresa , MacGillis Kyle
Published: June 2021

DNA methylation of cytosine (CpG) is the best understood epigenetic information that may silence the gene directly by inhibiting binding of transcription factors or indirectly by chromatin modification. This epigenetic variation may underlie hypertension and in fact it may explain the late onset, progressive and quantitative nature of this disease better than variation in DNA sequence. We hypothesized that epigenetic variations in CpG methylation are of genetic origin and contribute to the development of hypertension in Dahl salt‐sensitive (SS) rats. Genome‐wide methylation patterns in the SS and normotensive Brown Norway (BN) rats were assessed in a high throughput manner by combining methylated DNA immunoprecipitation with a competitive hybridization on a high‐density microarray (NimbleGene) covering 2.7 kb promoter area of 21,600 genes. More than 1,000 targets were hypermethylated in the kidney of SS rats as compared to BN. The hearts methylomes were more similar between the strains. Hypermethylation of the SS genome was confirmed by LUMA. We then zoomed in on the known hypertension QTLs, congenic areas and further narrowed down the hypermethylated targets, which included renin. Using pyrosequencing of bisulfite converted DNA we determined quantitatively that comparing to BN, over ten CpGs were significantly (p<0.05) hypermethylathed in the renin promoter of the SS kidney, consistent with the reduced renin expression in this strain. Thus, we identified genome‐wide differentially methylated genomic regions between hypertensive and normotensive genomes and suggest that epigenetic mechanism may underlie the salt sensitive hypertension. Support: NIH HL 082798, AHW MCW.
Impaired arterial β‐adrenergic receptor function in DOCA‐salt hypertension
Authors: Xu Hui , Galligan James J
Published: June 2021

Vascular tone is regulated by constrictor α1‐adrenergic receptors (ARs) and β‐AR‐mediated dilation. Increased constrictor reactivity for norepinephrine (NE) in mesenteric arteries (MA) contributes to increased vascular tone in DOCA‐salt hypertension. These studies examined if these alterations are due to reduced β‐AR function. The MA from sham and DOCA‐salt hypertensive rats were pressurized at 80 mmHg, α1‐ and β‐AR function was tested using computer‐assisted video‐microscopy to measure agonist induced changes in diameter in vitro. Constrictor activity of NE was similar in sham and DOCA‐salt MA, but EC50s for NE were reduced in sham, but not DOCA‐salt MA after blockade of β‐ARs by propranolol. β‐AR dilator function was tested in serotonin‐preconstricted MA, α1‐ARs were blocked by prazosin. Agonist‐induced relaxation was normalized to that caused by papaverine. In DOCA‐salt MA, EC50s for NE, epinephrine, and isoproterenol were similar to sham MA; but Emax for all agonists was reduced. Western blot and immunohistochemical studies in isolated smooth muscle cells revealed similar expression of β1‐ and β2‐AR in MA from sham and DOCA‐salt rats. Our results indicate that there is reducedβ‐AR vasodilator activity in DOCA‐salt hypertension. Reduced β‐AR function is not due to reduced β‐AR expression. Reduced β‐AR dilator function may contribute to increased sympathetic vasoconstrictor tone known to occur in DOCA‐salt hypertension.
Dietary calcium restriction induces salt‐sensitive hypertension in normal rats
Authors: Beierwaltes William H. , Pavle Alexis , Potter D’Anna L.
Published: June 2021

Numerous studies report an inverse relationship between blood pressure and calcium (Ca) intake, such that increased dietary Ca retards the development of salt‐sensitive hypertension in humans and animal models. We hypothesized the converse; that dietary Ca restriction would exaggerate the increase in blood pressure in response to a high sodium (Na) diet. 18 young Sprague Dawley rats were placed on isocaloric diets over 7 weeks, containing either normal Na (0.22%)/low Ca (0.15%), high Na (3.2%)/ normal Ca (1.36%), or high Na/low Ca. Systolic blood pressure (SBP) was measured by tail‐cuff plethsmography. All rats grew at the same rate (32 ±1 g/week). Baseline SBP was 129 ±2 mmHg. In normal Na/low Ca rats SBP increased 9 ±3 mmHg over the first 3 weeks, then returned to baseline. In high Na/normal Ca rats, SBP increased by 20 ± 4 mmHg over the first 3 weeks (p<0.05), returning to baseline by week 6. The combination of high Na/low Ca evoked a 26 ±3 mmHg increase in SPB over the first 3 weeks (p<0.05) and remained elevated by 15 ±2 mmHg after 7 weeks on this diet (p<0.05). Plasma renin activity was supressed by high Na diet similarly without or with low Ca (4.3 ±1.0 vs. 1.4 ±0.3* vs. 1.2 ±0.3* ngAngI/ml/hr, respectively, *p<0.005). Our findings suggest that dietary Ca restriction exaggerates and sustains the hypertensive response to excess dietary Na, inducing a salt‐sensitivity in otherwise normal rats.
Dynamic renal autoregulation in conscious, freely moving mice
Authors: Iliescu Radu , Cazan Radu , McLemore Gerald R. , Reckelhoff Jane F. , Ryan Michael J.
Published: June 2021

Myogenic (MYO) and tubuloglomerular feedback (TGF) mechanisms control renal blood flow (RBF) autoregulation. MYO and TGF operate with specific dynamic properties to regulate RBF in response to spontaneous fluctuations in blood pressure (BP). Developing a method to evaluate MYO and TGF control systems in conscious mice would allow RBF autoregulation to be studied in available genetic models. Using spectral analysis of BP and RBF, we identified dynamic operational characteristics of MYO and TGF in conscious, freely moving mice (NZW, n=10), instrumented with ultrasound flow probes and arterial catheters. We found that MYO generates a distinct resonance peak in transfer gain at 0.31±0.01 Hz. MYO‐dependent attenuation of RBF oscillations, indicative of active autoregulation, is apparent as a trough in gain below 0.3 Hz (−6.3±1.5 dB) and a strong positive phase peak (87±8 deg), which are abolished by amlodipine infusion (3.3±1.1 dB p<0.01; 14±1 deg p<0.01). Operation of TGF produces a local maximum in gain at 0.77±0.8 Hz and a positive phase peak (60.4±14.3 deg), both of which are removed by infusion of furosemide. Administration of amlodipine eliminated both MYO and TGF signature peaks, whereas furosemide did not alter MYO operational characteristics. These data indicate that MYO and TGF dynamics may be used to investigate the effectiveness of renal autoregulatory mechanisms in conscious mice. (Supported by R01HL085907 MJR, R01HL069194 JFR)
Plasma catecholamine responses are consistent among common laboratory stressors
Authors: Elvebak Rachel L. , Liu Z. , Hesse C. , Marrero FA Ramirez , Pike TL , Gleich SJ , Eisenach JH
Published: June 2021

Cardiovascular reactivity to stress may be predictive of cardiovascular disease. Sympathoexcitatory responses to mental stress (MS), cold pressor test (CPT), isometric handgrip (HG) and head‐up tilt (HUT) target different physiological mechanisms, but the correlation between response variables is unclear. Our purpose was to test the hypothesis that pressor and catecholamine responses between these stressors would be correlated within subjects. Healthy, normotensive young adults (n=76) underwent MS, CPT, HUT and HG to fatigue. Arterial pressure and HR were monitored, and arterial Epi and NE were sampled during the final 30 sec of each stressor. Percent change from baseline was averaged during MS, CPT, HUT and the final 20% time segment of HG. MAP and SBP were positively correlated between CPT and HG (r = 0.46, 0.52, resp, p < 0.001) but not among other tests. Interestingly, the % increase in NE response to all 4 maneuvers showed robust correlations (MS v CPT r = 0.65; MS v HG r = 0.74; MS v HUT r = 0.53; CPT v HG r = 0.72; CPT v HUT r = 0.68; HG v HUT r = 0.59; p < 0.001 for all). Similar correlations were present for Epi except for comparisons with HUT. This suggests that among the 4 tests, HR and BP responses are generally variable, but NE and Epi responses are remarkably consistent (except Epi for HUT), implying that lab stressors may evoke sympathoexcitation more similarly than previously thought.

Support: K23‐RR17520, HL 46493.
Soluble Flt‐1 induces hypertension and vascular dysfunction in pregnant rats
Authors: Bridges Jason P , Gilbert Jeffrey S , Colson Drew , Dukes Matthew , Babcock Sara A , Ryan Michael J , Granger Joey P
Published: June 2021

The balance between pro‐ and anti‐angiogenic factors such as vascular endothelial growth factor (VEGF) and soluble Flt‐1 (sFlt‐1) is altered in preeclampsia (PE) and may be a contributing factor in the development and progression of the syndrome. We hypothesized chronic infusion of sFlt‐1 into pregnant rats to mimic the increase observed in PE would reduce plasma VEGF concentrations, restrict feto‐placental growth, increase arterial pressure (AP) and cause vascular dysfunction in the pregnant rat. Concentration of plasma sFlt‐1 was increased (299±33 vs. 100±16 pg/ml; P<0.01) and free VEGF (570 ± 77 vs. 780 ± 48 pg/ml; P<0.01) decreased in sFlt‐1 compared to vehicle infused dams. AP was increased (117±6 vs. 98±4 mm Hg; P<0.01) while fetal (1.4±0.03 vs. 1.6±0.04, P<0.01) and placental (0.50±0.01 vs. 0.45±0.01, P<0.01) weight were decreased in the sFlt‐1 group compared to the vehicle group. Vasorelaxation to acetylcholine (ACh) and sodium nitroprusside were decreased (P<0.05) and the Hill slope of the ACh dose response curve was altered (P<0.05) in the sFlt‐1 group compared to the vehicle group. These findings support our hypothesis that chronic VEGF antagonism via sFlt‐1 results in vascular dysfunction and increased AP. These data indicate that elevated maternal concentrations of sFlt‐1 during pregnancy have deleterious effect on vascular function and may contribute to the hypertension observed in preeclampsia.
The ischemic placenta of pregnant rats elaborates factors that inhibit endothelial tube formation
Authors: Dukes Matthew P , Roberts Lyndsay , LaMarca Babbette B , Gilbert Jeffrey S , Granger Joey P
Published: June 2021

Dysregulation of the balance between pro‐ and anti‐angiogenic factors such as vascular endothelial growth factor and soluble fms‐like tyrosine kinase 1 (sFlt‐1) may be important in the pathogenesis of preeclampsia. We hypothesized that factors, such as sFlt‐1, emanate from the ischemic placenta and cause endothelial cell (EC) dysfunction such as decreased tube formation, a marker of angiogenesis. To this end, we collected serum from our well characterized rat model of reduced uterine perfusion pressure (RUPP), normal pregnant + sFlt‐1 infused via osmotic mini‐pump (sFlt‐1), and normal pregnant (NP) rats. To determine the effects of placental ischemia and sFlt‐1 on EC tube formation, HUVECs were plated onto 96 well Matrigel coated plates and exposed to 50% media/serum from NP, RUPP and sFlt‐1 rats for six hours. Tubes were counted and expressed as tubes/section/well. Both RUPP and sFlt‐1 infused rats demonstrated ∼3 fold elevations of serum sFlt‐1 concentrations compared to NP dams. EC tube formation was decreased 76% and 48% by RUPP, and sFlt‐1serum compared to control media. These data indicate that both RUPP and sFlt‐1 infused serum inhibit EC tube formation to different degrees. Thus, it appears that the ischemic placenta generates additional factors that work in concert with sFlt‐1 to impair tube formation. We conclude that placental ischemia leads to endothelial dysfunction that is mediated in part by sFlt‐1.
Transcriptional repression by non‐coding RNA regulators of the mammalian heat shock response
Authors: Goodrich James A , Mariner Peter , Walters Ryan D , Wagner Stacey D , Yakovchuk Petro , Espinoza Celso A , Kugel Jennifer F
Published: June 2021

We have found that mouse B2 RNA and human Alu RNA are transacting transcriptional repressors of RNA polymerase II. In response to heat shock, these ncRNAs are transcribed by RNA polymerase III from short interspersed elements (SINEs) that are abundant in the genomes and prototypically considered ‘junk’ DNA. B2 RNA and Alu RNA bind to core RNA polymerase II with high affinity and enter stable complexes at promoters of specific genes, where they repress transcription. Structure/function studies revealed that B2 RNA and Alu RNA contain two types of functionally distinct domains: Pol II binding domains and transcriptional repression domains. Our studies lead to a model in which Pol II binding domains in ncRNA transcriptional repressors bind a high affinity docking site on RNA polymerase II, thereby tethering distinct repression domains to the polymerase in order to block transcription. Surprisingly, the functional domains in SINE ncRNAs, much like those of classical protein transcriptional regulators, are modular and can be mixed and matched to create chimeric ncRNA transcriptional repressors. Thus, SINE‐encoded ncRNAs resemble prototypical protein regulators of eukaryotic transcription. Our work provides an explanation for how RNA polymerase II transcription is repressed during heat shock in mouse and human cells, and provides an evolutionary rationale for the maintenance of SINEs in the mouse and human genomes.