bluuebunny/crossref_metadata_2025 huggingface dataset:
The 5‐HT1A receptor and sympathetic neurotransmission to mesenteric blood vessels in salt‐sensitive hypertension
Authors: Bomar Jonathan David , Galligan James J.
Published: June 2021

Salt‐sensitive hypertension is prevalent U.S. and elsewhere. Salt acts in the brain to activate sympathetic nerves which supply the heart and blood vessels. Increases in sympathetic nerve activity cause high blood pressure (hypertension). Serotonin modulates blood vessel tone in the mesentery, a major contributor to peripheral resistance and venous return. Previous studies showed that sympathetic α2‐adrenergic autoreceptor function is impaired leading to increased norepinephrine release and vasoconstriction in deoxycorticosterone acetate (DOCA)‐salt rats. We tested the hypothesis that there is also impaired function of 5‐HT1A receptors on sympathetic nerve terminals in the mesentery leading to increased vasoconstriction. We used computer assisted video microscopy to measure in vitro changes in mesenteric blood vessel diameter caused by transmural nerve stimulation in the presence of 8‐OH‐DPAT, a 5‐HT1A receptor agonist. In sham (normotensive) arteries and veins, 8‐OH‐DPAT produced a dose‐dependent inhibition of constriction. There was a significant rightward shift in response in DOCA‐salt arteries and veins (p<.05). These findings suggest that impaired 5‐HT1A receptor function in the mesentery may contribute to a loss in regulation of sympathetic activity. This may be a mechanism by which pathology is maintained in salt‐sensitive hypertension. (Supported by P01HL070687)
Glucoprivation of hypothalamic neurons elicits the counter‐regulatory response in rats
Authors: Verberne Tony J , Korim Willian S
Published: June 2021

Whether the sympathetic counter‐regulatory response to hypoglycaemia is activated by glucose sensing neurons in the fore‐or hind‐brain remains controversial, since systemic hypoglycaemia excites neurons located in both areas. Here, we hypothesize that neurons in the perifornical hypothalamus (PeH), but not in the rostral ventrolateral medulla (RVLM), sense the reduction in glycaemia and increase the adrenal sympathetic nerve activity (ASNA). Experiments were conducted in anaesthetized (urethane, 1.2 g/kg i.v.), paralysed (pancuronium bromide, 1 mg/kg i.v.), and artificially ventilated male Sprague‐Dawley rats. Local glucopaenia, induced by bilateral microinjections of 2‐deoxy‐D‐glucose (2DG; 15 ng/50 nl) into the PeH, increased ASNA (101±1% vs 175±11%, N=10; P<0.05) and blood glucose (6.6±0.3 vs 7.6±0.3 mmol/l, N=9; P<0.05). A higher dose of 2DG (150 ng/50 nl) further increased ASNA (157±7% vs 280±59%, N=6; P<0.05) and blood glucose (7.5±0.5 vs.9.6±0.5 mmol/l, N=4; P<0.05). Microinjections of 2DG (15 ng/50 nl) into the RVLM (N=6) neither changed ASNA (101±1% vs 84±14%) nor blood glucose (6.4±0.1 vs 6.9±0.1 mmol/l). Subsequent intravenous infusion of 2DG (250 mg/kg) increased ASNA (101±1% vs 183±8%; P<0.05). The results indicate that neurons in the PeH, but not in the RVLM, are glucose sensitive and their excitation produces adrenaline release in response to glucoprivation.
Taste without calories is insufficient to drive conditioned flavor preferences
Authors: Stratford Jennifer M , Finger Thomas E
Published: June 2021

The preference for a food is dependent primarily upon the interplay between oral and post‐oral factors. The relative contribution of these two systems in driving food intake is not fully explored. We previously found that mice that lack taste capabilities, via genetic deletion of the P2X2 and P2X3 purinergic receptor subunits (P2X‐KO), can form a preference for a monosodium glutamate (MSG) solution using only post‐ingestive cues (Stratford et al., 2011). Whether this effect depends upon caloric content or other nutritive qualities is unknown. Therefore, the current study determined whether P2X‐KO mice can form a preference for the calorie‐free artificial sweetener, SC45647. To test this, WT and P2X‐KO mice were given eight training sessions on alternating days either with an odor alone or with a different odor mixed with 0.5 mM SC45647. Then all animals were given two‐bottle preference tests with the bottles each containing one odor but neither containing SC45647. Throughout training, WT animals drank more SC45647 than did P2X‐KO mice, suggesting that WT, but not P2X‐KO mice, can taste SC45647. However, neither WT nor P2X‐KO animals preferred the odor that had been paired previously with SC45647. Thus, taste, in the absence of caloric content, is not sufficient to drive a conditioned food preference. Supported by NIH and 3ARP grants to TEF, JMS.
Carotid artery intima‐media echogenicity and aortic stiffness in healthy middle‐aged and older humans
Authors: Aizawa Kunihiko , Elyas Salim , Adingupu Damilola D , Casanova Francesco , Gooding Kim M , Mawson Dave M , Shore Angela C , Strain W David , Gates Phillip E
Published: June 2021

Echogenic carotid plaque has been shown to be related to aortic stiffness. However, whether a similar relationship is observed between carotid artery intima‐media complex echogenicity (IM‐GSM) and aortic stiffness is not known. This study examined the relationship between IM‐GSM and aortic stiffness in 94 healthy middle‐aged and older individuals (62.9±8.2 yrs, 50F). B‐mode ultrasound images of the common carotid arteries were analyzed by semi‐automated software for IM‐GSM and intima‐media thickness (IMT), and aortic stiffness was assessed by carotidfemoral pulse wave velocity (cfPWV) using applanation tonometry. In univariate analysis, age (r=−0.22), body mass index (r=−0.31), IMT (r=−0.56), mean arterial pressure (r=−0.35), fasting glucose (r=−0.31), and cfPWV (r=−0.41) were all inversely associated with IM‐GSM (all p<0.05). In multivariate analysis, cfPWV was an independent determinant of IM‐GSM together with IMT, body mass index and fasting glucose (total R2=0.381). These results demonstrate that IM‐GSM is inversely associated with aortic stiffness, suggesting an interaction between arterial stiffness and the composition of the carotid artery intima‐media complex with ageing.
Measurement of heart function for myocardial infarction model of rat by micro‐CT
Authors: Matsushita Satoshi , Naito Mayuko , Amano Atsushi
Published: June 2021

Materials and Methods

We demonstrated functional measurement with no (normal) and mild or severe myocardial infarction of the rat heart using micro‐computed tomography (μCT) with administration of contrast agent. The hearts were sectioned to assess pathological evaluation of the heart following with CT acquiring.

Results

The plot curve represented by each disk volume in normal heart revealed that contraction force was peaked at middle of the heart. In the mild infarcted heart, the volume plotting curve clearly demonstrated that infarcted area was limited at the apex of the heart, whereas that in severe infarction was in larger area. The left ventricular ejection fraction in normal, mild and severe was 68.6%, 40.0% and 16.4%, respectively. In addition, the volume analysis in severe infarction at seven days after injury demonstrated ventricular dilatation, which is consisted with histological evaluation.

Conclusions

Functional analysis of rat heart by μCT was accurate. This method is useful especially in small animal with heterogeneous dysfunction of the heart, since it can be analyzed with whole heart data.
β‐arrestin‐mediated signal transduction participates in laminar shear stress‐induced production of nitric oxide in endothelial cells
Authors: dos Santos Ana Paula Carneiro , Barauna Valério Garrone , Alaniz Miriam Helena Fonseca , Girardi Adriana Castello Costa , Krieger José Eduardo
Published: June 2021

Endothelial cells are capable of converting mechanical stimuli into intracellular signals generating vasoactive factors such as nitric oxide (NO). A great body of evidence suggests that β‐arrestins play a role not only on GPCR desensibilization but also in mechanotransduction. The aim of this study was to test the hypothesis that laminar shear stress (SS)‐induced NO production by endothelial cells requires the activation of the β‐arrestin signaling pathway. Towards this end, human saphenous vein endothelial cells (SVEC) were transfected with siRNA against the isoforms 1 and 2 of β‐arrestins and subsequently subjected to SS (15 dynes/cm2) for 10 minutes. We found that the SS‐induced production of nitrite in the cell culture medium was significantly decreased in SVEC silenced for β‐arrestin 1 and 2 compared to control cells (326±44 vs. 166±17%; P < 0.001, n=5). Furthermore, silencing of β‐arrestin 1 and 2 significantly attenuated the phosphorylation levels of AKT at the serine residue 473 (99±22 vs. 293±42% in control; P <0.001, n=5) and led to a non‐significant trend towards an increase of ERK phosphorylation (157±71 vs.107±5% in control; P= 0.3670, n=5). Altogether, we provide evidence that β‐arrestins play a role on laminar SS‐induced generation of NO associated with AKT activation in endothelial cells.

Supported by FAPESP, CAPES and CNPq.
Adipose tissue decrease by exercise training is associated with blood pressure reduction in old fructose‐fed female rats
Authors: Machi Jacqueline Freire , Bernardes Nathalia , Dias Danielle Silva , Santos Fernando , Moraes‐Silva Ivana C , Sartori Michelle , Wichi Rogério Brandão , De Angelis Kátia , Irigoyen Maria Claudia
Published: June 2021

The aim of the present study was to investigate the effects of exercise training (ET) in old female Wistar rats (24 months old). They were ovariectomized, fed with fructose (100g/L) in drinking water and divided in 2 groups (n=6): sedentary (S) or trained (T). ET was performed on a treadmill (1 h/day; 5 days/wk for 8 wk). Glycemia, triglycerides, adipose tissue, mean blood pressure (MBP), and heart rate variability were evaluated at the end of the protocol. The metabolic results showed that ET decreased only the adipose tissue (T: 3.45±0.45 vs. S: 8.43±1.23 g). ET reduced MBP (T: 106±1.4 vs. S 117±3.8 mmHg) and increased the pulse interval variance (T:42.1±11 vs. S:33.2±5.5 ms). Cardiac sympathetic modulation (LF) was decreased in T (16.0±5.6 vs. S: 31.5±6.5 n.u.) while cardiac vagal modulation (HF) was increased (T: 84±5.6 vs. S: 68±6.5 n.u). Consequently, sympatho‐vagal balance was decreased in T (0.21±0.09) vs. S rats (0.56±0.16). A similar behavior was observed for sympathetic modulation of blood pressure (T: 5.12±1.5 vs. S: 14.9±8.1 mmHg2). Finally, we observed a direct and positive correlation between adipose tissue and MBP (r2=0.8), LF of pulse interval (r2=0.45) and LF of blood pressure (r2=0.4), while HF of pulse interval was inversely correlated (r2=0.43). In conclusion, adipose tissue decrease induced by ET is a key factor to hemodynamic benefits in old fructose‐fed female rats with ovarian hormone deprivation.
Whole‐body sweat sensitivity in burn survivors following heat acclimation
Authors: Schlader Zachary J. , Lucas Rebekah A.I. , James Pearson , Ganio Matthew S. , Crandall Craig G.
Published: June 2021

This study tested the hypotheses that heat acclimation (HA) improves whole‐body sweat sensitivity in burn survivors and this improvement is related to the percent of body surface area of grafted skin (%BSA). 28 burn survivors were stratified into highly grafted (>;40 %BSA; N=10) and moderately grafted (17–40% BSA; N=18) groups. Seven uninjured subjects served as controls. Each underwent a 7 day HA regimen consisting of 90 min of daily exercise in 40°C, 30% RH. Sweat rate (SR; pre‐to‐post nude weight), change in core (intestinal) temperature (ΔTC) and sweat sensitivity (SR/ΔTC) to identical exercise bouts were measured on the first and last day of HA. HA attenuated ΔTC in all groups (P<0.01), with a greater attenuation occurring in the grafted groups (~0.4°C) compared to control (~0.1°). In all groups, SR was unaffected by HA. Between the first and last day of HA, sweat sensitivity was not different in the high (from 6.0 ± 2.6 to 9.4 ± 3.3 ml/min/°C; P=0.22) or moderate (from 12.3 ± 7.5 to 16.0 ± 7.5 ml/min/°C; P=0.07) grafted groups, or in the controls (from 17.8 ± 6.2 to 18.9 ± 4.5 ml/min/°C; P=0.75). Changes in sweat sensitivity to HA were unrelated to %BSA (r = 0.05, P=0.80), but sweat sensitivity pre‐HA was related with %BSA (r = −0.46, P<0.01) and this relationship was maintained post‐HA. These data indicate that sweat sensitivity in burn survivors is related to %BSA, but is minimally affected by HA.

Supported by NIH Grant GM068865
The Effects of Continuous‐flow Left Ventricular Assist Devices on Peripheral Vascular Function
Authors: Witman Melissa A.H. , Groot H. Jonathan , Gifford Jayson R. , Trinity Joel D. , Stehlik Josef , Drakos Stavros G. , Richardson Russell S.
Published: June 2021

Peripheral endothelial function is known to be impaired in patients with heart failure (HF), but the vascular effects of continuous‐flow left ventricular assist device (LVAD) implantation, now employed as either a bridge to transplantation or as destination therapy, remain unclear. Using flow‐mediated vasodilation (FMD) and passive leg movement, this study aimed to provide greater insight into LVAD‐induced changes in vascular function. 8 healthy age‐matched controls (61 ± 3 yrs), 10 HF patients (61 ± 2 yrs), and 12 LVAD patients (57 ± 3 yrs) underwent FMD testing of the brachial artery and passive leg movement‐induced hyperemia assessment, with blood flow velocity and artery diameters assessed by ultrasound Doppler. FMD was significantly lower in HF (4.7 ± 0.7%) compared to the healthy controls (7.1 ± 1.2%) and lower than both of these groups in the LVAD patients (2.7 ± 0.8%). The change in leg blood flow from baseline to peak during passive movement followed a similar pattern. The hyperemic response in patients with HF was significantly attenuated compared to the controls (90 ± 9%Δ and 160 ± 27%Δ, respectively) and the response in the LVAD patients was significantly lower than both groups (45 ± 10%Δ). These results suggest that despite circulatory assistance, peripheral vascular function and hemodynamic regulation are attenuated in patients following LVAD implantation.
Nitric oxide‐mediated vascular function in response to limb movement: the impact of age
Authors: Trinity Joel Douglas , Groot H. Jonathan , Layec Gwenael , Rossman Matthew J , Ives Stephen J , Morgan David E , Gmelch Ben S , Bledsoe Amber , Richardson Russell S
Published: June 2021

In young men passive movement‐induced increases in leg blood flow (LBF) and leg vascular conductance (LVC) are predominantly nitric oxide (NO)‐dependent, however the contribution of NO to the attenuated response with age remains unknown. Therefore, this study sought to determine the contribution of NO to movement‐induced hyperemia and vasodilation in the elderly. Fourteen male subjects (7 young, 24 ± 1 yrs, and 7 old, 75 ± 3 yrs) underwent passive knee extension with and without NO synthase (NOS) inhibition via intra‐arterial infusion of NG‐monomethyl‐L‐arginine (L‐NMMA). Leg blood flow (LBF) and central hemodynamics were determined by Doppler ultrasound and finger photoplethysmography, respectively. NOS inhibition blunted the peak increase in LBF in the young (control; 668 ± 106, L‐NMMA; 431 ± 95 ml/min, p = 0.03), but had no effect on the already attenuated hyperemic response of the old (control; 266 ± 98, L‐NMMA; 251 ± 92 ml/min, p = 0.59). Similarly, the magnitude of change in the area under the curve over time for the LBF and LVC responses to NOS inhibition was less in the old (LBF; −31 ± 18 ml, LVC; −0.4 ± 0.2 ml/mmHg) than the young (LBF; −129 ± 21 ml, p < 0.01, LVC; − 1.5 ± 0.2 < 0.01). Thus, age‐associated reductions in hyperemia and vasodilation in the elderly are primarily due to a reduced contribution of NO and support passive limb movement as a novel approach to assess NO‐mediated vascular function across the lifespan.
Polyphenols prevent obesity‐induced aortic vascular stiffness in C57BL/6 mice
Authors: Weisbrod Robert Mark , Fry Jessica , Chauhan Pratibha , Cohen Richard A , Seta Francesca
Published: June 2021

Vascular stiffness and hypertension (HTN) increase in humans with aging, diabetes, and obesity. Our lab has shown that vascular stiffness precedes by 4–5 months the development of HTN in a model of high fat high sucrose (HFHS) diet‐induced obesity by measuring aortic pulse wave velocity (PWV) and blood pressure. Polyphenols may improve blood vessel function by reducing oxidants, inflammation, and vasodilation.

Purpose

To determine if polyphenols can prevent HFHS diet‐induced vascular stiffness in a mouse model.

Methods

Male C57BL/6 mice were fed HFHS diet for 8 months with a synthetic polyphenol, S17834, or resveratrol (130 mg/kg/day). PWV was calculated from the difference in pulse wave transit times between two sites along the aorta using ultrasound.

Results and conclusion

After 4 months HFHS diet significantly increased BW by 33% and PWV by >;2‐fold (2.1±0.1 vs 4.8±0.7 m/s, n=6). Both polyphenols prevented the increase in PWV, but only one, S17834, slowed the increase in body weight. Similar results were found over 8 months. These results demonstrate that HFHS diet causes a significant increase aortic stiffness associated with obesity. Both S17834 and resveratrol inhibited the development of aortic stiffness caused by HFHS diet suggesting polyphenols may have therapeutic value in reducing vascular stiffness and subsequent HTN.

Source:

NIH RO1 HL105287, Servier.
Vascular Aging: Association between Endothelial Function and Arterial Stiffness
Authors: Heldens Manon , Tarumi Takashi , Ayaz Muhammad , Parker Rosemary , Tinajero Cynthia , Hill Candace , Tseng Benjamin Y , Liu Jie , Zhang Rong
Published: June 2021

Endothelial dysfunction and arterial stiffness are independent risk factors for cardiovascular disease. However, the relationship between these vascular parameters is not well understood during healthy aging. The purpose of this study was to determine: 1) age‐related differences in endothelial function and arterial stiffness and 2) the interrelationship between endothelial function and arterial stiffness across the lifespan from young to older adults.

Methods

48 healthy subjects aged between 22–78 years underwent the measurements of brachial artery flow mediated dilation (FMD) and central and peripheral pulse wave velocities (PWV).

Results

FMD, measured as percent change from baseline to maximal dilatation, was inversely correlated with age (r=−0.75, P<0.001). In addition, there was a positive association between age and the time to reach peak vasodilation after cuff release (r=0.39, P<0.01). Aortic PWV exhibited a positive association with age (r=0.63, P<0.001) and was correlated inversely with FMD (r=−0.40, p=0.004).

Conclusions

Endothelial dysfunction and increases in central arterial stiffness are correlated with advancing age. Moreover, the observed inverse relationship between endothelial dysfunction and increases in arterial stiffness suggests the presence of common underlying mechanisms for changes in vascular function with aging.
cGMP‐dependent protein kinase I (PKGI) undergoes post‐translational modifications in the Golgi apparatus (GA)
Authors: Kato Shin , Roberts Jesse David
Published: June 2021

PKGI mediates how nitric oxide and cGMP regulate vascular sooth muscle cell phenotype and pulmonary artery structure through incompletely understood mechanisms. Recently we determined that trans‐Golgi network (TGN)‐resident proprotein convertases, such as furin, stimulate PKGI proteolysis and regulate the nuclear localization of its kinase domain, PKGIγ. The current project tested whether PKGI undergoes proteolysis and glycosylation in the GA.

Using specific antibodies and confocal microscopy, we observed that PKGI is colocalized with furin and TGN38, TGN‐resident proteins, in the peri‐nuclear compartment of BHK cells. Moreover, using this method and also immunoblotting, we noted that IP3 receptor‐associated cGMP kinase substrate (IRAG), overexpressed in RFL‐6 cells tethers PKGIβ to the endoplasmic reticulum and decreases PKGI proteolysis and PKGIγ nuclear translocation. Also, immunoblotting showed that brefeldin A and monensin, which decrease pre‐and intra‐GA protein transport respectively, inhibit PKGI proteolysis. Lastly, the resistance to endoglycosidase H and maintenance of PNGase F digestion of PKGIγ immunopurified from BHK cells indicated that PKGIγ is N‐glycosylated in the GA.

These data demonstrate for the first time that the Golgi apparatus plays an important role in the regulation of PKGI post‐translational modifications.

These studies were supported by NIH grant R01HL096779.
Mitochondrial catalase expression protects against hypoxia‐induced pulmonary hypertension
Authors: Adesina Sherry Eniola , Hart C. Michael , Sutliff Roy L.
Published: June 2021

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH‐associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of ROS in the vasculature. The crosstalk between these two ROS sources under hypoxic conditions is unknown.

The antioxidant catalase attenuates H2O2. A mouse model with mitochondria‐targeted catalase expression (mCAT) was used to assess the role of mitochondrial H2O2 in hypoxia‐induced PH. We hypothesize that mCAT expression will reduce mitochondria‐derived ROS and hypoxia‐induced PH pathogenesis.

Control and mCAT mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks. PH development was assessed by measuring right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). ROS levels and Nox mRNA expression were measured by Amplex red assay and qRT‐PCR, respectively.

Compared to control mice, mCAT mice developed less hypoxia‐induced increases in RVSP, Nox mRNA, and lung H2O2 release. mCAT expression did not prevent hypoxia‐induced increases in RVH. These studies suggest that attenuation of mitochondria‐derived ROS may be protective against pulmonary vascular derangements involved in PH pathogenesis.

Research Service of the Atlanta VAMC and NIH grants DK074519/HL102167 (CMH and RLS).
Pressure‐dependent pulmonary arterieal tone following chronic hypoxia requires depolarization‐induced activation of epidermal growth factor receptor and NADPH oxidase
Authors: Norton Charles Elbert , Walker Benjimen R. , Resta Thomas C.
Published: June 2021

Small pulmonary arteries from rats with chronic hypoxia (CH)‐induced pulmonary hypertension exhibit pressure‐induced vascular smooth muscle (VSM) membrane depolarization and elevated tone without a change in intracellular Ca2+ ([Ca2+]i). In addition, the epidermal growth factor receptor (EGFR) and NADPH oxidase (NOX) contribute to depolarization‐induced Ca2+ sensitization in CH pulmonary arteries. Therefore, we hypothesized that pressure‐dependent pulmonary arterial tone following CH requires depolarization‐induced EGFR and NOX activation. To test this hypothesis we measured tone in response to increasing intraluminal pressure steps (5–45 mmHg) in isolated small intrapulmonary arteries (~150 μm diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were endothelium‐disrupted and loaded with fura‐2 AM to monitor [Ca2+]i. Significant tone was observed at 25, 35, and 45 mmHg in CH (p<0.05) but not control arteries. Consistent with our hypothesis, inhibition of EGFR (AG 1478; 1 μM), NOX (apocynin; 30 μM), or clamping VSM membrane potential with the K+ ionophore valinomycin (5 μM) (confirmed using sharp electrodes) abolished tone in CH arteries. Vessel wall [Ca2+]i was unaltered by AG 1478, apocynin, or by increased pressure. We conclude that CH causes pressure‐dependent pulmonary arterial tone through depolarization‐induced EGFR and NOX activation and independently of changes in [Ca2+]i.
Thioredoxin‐1 is necessary for hypoxia‐induced cell proliferation in human pulmonary artery smooth muscle cells
Authors: Jin Yi , Tipple Trent E , Chicoine Louis G , Nelin Leif D , Chen Bernadette
Published: June 2021

To test the hypothesis that hypoxia increases Trx‐1 in human pulmonary artery smooth muscle cells (hPASMC), resulting in an increase in cell proliferation, hPASMC were exposed to normoxia (21% O2) or hypoxia (1% O2) and Trx‐1 activity or expression were manipulated using an siRNA against Trx‐1 or the putative pharmacological inhibitor PX‐12. Trx‐1 protein expression was 2‐fold greater in the hypoxic hPASMC than in normoxic hPASMC at 48 h. Hypoxic exposure also resulted in significantly greater viable cell numbers after 120 h than in normoxic hPASMC. Trx‐1 expression was suppressed by Trx‐1 siRNA transfection and the hypoxia‐induced increase in cell proliferation was prevented by Trx‐1 siRNA. The pharmacological Trx‐1 inhibitor PX‐12 also inhibited the hypoxia‐induced increase in hPASMC proliferation. The hypoxia‐induced increase in HRE‐luciferase activity was prevented by treatment with either the Trx‐1 siRNA or PX‐12. Akt was phosphorylated at 2h and 24h in the hypoxic hPASMC, and Akt phosphorylation was prevented by treatment with the Trx‐1 siRNA. Inhibition of the PI3K‐Akt pathway using LY294002 resulted in a decrease in proliferation in hPASMC. Our data demonstrates that hypoxia results in Trx‐1‐mediated cell proliferation in hPASMCs, at least partly through activation of HIF signaling and the PI3K‐Akt pathway. Trx‐1 may be a potential therapeutic target for pulmonary hypertension.
Caveolae regulate nitroxidative signaling via localized nitration of Src‐family kinase in endothelial cells
Authors: Singh Harinder , Rizzo Victor
Published: June 2021

Peroxynitrite (ONOO−)‐mediated tyrosine nitration of proteins has been implicated in vascular pathologies. The regulatory mechanism of cross‐reaction between O2− and NO for ONOOgeneration and subsequent nitroxidative signaling is poorly understood. Given our previous findings that endothelial cell caveolae localize NAPDH oxidase enzyme complexes (O2−) and eNOS (NO), we hypothesize that caveolae may act as compartments for radical generation and oxidative/nitrative signaling pathways that lead to endothelial dysfunction. Here, we report that caveolae‐localized Src‐family kinase (SFK's) become nitrated in a superoxide‐ and NO‐dependent manner in response to the proinflammatory cytokine, TNFα. We further tested whether SFK's nitration serves as an early, upstream mediator of endothelial dysfunction. We found a significant reduction in TNFα induced activation of the NFkB pathway and upregulation of adhesion molecule expression (ICAM‐1/VCAM‐1) in cells pretreated with an inhibitor to SFK's (PP2). Moreover, disruption of caveolae domains showed a similar decrease in the activation of these proinflammatory pathways. These findings were confirmed in arteries from Cav1KO mice where both general and SFK‐specific protein tyrosine nitration and adhesion molecule expression were attenuated compared to WT animals infused with TNFα. Our results indicate that caveolae act as reaction centers for nitroxidative signaling that contribute to endothelial dysfunction.
Differential effects of low pH on Ca2+‐induced ROS emission from mitochondrial complexes I and III
Authors: Lindsay Daniel P , Camara Amadou KS , Stowe David F , Aldakkak Mohammed
Published: June 2021

Mitochondrial emission of reactive oxygen species (ROS) is a critical factor in cardiac ischemic injury. Increased extra‐matrix Ca2+, decreased extra‐matrix pH, and altered substrate utilization are factors that modulate ROS emission during ischemia. Here we examined the combined effects of these factors on ROS emission from complexes I and III. Guinea pig heart mitochondria were suspended in experimental buffer (pH 7.15, 6.9, 6.5), pre‐incubated with increasing [CaCl2], and then energized with either pyruvate or succinate followed by adding either rotenone or antimycin A. H2O2 release rate and matrix volume were measured using pyruvate/rotenone or succinate/antimycin A with or without rotenone to mimic conditions that exist during early or late global ischemia, respectively. High buffer CaCl2 enhanced rotenone or antimycin A‐induced H2O2 release under all conditions. The largest increase in H2O2 release occurred at pH 6.9 or pH 6.5 with pyruvate/rotenone or succinate/antimycin A, respectively. The large increase in H2O2 release was associated with a significant mitochondrial swelling, and both measures were abolished with cyclosporine A, indicating opening of the mitochondrial permeability transition pore. These results highlight the significance of each respiratory complex in Ca2+‐induced ROS emission under different substrate/pH conditions that may mimic early and late ischemia. (NIH and VA)
Critical Role of Sox9 in the Differentiation of Mesenchymal Stem Cells to Endothelial Cells.
Authors: Agrawal Devendra K , Attairu Izuagie , Djossou Kokouvi P , Pankajakshan Divya
Published: June 2021

Objective

Mesenchymal stem cells (MSCs) have been shown to replenish and repair endothelial cells (ECs) of the cardiovascular system. However, mechanisms underlying differentiation is still unclear. Exposure of MSCs to vascular endothelial growth factor (VEGF) enhances in‐vitro differentiation of MSCs to ECs. In this study, we investigated a potential role of Sex‐determining region Y related protein (Sox9) signaling in the differentiation of MSC to EC in response to VEGF.

Method and Results

Porcine bone marrow MSCs, were differentiated in media containing 50 ng/mL VEGF that induced significant increase in EC markers, PECAM, VE cadherin and vWF in 10 days. Western blot analysis revealed a 3‐fold increase in Sox9 expression in VEGF‐treated differentiating MSCs compared to control MSCs. Flow cytometry results revealed that the transfection of MSCs with siRNA‐Sox9 decreased the expression of EC markers in VEGF‐treated MSCs. Overexpression of MSCs by transfecting Sox9 cDNA, followed by culture in MSC growth media, significantly increased the expression of EC markers in differentiated MSCs. Pre‐treatment of MSCs with PD173074, a VEGFR‐2 blocker, followed by culturing in the differentiating media decreased EC marker expression with no significant change in Sox9.

Conclusion

Sox9 plays a critical role in the differentiation of MSCs to ECs via cellular communication between phosphorylated VEGFR‐2 and Sox9.

Supported by NIH R01HL090580 and R01HL104516.
The role of increased Sarcolipin expression in neonatal development and in muscle disease
Authors: Pant Meghna , Bal Naresh C , Delfin Dawn A , Rafael‐Fortney Jill A , Periasamy Muthu
Published: June 2021

Skeletal muscle constitutes more than 40% of body mass and has been suggested to play an important role in thermogenesis and metabolism. Studies in our laboratory have shown that Sarcolipin (SLN) (31 amino acid trans‐membrane peptide) an inhibitor of SR Ca2+ ATP ase (SERCA) is critical for muscle‐based thermogenesis. Therefore we investigated the importance of SLN during early mouse neonatal development and in dystrophic muscle. We found that SLN expression is several fold higher at birth in most skeletal muscles studied, which then gradually decreases in adult fast twitch skeletal muscles but preserved in oxidative/slow twitch fibers. Gradual cold acclimatization of these neonatal mice extended the duration of SLN expression in all the muscles. We also observed that in SLN KO neonates the expression of key metabolic regulators involved in fatty acid and glucose oxidation were altered. When compared to skeletal muscle from dystrophic MDX mice, SLN expression was significantly upregulated in the utrophin‐dystrophin double knock out (DKO) mice. Interestingly, the DKO mice exhibit a higher level of whole body oxygen consumption but they show decreased mobility. These data together suggest that increased expression of SLN is important for maintaining thermogenesis and increased metabolic demand during early neonatal stages and as well in dystrophic muscle disease where muscle function is compromised. Research support: NIH, MDA.
A novel mechanism for UCP1‐independent thermogenesis
Authors: Rowland Leslie , Bal Naresh , Periasamy Muthu
Published: June 2021

Nonshivering thermogenesis (NST) via uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is a well‐established mechanism for temperature homeostasis in many mammals. The recent findings that BAT function is limited in adult humans and UCP1‐deficient mice can be cold adapted indicated that alternative sites of NST must exist. Ongoing studies in our laboratory have established that sarcolipin (SLN), an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), is involved in skeletal muscle‐based NST. Therefore, we hypothesized that SLN can compensate for UCP1 and that SLN and UCP1 play synergistic roles. To test this, we generated double knockout (DKO) mice deficient in both UCP1 and SLN and challenged them to acute cold (4°C). The DKO mice are severely cold‐intolerant, compared to UCP1‐KO and SLN‐KO. Furthermore, we show that SLN is upregulated in the skeletal muscle of UCP1‐KO mice and UCP1 is induced in SLN‐KO mice during cold adaptation. These data suggest that UCP1 and SLN play synergistic roles in cold adaptation and that they compensate for one another. Importantly, these data establish SLN as a critical component of NST and suggest that muscle‐based NST can replace BAT when BAT function is minimal as found in adult large mammals, including humans. Because NST mechanisms can be exploited to expend excess energy, these findings have novel implications for weight loss and obesity therapies. Research Support: NIH.
Pancreatic ACE2 shedding is associated with impaired glycemia in high fat diet‐fed mice.
Authors: Chhabra Kavaljit H. , Pedersen Kim B , Xia Huijing , Shenoy Vinayak , Raizada Mohan K , Lazartigues Eric
Published: June 2021

Angiotensin converting enzyme 2 (ACE2) activity decreases with the progression of type 2 diabetes and restoration of ACE2 appears to be beneficial in this disease. We hypothesized that shedding of pancreatic ACE2 might be associated with impaired glycemia in high‐fat diet (HFD)‐fed mice. Pancreatic ACE2 and ADAM17 (a sheddase) activities (FU/min/μg protein) were assessed in 6‐month old C57BL/6J mice (N=5/group) fed with HFD or regular diet (RD) for 16 weeks. HFD‐fed mice showed lower pancreatic ACE2 activity (144 ±17 vs. 218 ±20; P<0.05) associated with higher ADAM17 activity (60.5 ±0.4 vs. 53.1 ±1.8; P<0.05) compared to RD mice. To restore ACE2 activity, we treated HFD‐fed mice with a lentivirus coding for mACE2 or GFP (pancreas injection, 10^6 TU/ml in 100 μl saline) at the 13th week of HFD‐feeding. Three weeks later, ACE2‐treated mice not only exhibited higher pancreatic ACE2 (225.4 ±18.3) and reduced ADAM17 (55.5 ±1.4) activity but also reduced Ang‐II levels (2.1 ±0.3 vs. 3.4 ±0.2 ng/g of pancreas) and improved glucose tolerance (AUC= 46153 ±1646 vs. 56045 ±2552 mg/dl*min.) versus GFP‐injected mice (P<0.05). Altogether, our data suggest that pancreatic ACE2 shedding might be a possible mechanism contributing to impaired glycemia in HFD‐fed mice. Hence, restoration of ACE2 could be a promising therapeutic target for managing HFD‐induced type 2 diabetes.
Metformin, Aerobic Exercise, and the Combination in the Treatment of Type 2 Diabetes and NAFLD in OLETF rats
Authors: Linden Melissa A , Meers Grace M. , Fletcher Justin A. , Siddique Sameer , Laughlin M. Harold , Booth Frank W. , Ibdah Jamal A. , Thyfault John P. , Rector R. Scott
Published: June 2021

Exercise and metformin are common therapies for type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD). However, it is unclear whether combining therapies offers greater benefit for T2D and NAFLD outcomes. We randomly assigned type 2 diabetic, Otsuka Long‐Evans Tokushima Fatty (OLETF) rats (age 20 wks; n=10/group) to sedentary (SED), SED + metformin (SED+MET; 300 mg/kg/day), moderate intensity exercise training (EX; 20 m/min, 60 min/d, 5 d/wk treadmill running), or EX+MET groups for 12 weeks. MET, EX and EX+MET were effective in the management of T2D and all three treatments lowered hepatic steatosis and serum markers of liver injury; however, EX lowered liver TAG content more than MET with no added benefit of combining EX + MET. Only EX animals exhibited increases in hepatic mitochondrial fatty acid oxidation, hepatic citrate synthase activity, and hepatic β‐HAD activity compared with SED (p<0.05). Furthermore, EX attenuated hepatic de novo lipogenesis markers acetyl‐CoA carboxylase, fatty acid synthase, and stearoyl‐CoA desaturase‐1 to a greater extent than MET, with no further reductions in the EX+MET group. In conclusion, EX may be a more effective treatment for NAFLD than MET and the addition of MET to EX does not appear to illicit greater improvements in the hyperphagic, obese rodent model of T2D and NAFLD. Supported by: NIH T32 AR 048523–07, DK088940, RO1HL036088, and VHA‐CDA2 IK2BX001299–01
A Cav1.2 Ca2+ channel mutation that reduces intestinal smooth muscle contractility
Authors: Duffey Michael E , Helman Justin M , Gugino Sylvia F , Russell James A , Bett Glenna CL , Rasmusson Randall L
Published: June 2021

Ca2+ entry through Cav1.2 L‐type voltage‐gated Ca2+ channels is a Ca2+ source for intestinal smooth muscle contraction. We examined Cav1.2 in a Timothy Syndrome (TS) mouse with a TS2 G406R mutation and a neo cassette suppressor of Cav1.2 expression. Isometric force was measured in rings of proximal colon from TS2 positive (TS2NEO) and negative (LM) littermate mice. Spontaneous contractile activity occurred in all tissues, but treatment with Nω‐nitro‐L‐arginine (10−3 M, L‐NA), a nitric oxide synthase antagonist, caused rhythmic contractions (LM, 1.37±0.62 vs. 0.02±0.16 A(area under response curve)/min/g tissue, untreated, n=3). Electric field stimulation (EFS, 0.5–20 Hz) produced frequency‐dependent tension in all tissues, suggesting the release of multiple neurotransmitters. An increase in tension was seen when stimulation was stopped, which was significantly less in TS2NEO mice (5 Hz, LM, 0.24±0.05; TS2NEO, 0.11±0.04 g tension/g tissue, n=5). Tissues relaxed when stimulated by 20 Hz EFS, but contracted at 20 Hz if pre‐exposed to L‐NA, and tension was significantly less in TS2NEO mice (LM, 5.44±1.08; TS2NEO, 1.61±0.44 A/min/g, n=5). L‐NA‐induced tension was blocked by the muscarinic antagonist, atropine (10−3 M), suggesting nitronergic and cholinergic control of muscle activity. These data show that mutations associated with TS result in reduced contractile activity. (DOD AR080107, NIH R21HL088058)
Role of Na‐K‐2Cl symporter in GABA‐evoked excitation in rat enteric neurons
Authors: Liu Sumei , Ji Tuo , Ren Wei , Qu Mei‐Hua , Zhu Jin‐Xia , Wood Jackie D
Published: June 2021

Background and Aims

Gamma‐aminobutyric acid (GABA) inhibits neurons in the brain and spinal cord. GABA does the opposite in neurons of the enteric nervous system (ENS) where its mechanism of excitation is unclear. We aimed to test a hypothesis that the Na+‐K+‐2Cl− symporter (NKCC) maintains high intracellular Cl− and contributes to GABA excitation in ENS neurons.

Methods

Intracellular electrophysiological recording was used to record GABA‐evoked responses in the myenteric plexus of the rat colon. Immunofluorescence and Western blot analysis were used to study the expression of NKCC.

Results

Both NKCC1 and NKCC2 proteins were expressed in ENS neurons in postnatal and adult rats. NKCC1 and NKCC2 were coexpressed with the GABAA receptor. The K+‐Cl− cotransporter 2 (KCC2), a Cl− extruder, was not expressed. GABA depolarized the membrane potential in ENS neurons with a reversed potential of −20 mV, which is near the Cl− equilibrium potential. Blockade of NKCC by bumetanide or furosemide suppressed GABA‐evoked depolarization and shifted the reversal potential in the hyperpolarizing direction. Neither the KCC blocker nor the Cl−/HCO3− exchange inhibitor suppressed GABA‐evoked depolarization.

Conclusions

NKCCs contribute to the elevation of intracellular Cl− and resulting excitatory actions of GABA in ENS neurons.
Cystathione‐γ‐lyase deficiency protects mice from galactosamine/LPS‐induced acute liver failure
Authors: Shirozu Kazuhiro , Tokuda Kentaro , Marutani Eizo , Lefer David , Wang Rui , Ichinose Fumito
Published: June 2021

Introduction

Acute liver failure is a fatal syndrome attributed to massive hepatocyte death. Hydrogen sulfide (H2S) has been reported to exert cytoprotective effects in variety of organs. Here, we examined the role of cystathione‐γ‐lyase (CSE, an enzyme produces H2S) in the acute liver failure induced by galactosamine (Gal) and lipopolysaccharide (LPS).

Methods

Wild‐type (WT) and CSE‐deficient (CSEKO) mice were challenged with Gal/LPS.

Results

CSEKO exhibited better survival rate and lower plasma alanine aminotransferase (ALT) levels compared with WT after Gal/LPS challenge. Administration of DL‐propargylglycine (PAG, a CSE inhibitor) also prevented Gal/LPS‐induced increase of plasma ALT in WT. CSE deficiency markedly attenuated Gal/LPS‐induced upregulation of inflammatory cytokines and activation of caspase 3 and PARP in liver tissue. On the other hand, CSE deficiency increased nuclear translocation of Nrf2 and upregulated gene expression of anti‐oxidant proteins in the liver. Liver levels of thiosulfate was markedly increased in CSEKO compared to WT. Finally, administration of sodium thiosulfate attenuated the Gal/LPS‐induced increase of plasma ALT in WT.

Conclusions

These results suggest that CSE deficiency increases liver levels of thiosulfate and protects mice from acute liver failure at least in part by inhibition of caspase 3 activation and by augmentation of Nrf2 signaling pathway.
The Dissociation of the Soluble Prorenin Receptor in Plasma and Urine of Diabetic Patients
Authors: Arita Danielle Y , Luffman Christina , Bourgeois Camille R T , Theti Tina , Prieto Minolfa C
Published: June 2021

Diabetic patients display low‐to‐normal plasma levels of renin but increased prorenin levels. In contrast, their urine contains high levels of “active” renin but low prorenin. Our hypothesis is that the soluble form of the prorenin receptor (sPRR) contributes to the enhancement renin activity in the urine of diabetic patients. We measured plasma and urinary sPRR levels by enzyme‐linked immunosorbent assays (ELISA; IBL, Japan) in 60 patients, including: Controls (CT; n=20), Diabetics (DM; n=20) and Diabetics with hypertension (DM+HTN; n=20). Plasma sPRR levels were augmented in DM compared to CT and DM+HTN (DM: 9849±828 vs. CT: 7310±715 and DM+HTN: 7121±373 pg/mL; p<0.05). In contrast, in the urine of DM and DM+HTN patients, sPRR levels were decreased (DM: 576.5±130.9 and DM+HTN: 486.1±81.64 vs. CT: 1248.0±345.6 pg/mg; p<0.05) but urinary renin content was increased (DM: 130±14; DM+HTN: 162±31 vs. CT: 10±1.3 ng AngI/mL/hr; p<0.0001). uCre levels were similar among groups. The dissociation between plasma and urine levels of sPRR suggests that lower pH in the urine of diabetic patients may increase the capability of sPRR to enhance renin enzymatic activity. Further studies will be performed to verify it the sPRR is bound or unbound to renin/prorenin in the urine of these patients.
Aspirin influence on mice sex genders during cutaneous wound healing
Authors: Santos Jeanine Salles , Costa Andréa Monte Alto
Published: June 2021

The aim of this study was to evaluate whether aspirin, and thus cyclooxygensase (COX) inhibition, affects cutaneous wound healing in distinctly female and male mice. Female and male Balb/c mice were treated with aspirin (25 mg/kg/day) until euthanasia. The control group received vehicle only. A full‐thickness excisional lesion was performed, and macroscopic, histological and biochemical parameters were evaluated. Sections were stained and immunostained for microscopic analysis. Myeloperoxidase activity, hydroxyproline quantity and protein expression of von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF) were also measured. Female control and treated groups presented delayed contraction and reepithelialization when compared to male control and treated group, respectively. Female control group presented reduced myeloperoxidase activity and MIF‐positive cells compared to male control group. In female treated group, myeloperoxidase activity and F4/80‐positive macrophages quantity was greater than in control group. Collagen was reduced only in female treated group. vWF and VEGF protein expression were increased in female‐treated group. In summary, aspirin administration impaired wound healing process in Balb/c females but not in males.
A novel AMPK activator, PT‐1, increases gamma1 AMPKassociated activity, but not gamma3 AMPK‐associated activity or glucose transport
Authors: Jensen Thomas Elbenhardt , Birk Jesper B. , Richter Erik A.
Published: June 2021

AMP activated protein kinase (AMPK) is a heterotrimeric αβγ complex. Selective pharmacological activation of specific AMPK‐complexes through targeting of its regulatory subunits is an attractive strategy to confine AMPK activation to select tissues to combat diseases such as type 2 Diabetes. Furthermore, it is a useful tool to study specific AMPK complexes in vivo. Here, a putative direct α‐AMPK specific activator, PT‐1, was found to selectively increase γ1, but not γ3 AMPK‐associated activity in incubated mouse muscles. In contrast, the AMP‐mimetic AMPK activator, AICAR stimulated both γ1 and γ3 AMPK activities. Both PT‐1 and AICAR augmented AMPK Thr172 phosphorylation when measured in whole muscle lysates. However, unlike AICAR, PT‐1 did not increase TBC1D1 Ser237 or ACCbeta Ser221 phosphorylation, suggesting that these phosphorylations are catalyzed by γ3 but not γ1‐AMPK complexes. Unlike AICAR, known to require α2,β2 and γ3 AMPK to increase glucose transport, PT‐1 failed to stimulate this process. In summary, this study demonstrates for the first time γ1‐ but not γ3‐AMPK activation by PT‐1. TBC1D1 Ser237, ACCbeta Ser221 and glucose transport‐stimulation reflect α2β2γ3‐ but not γ1‐AMPK activity in mouse muscle. Funded by Danish Research Council and Novo Nordisk Foundation.
Cockroach Allergen Proteinases Regulate Proteinase‐Activated Receptor‐1 (PAR1) Signalling
Authors: Polley Danny , Mihara Koichiro , Ramachandran Rithwik , Vliagoftis Harissios , Boitano Scott , Hollenberg Morley D. , Daines Michael
Published: June 2021

Objective

To assess potential pro‐fibrotic PAR1 MAPKinase signaling by cockroach allergen‐derived trypsin‐like proteinases.

Methods

Cockroach allergen‐derived trypsin‐like enzymes, known to cause lung inflammation, were identified with serine proteinase activity‐based probes (ABP). Enzymes were purified (ion‐exchange chromatography) and analyzed for their ABP reactivity & substrate‐inhibitor kinetic profiles. Each enzyme was evaluated for causing calcium (Fluo‐4) and MAPKinase signaling (western blot) in PAR1‐expressing KNRK cells. Additionally, the cleavage‐internalization of fluorescently‐tagged PAR1 constructs was monitored as an index of receptor activation.

Results

Cockroach allergen contained three ABP‐labeled 20–26 kDa MW proteinases with distinct enzyme kinetics. The allergen‐derived enzymes activated PAR1 MAPK and calcium signaling, but with distinct concentration‐effect kinetics. PAR1 cleavageinternalization caused by the allergen proteinases was similar to that caused by trypsin, but NOT thrombin.

Conclusions

Cockroach allergen contains three different trypsin‐like proteinases that can activate “biased” PAR1 pro‐fibrogenic MAPKinase (but not Ca++) signaling pathways. We suggest that the allergen proteinases can act as pro‐fibrotic factors in the setting of airway allergic inflammation and asthma. Support: CIHR, NIH USA and Lung Assn. of AB & NWT