bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapamycin Activates TGF Receptor Independently of Its Ligands: Implications for Endothelial Dysfunction
Authors: Girao‐Silva Thais , Miyakawa Ayumi , Krieger Jose , Edelman Elazer
Published: June 2021

Rapamycin, the active drug in drug‐eluting stents, has a well‐recognized antiproliferative action: it binds to FKBP12 and the rapamycin‐FKBP complex inhibits the mTOR pathway inducing cell cycle arrest. FKBP12 is bounded to TGF receptor‐type I protecting its spontaneous activation. We hypothesize that rapamycin can displace FKBP12 from TGFRI promoting its activation. Human umbilical vein endothelial cells (HUVEC) were treated with rapamycin (10nmoL/L) and/or TGF‐β RI kinase inhibitor (TGFRIi, 100nmoL/L) for 24 hours. Rapamycin induced Smads phosphorylation (Smad1, Smad2 and Smad5) and PAI‐1 up‐regulation, which was specifically abrogated by Smad2 knockdown. TGFRIi efficiently blocked phosphorylation of Smad2, but not Smad1/5. TGF‐β secretion was not influenced by rapamycin indicating that Smad activation is independent of the ligand. Interestingly, rapamycin induced endothelial‐to‐mesenchymal transition was potentiated by IL‐1β, and efficiently blocked by TGFRIi. In vivo, the pro‐thrombogenic effects of rapamycin decreased by TGFRIi treatment and rapamycin‐induced PAI‐1 expression in carotid arteries were decreased by TGFRIi. Altogether, we provide evidence that rapamycin activates TGF receptor independent of its ligand TGF‐β, promotes PAI‐1 expression and induces changes in endothelial phenotype. These undesirable effects, the prothrombogenic state and activation of EndMT, are Smad 2‐dependent and independent of the therapeutic rapamycin‐induced cell proliferation arrest.
Single LDL‐Cholesterol Apheresis Treatment Does Not Improve Vascular Endothelial Function in Hypercholesterolemic Patients
Authors: Ballard Kevin , Taylor Beth , Beam Jo Ellen , Mah Eunice , Guo Yi , Bruno Richard , Polk Donna , Thompson Paul
Published: June 2021

Low‐density lipoprotein cholesterol (LDL‐C) apheresis is an effective therapy to reduce CVD risk in hypercholesterolemic patients who are refractory or intolerant to lipid‐lowering therapy. We hypothesized that a single treatment of LDL‐C apheresis would increase vascular endothelial function by decreasing oxidative stress responses that limit nitric oxide (NO) bioavailability. Patients (n=5; 52±11 y) were actively receiving bi‐weekly LDL‐C apheresis treatments (duration=54±41 mo). Brachial artery flow‐mediated dilation (FMD), plasma lipids, vitamin E (α‐ and γ‐tocopherol), markers of oxidative stress (nitro‐γ‐tocopherol, malondialdehyde (MDA)), and regulators of NO metabolism (arginine (ARG), asymmetric dimethylarginine (ADMA)) were assessed prior to (Pre) and immediately following LDL‐C apheresis (Post) and at 1, 3, 7, and 14 d Post. Relative to Pre, total C (300±58 mg/dL) and LDL‐C (239±46 mg/dL) at Post were 61% and 70% lower (P<0.01), respectively, and remained 46‐56%, 27‐34%, and 14‐16% lower at 1, 3, and 7 d, respectively. Plasma α‐, γ‐, and nitro‐γ‐tocopherol concentrations were 52‐69% lower at Post (P<0.01), and α‐tocopherol remained 36% lower at 1 d while nitro‐γ‐tocopherol remained 41% lower at d 3. Brachial FMD responses (6.9±3.6%) and plasma MDA, ARG, and ADMA concentrations were unaffected following LDL‐C apheresis. A single LDL‐C apheresis session did not alter FMD in patients treated chronically for hypercholesterolemia, a finding potentially attributed to the effect of chronic treatment or the decline in plasma antioxidants induced by cholesterol lowering. Funded by Kaneka Pharma, LLC
Effects of Ageing on Gross and Cortical Cerebral Autoregulatory Upper Limits
Authors: Thompson Emma , Coney Andrew , Marshall Janice
Published: June 2021

Ageing leads to impaired endothelium‐dependent vasodilatation and sympathetically‐mediated vasoconstriction in many tissues including brain. We hypothesised cerebral autoregulation is modulated by these influences and ageing is associated with impaired autoregulation at the upper limit (UL).

In anaesthetised young (Y) male Wistar rats (6‐8 weeks, n=25), basal arterial blood pressure (ABP) was 127±2mmHg, whereas in old (O) rats (52‐58 weeks, n=12), ABP was 148±2mmHg* (*=P<0.05).

Phenylephrine was infused (0.1–200µg/kg/min i.v.) to raise ABP and gross cerebral blood flow (CBF) was recorded from common carotid after external carotid ligation. Dual‐line linear regression of gross CBF vs ABP, gave an UL for Y rats of 168±2mmHg. In 8 Y rats, cortical red cell flux (cRCF) was simultaneously monitored by laser speckle imaging; in these, gross CBF UL was 166±4mmHg, but cCBF UL was higher at 170±5mmHg*. By contrast in O rats, gross CBF UL was 180±3mmHg* (vs Y rats), and cCBF UL was 179±1mmHg (NS vs gross CBF UL in O rats, or cCBF UL in Y rats).

We suggest in Y rats, a higher UL for cCBF vs gross CBF reflects further capacity for autoregulatory constriction of smaller cerebral vessels even when larger, proximal vessels have reached their UL. This may protect cortical tissue circulation. Since O rats were hypertensive compared to Y rats and hypertension has been shown to increase the UL, this may explain the higher gross CBF UL in O rats. However, as the UL for cCBF and gross CBF were similar in O rats, it seems the extended autoregulatory range for smaller cerebral vessels is lost with ageing. This could result in fragile cortical vessels being exposed to increased pressure when ABP rises and increase the risk of haemorrhagic stroke.

British Heart Foundation funding is gratefully acknowledged.
Development of Capabilities for Simultaneous Assessment of Pulmonary and Systemic Hemodynamics in Conscious Rat to Identify Therapies with Pulmonary Selectivity
Authors: Zhao Huawei , Shen Xiaolan , Hong Xuening , Small Kersten , Madwed Jeffrey
Published: June 2021

Novel therapies for pulmonary arterial hypertension (PAH) that specifically dilate the pulmonary vasculature are desired. Our aim was to establish capabilities to monitor pulmonary and systemic pressures and cardiac output simultaneously in a conscious rat model of PAH to identify those therapies that show pulmonary selectivity. To achieve this capability, HD‐S21 dual pressure transmitters from Data Science International were surgically implanted to male Sprague‐Dawley rats with one catheter placed in the pulmonary artery and other in the abdominal aorta to measure pulmonary and systemic arterial pressure (PAP, SBP) simultaneously. Rats were also instrumented with flow probes (Transonic Systems Inc) around the ascending aorta to monitor cardiac output (CO) and to calculate intra‐rat pulmonary and systemic vascular resistance (PVR, SVR). Rats were exposed to hypoxia (10% O2) for two weeks to create a clinically relevant PAH disease model. The increase in mean PAP was 18 ± 1 to 50 ± 2 mmHg which was mainly due to the increase in PVR (0.14 ± 0.01 to 0.57 ± 0.06) mmHg/ml/min. The methods were validated using Tadalafil, a standard PAH therapy inhibiting PDE 5 at 1, 3 and 10 mg/kg which showed dose dependent decreases in PAP and PVR to a greater extent than the SBP and SVR. In conclusion, innovative capabilities to determine PVR and SVR simultaneously have been established in conscious rodent which allows us to directly compare pulmonary and systemic hemodynamic activities in the same animal minimizing cross‐study variability with different cohort of animals. These methods have provided us with a powerful approach to identify therapies for PAHs with pulmonary selectivity.
Carotid Artery IMT, Blood Pressure, and Fasted Glucose and Cholesterol Levels in Males and Females
Authors: Cromwell Christina , Lenzo Jessica , Oakman Joyann , Aichele Kristin , Neal Michael , Bye Naomi , Chavis Lauren , Evans Rachel , Getty Allyson , Hill Jessica , Louth Victoria , Perez Avery , Santaniello Erica , Thiele Karla , Wisdo Tia , Feairheller Deborah
Published: June 2021

Previous studies have investigated carotid artery intima‐media thickness (IMT) and blood pressure (BP) and found a direct correlation between the two. Also, studies have examined IMT and blood glucose and cholesterol levels. It is known that young adult females have better cardiovascular health than males. To the best of our knowledge, no prior study has investigated the carotid artery IMT, BP, and blood glucose/cholesterol levels in relation to gender differences in young healthy adults. On three separate days, 37 adults (25.35 ±10.9yrs; 22M, 15F) underwent carotid IMT ultrasound, flow‐mediated dilation (FMD), fasted glucose and cholesterol test, 24hr ambulatory BP monitoring, VO2max test, and body composition measurement (bioelectrical impedance). We found that females have lower systolic BP, lower LDL/HDL ratios, lower body mass index and a higher HDL count than males (p < 0.01 for all), all of which are markers of increased cardiovascular health. However, we found no gender differences in vascular health measures, IMT and FMD (p >0.05) Our results suggest that while adult females have better cardiovascular health than males, endothelial function may not yet be affected in the young adult years.
20‐SOLA, a Novel Water Soluble 20‐HETE Antagonist, Reduces Blood Pressure Through Regulation of Vascular ACE Expression via an IKK Dependent Pathway
Authors: Garcia Victor , Shkolnik Brian , Pandey Varun , Capdevila Jorge , Falck John , Schwartzman Michal
Published: June 2021

Increased vascular 20‐HETE has been associated with hypertension and activation of the renin‐angiotensin system through induction of vascular angiotensin converting enzyme (ACE) expression. CYP4a14 knockout (KO) mice exhibit male‐specific androgen‐sensitive 20‐HETE‐dependent hypertension. A novel water‐soluble 20‐HETE antagonist, 2,5,8,11,14,17‐hexaoxanonadecan‐19‐yl 20‐hydroxyeicosa‐6(Z),15(Z)‐dienoate (20‐SOLA) was administered to CYP4a14 KO male and female mice (10mg/kg/day in drinking water). 20‐SOLA normalized blood pressure in hypertensive CYP4a14 KO male mice over the course of 10 days (124±1 vs 153 ±2 mmHg in untreated littermates) and had no effect on blood pressure of CYP4a14 KO female mice (126±1 vs 124±1 mmHg). Renal preglomerular microvessel (PGMV) ACE expression was significantly elevated in CYP4a14 KO males (3.57 ± 0.6 fold) compared to normotensive female mice and treatment with 20‐SOLA normalized vascular ACE expression. 20‐HETE‐mediated induction of vascular endothelial ACE expression has been demonstrated to be IKK‐dependent. PGMV of CYP4a14 KO male mice expressed a 2.6‐fold increase in phospho‐IKK compared to female KO mice and this increase was significantly reduced by 20‐SOLA (2.6±0.2 vs. 1.3±0.3 fold, respectively). These results suggest that the water soluble 20‐HETE antagonist 20‐SOLA reduces blood pressure in the male CYP4a14 KO mice, at least in part, through regulation of vascular ACE expression.
Baroreflex Function in Young Women with a Family History of Hypertension
Authors: Sebzda Kelly , Matthews Evan , Wenner Megan
Published: June 2021

A positive family history of hypertension (+FH) is a risk factor for the future development of hypertension. Reductions in cardiovagal baroreflex sensitivity (BRS) are also associated with hypertension and cardiovascular disease. Therefore, we hypothesized that young women with +FH (n=13, 22±1 yrs, 21±1 kg/m2, 84±2 mmHg resting mean blood pressure (BP)) would have lower BRS compared to young women without a family history of hypertension (‐FH; n=11, 21±1 yrs, 22±1 kg/m2, 81±3 mmHg resting mean BP, all P>0.05 between groups). We examined BRS as the relationship between systolic BP and R‐R intervals during phases II and IV of the Valsalva maneuver. The slope of the relationship between systolic BP and R‐R interval was determined using linear regression, and used as an index of cardiovagal BRS. We accepted linear regressions with an r > 0.70 for analysis. Cardiovagal BRS was similar during phase II of the Valsalva maneuver (Mean slope: +FH 6.1±0.8 vs. ‐FH 6.4±1.6, P=0.87). However, cardiovagal BRS during phase IV of the Valsalva maneuver tended to be lower in the +FH group (+FH n=10, ‐FH n=8, mean slope: +FH 9.3±2.2 vs. ‐FH 15.3±2.2, P=0.07). Mean BP (+FH 80±1 mmHg, ‐FH 78±2 mmHg) and resting heart rate (+FH 63±3 bpm, ‐FH 66±2 bpm) during the experimental visit were similar between groups. These preliminary data suggest that women with a positive family history of hypertension may have reduced cardiovagal BRS. This may be one mechanism contributing to the development of future hypertension in those with a family history. This work was supported in part by NIH Grant U54‐GM104941 (PI: Binder‐Macleod).
Paradigms of Cardiovascular Control in Response to Sympathetic Stimulation by Simulated Orthostatic Stress
Authors: Bronzwaer Anne‐Sophie , Verbree Jasper , Stok Wim , van Buchem Mark , Daemen Mat , van Osch Matthias , van Lieshout Johannes
Published: June 2021

Background

Sympathetic stimulation by lower body negative pressure (LBNP) as surrogate of orthostatic stress augments heart rate (HR) and total peripheral resistance (TPR) in maintaining blood pressure (BP). This study addressed the variation in cardiovascular response to LBNP within and between subjects.

Methods

In 10 healthy subjects (5 female), BP (Nexfin), HR, stroke volume (SV), cardiac output (CO), TPR, BP/HR variability, and frequency domain baroreflex HR control (BRS) were quantified during three consecutive 5 minute runs of ‐50 mmHg LBNP (repeated after one week) to determine reproducibility.

Results

In response to repeated LBNP runs, SV and HR demonstrated excellent and CO and TPR good within‐subject reproducibility. The cardiovascular response between subjects differed. Subjects with a marked decrease in SV (ΔSV>31%) demonstrated a higher resting HF/Ptot ratio as surrogate of parasympathetic activity and a more HR mediated response to LBNP. Contrary, a small SV decrease (ΔSV蠄31%) paralleled a higher resting LF/HF ratio as surrogate of sympathetic activity and a predominant increase in TPR in response to LBNP. BRS did not differ between groups.

Conclusion

Considerable between‐subject variation in cardiovascular responses to LBNP indicates differences in blood pressure regulation and suggests subject‐specific strategies of autonomic cardiovascular control in young healthy individuals.
Role of Adenosine Receptors in Evoking Venous Distension Reflex in Humans
Authors: Cui Jian , Blaha Cheryl , Sinoway Lawrence
Published: June 2021

Our prior work has shown that volume infusion (saline or albumin solution) into the veins of an occluded forearm induces venous distension and evokes reflex increases in muscle sympathetic nerve activity (MSNA) and blood pressure (BP) in humans (termed venous distension reflex). The receptors involved in this reflex are unclear. We hypothesized that stimulation of adenosine receptors might contribute to the venous distension reflex. BP (Finapres), heart rate, and MSNA (microneurography) were assessed in 11 young healthy subjects in 3 separated visits. In the saline (control) trial, 40 ml saline was infused into veins of an arterially occluded arm (N=11). In the second experiment, 6 mg adenosine in 40 ml saline was infused into veins of an arterially occluded arm (N=4). In the third experiment, 40 mg aminophylline (adenosine receptor antagonist) in 40 ml saline was infused into veins of an arterially occluded arm (N=11). MSNA and BP significantly rose towards to the end of infusion of saline. Infusion of adenosine solution induced dramatic increases in MSNA and BP. The BP response in the adenosine trial was significantly greater than that in the saline trial (P<0.05). MSNA and BP did not significantly change in the aminophylline trial. The MSNA response in aminophylline trial was significantly lower than that in saline trial (Δ‐0.8±1.9 to Δ11.6±3.3 bursts/min, P<0.05). These observations indicate that stimulation of adenosine receptors accentuated the responses, and blockade of adenosine receptors attenuated the responses. The results suggest that adenosine receptor play a role in evoking the venous distension reflex in humans.
Acetate is an Active Metabolite of Ethanol: Increases Firing and Evokes Inward Currents through Activation of NMDA Receptors in RVLM Projecting CeA Neurons
Authors: Chapp Andrew , Huber Michael , Gu Mingjun , Carter Jason , Chen Qing‐Hui
Published: June 2021

We have recently reported that activation of central nucleus of amygdala (CeA) is involved in the in vivo sympathoexcitatory and pressor responses to acute challenge of acetate, the ethanol metabolite. However, the underlying neural mechanisms have not been determined. Here we investigated the role of acetate in regulating the in vitro excitability of CeA neurons with axon projecting to rostral ventrolateral medulla (CeA‐RVLM) under brain slice preparation. In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +250 pA injections and averaged 19 ± 1 Hz (n=8). Bath application of acetate (0, 7.5, 37.5 and 75 mM) increased the excitability of CeA‐RVLM neurons in a dose‐dependent manner. Maximum spike discharge in the presence of acetate (75 mM) was 44 ± 5 Hz (n=7). Pre‐treatment with AP5 (60 µM, n=7), the NMDA receptor competitive blocker or memantine (30 µM, n=9) a NMDA receptor channel pore blocker, significantly attenuated the increased excitability elicited by acetate (34 ± 4 Hz for AP5 and 20 ± 5 Hz for memantine respectively, p<0.05 vs 75 mM acetate alone). AP5 or memantine alone did not alter baseline excitability. In voltage‐clamp recordings, acetate application produced inward currents (22.4 ± 2 pA, n=6) which were completely abolished by pre‐treatment with AP5 (n=4) or memantine (n=6). Immunohistochemistry study demonstrates expression of NMDA NR1 in CeA‐RVLM neurons. Our data indicate that activation of NMDA receptors by acetate contributes to the increased excitability of CeA‐RVLM neurons, which may underlie the mechanism of sympathoexcitation and pressor response in vivo. AHA2640130 (QHC)
Acute oral ingestion of alcohol modulates muscle sympathetic neural activity differently in Caucasians and African Americans
Authors: Fonkoue Ida , Chen Qing‐Hui , Carter Jason
Published: June 2021

Recent studies consistently report that acute alcohol consumption increases muscle sympathetic nerve activity (MSNA) in humans. Despite the increased prevalence of hypertension and alcohol consumption in African Americans (AA), no studies have compared MSNA responses to alcohol consumption in AA and Caucasians (C). We hypothesized that alcohol consumption would increase MSNA in both AA and C, but that sympathoexcitatory responses would be augmented in AA. Five minutes of supine heart rate (HR), blood pressure, MSNA, and forearm blood flow were recorded in 8 AA (age, 23±1 yr) and 8 C (age, 22±1 yr) before and 45 min after consumption of 2.5ml of vodka per kg body mass. Increases in blood alcohol content (BAC) were greater in AA (0.105%) compared to C (0.087%; P<0.05). Alcohol elicited similar increases of HR in AA (62±5 to 70±4 beats/min; p<0.001) and C (62±5 to 68±5 beats/min; p<0.001). Contrary to our initial hypothesis, alcohol consumption increased MSNA in C (14±3 to 27±5 bursts/100 heart beats; P<0.05), but did not alter MSNA in AA (28±7 to 29±6 bursts/100 heart beats). Forearm vascular conductance (FVC) was not altered by alcohol consumption in AA or C. However, when sympathetic vascular transduction was calculated (i.e., FVC/MSNA), alcohol consumption reduced sympathetic vascular transduction in C (2.4±0.4 to 1.1±0.2 units; p<0.01), but not AA (1.3±0.4 to 1.3±0.4 units). These findings suggest acute alcohol consumption influences resting MSNA and MSNA‐vascular coupling differently in AA and C, but it is unclear if the different levels of BAC, and perhaps rates of alcohol metabolism, are contributing to these racial differences.
Premature Ventricular Contractions Trigger Non‐Baroreceptor Mediated Sympathetic Bursting
Authors: Ratcliffe L , Nightingale A , Paton J , Hart E
Published: June 2021

Premature ventricular contractions (PVC) trigger muscle sympathetic nerve activity (MSNA) of greater amplitude and duration compared to MSNA in normal sinus rhythm (NSR). The increase is usually attributed to a fall in diastolic blood pressure (DBP) that unloads arterial baroreceptors evoking a MSNA burst. We describe a case of spontaneous bigeminy where increased MSNA was not provoked by a fall in DBP (as most PVC caused systolic ejection) suggesting other pathways controlling MSNA during bigeminy.

In a normotensive woman (53 years), MSNA (peroneal microneurography), blood pressure and electrocardiogram (ECG) were recorded. Data was analysed according to ECG rhythm: NSR, NSR with PVC, and bigeminy.

Average DBP was highest in bigeminy (61 ± 0.4 mmHg, n=26 p<0.05) compared to NSR (57 ± 0.5 mmHg, n=35), and NSR with PVC (56 ± 0.6 mmHg, n=45). Total MSNA activity (arbitrary units per 10 heart beats, AU/10hb), was greatest in bigeminy (224.1 AU/10hb), and similar in both NSR (196.8 AU/10hb) and NSR with PVC (199.2 AU/10hb). Higher temporal resolution analysis demonstrated that MSNA burst area was highest in NSR with PVC (42.3 ± 1.7 AU). Interestingly, despite the DBP being highest in bigeminy the MSNA burst area was also higher in bigeminy (34.5 ± 0.1 AU) compared to NSR (12.7 ± 1.3 AU). PVC generating a systolic ejection were associated with a higher DBP compared to PVC not generating a systolic ejection (54.4 ± 0.4 vs. 48.8 ± 2.6 mmHg) but a trend suggests that the area of the post‐PVC MSNA burst was not different (51.5 ± 5 AU vs. 43 ± 1 AU p=0.07).

Our data suggest that DBP during a PVC is not the only factor determining MSNA burst size. We propose that early systolic emptying and left ventricular &/or coronary artery mechanoreceptor reflexes may be important in regulating MSNA burst size during a PVC.

BHF supported.
Age‐dependent Changes of CO2 Chemoreflex and Blood Pressure (BP) in Juvenile and Adult Spontaneously Hypertensive Rats (SHRs)
Authors: Li Aihua , Nattie Gene
Published: June 2021

Activation of central chemoreceptors by CO2 increases BP, sympathetic nerve activity (SNA), and breathing. Experiments using SHRs, a well‐established neurogenic hypertensive model, have shown that the high BP is associated with elevated SNA and peripheral chemoreflexes, and an impaired baroreflex. Earlier we showed that adult SHRs have enhanced ventilatory (VE) and BP responses to hypercapnia, and blocking both orexin receptors can normalize such enhanced responses and markedly lower BP. Here, using juvenile SHRs (P42‐60), we report that: 1) VE responses to normoxic‐hypercapnia and hyperoxic‐hypercapnia (179±25 & 162±38 %increase respectively) are higher than that of age‐matched normal rats (118±22 & 113±26 %increase, respectively), but lower than that of adult SHRs (226±39 % increase)(P<0.05); 2) resting BP is higher than of age‐matched normal rats (130±12 & 99±5mmHg respectively), but lower than that of adult SHRs (157±12mmHg)(P<0.05). Summary, in SHRs 1) significant increases in both CO2 chemoreflex and BP are measureable in juveniles; 2) the enhanced CO2 chemoreflex persists while peripheral chemoreceptors were inhibited by hyperoxia indicate an important role for central chemoreflex in neurogenic hypertension. We suggest that the enhanced CO2 chemoreflex in early life may be causally linked to the development of neurogenic hypertension in SHRs, and modulation of the overactive CO2 chemoreflex could be beneficial in treating neurogenic hypertension. (HL 28066)
Physical Activity Motivation: are Nucleus Accumbens Dopamine and Opioid Signaling Involved
Authors: Ruegsegger Gregory , Toedebusch Ryan , Booth Frank
Published: June 2021

Neuro‐molecular mechanisms that regulate physical activity motivation are poorly understood. The mesolimbic dopamine system has been postulated to influence rewarding behaviors and may be involved in the central control of physical activity motivation. Therefore, we investigated 1) differences in nucleus accumbens (NAc) mRNA expression of dopamine‐related genes in rats selectively bred for low motivation for voluntary running (LVR) versus high motivation for voluntary running (HVR) behavior after 9 weeks of voluntary wheel running or being sedentary (n=8 per group), and 2) how NAc opioid receptor mu1 (Oprm1) modulation impacts nightly running behavior in LVR rats (n=7). On average, HVR with wheels ran significantly further (23.88 ± 6.69 vs. 2.56 ± 0.45 km/day), longer (387.60 ± 14.94 vs. 60.48 ± 9.91 min/day), and faster (61.73 ± 1.25 vs. 41.91 ± 2.45 m/min) than LVR. Relative mRNA levels of the dopamine‐related genes dopamine receptor1 (Drd1), Drd2, Drd5, tyrosine hydrolase, and protein kinase inhibitor a were significantly decreased in LVR vs. HVR, regardless of running wheel availability. Similarly, Oprm1 mRNA level was ~4.3‐fold lower in the LVR vs. HVR (p < 0.001). Surprisingly, no differences in other dopamine‐target genes were observed between LVR and HVR. In LVR, NAc injection of the Oprm1 agonist DAMGO decreased running for the entire dark‐cycle and for hours 4‐12 of the dark cycle (p < 0.05) at a dose of 2.5μg per side, but not with 0.25μg per side. Oprm1 antagonist naltrexone injection (5, 10, and 20μg per side) had no significant impact on running behavior. In conclusion, mRNA level differences related to NAc dopamine and opioid function between LVR and HVR may account for differences in physical activity motivation, and modulation of opioid signaling may alter voluntary running behavior.
Hypoxia Activates Extracellular Signal‐Regulated Kinase (ERK) via EGFR Activation in Human Pulmonary Microvascular Endothelial Cells
Authors: White Hilary , Jin Yi , Chicoine Louis , Chen Bernadette , Nelin Leif
Published: June 2021

We have previously shown that hypoxia‐induced proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on epidermal growth factor receptor (EGFR)‐mediated arginase II induction. We hypothesized that hypoxia‐induced EGFR activation leads to phosphorylation of extracellular signal‐regulated kinase (ERK) which then leads to increased arginase II expression in hPMVEC. Cells were incubated in hypoxia (5% CO2, 1% O2) for 0, 2, or 24 hours and western blotting was performed for phospho‐ERK and total ERK. We found that hypoxia resulted in greater levels of phospho‐ERK protein with little effect on total ERK protein levels at both 2 and 24 hours. hPMVECs were transfected with an siRNA against EGFR or scramble for 24 hours, allowed to recover for 24 hours, and then incubated in hypoxia or normoxia (5% CO2, 20% O2) for 24 hours. Cells incubated in hypoxia and treated with EGFR siRNA had lower phospho‐ERK levels than hypoxic cells treated with scramble. Furthermore, hypoxic hPMVECs treated with the EGFR siRNA had lower levels of arginase II protein than did hypoxic cells treated with scramble. In the normoxia exposed cells there were lower levels of arginase II protein than in hypoxia exposed cells and there was little effect of the EGFR siRNA on arginase II protein levels. Our findings suggest that in hPMVEC hypoxia activates EGFR which in turn phosphorylates ERK eventually leading to increased levels of arginase II. We speculate that ERK may be a potential therapeutic target to prevent or reverse the vascular remodeling seen in pulmonary hypertension associated with hypoxia.
The Src Family Tyrosine Kinases yes and src Have Divergent Effects on Cytokine‐Induced Apoptosis in Pulmonary Endothelial Cells Through Divergent Downstream Effects on PI3K and ERK Pathways
Authors: Nelin Leif , Jin Yi , White Hilary , Chen Bernadette , Liu Yusen
Published: June 2021

In pulmonary endothelial cell culture studies we found that the Src family tyrosine kinase (STK) yes activated caspase 3, while the STK src inhibited caspase 3 activation. We hypothesized that the divergent effects of yes and src were due to their differential effects on the downstream activation of extracellular signal‐regulated kinase (ERK) and/or phosphatidylinositol‐3‐kinase (PI3K) pathway. To test this hypothesis, human pulmonary microvascular endothelial cells (hPMVEC) were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls for 24 hours. After a 24 hour recovery period the hPMVEC were treated with cytomix (LPS, IL‐1β, TNF‐α, and IFN‐γ). Cytomix treatment increased levels of phospho‐ERK (pERK). Treatment with siRNAyes enhanced cytomix‐induced pERK levels, while siRNAsrc blunted the cytomix‐induced pERK levels. Inhibiting the ERK pathway using U0126 prevented the cytomix‐induced increase in pERK and increased cytomix‐induced caspase 3 activity in hPMVEC. Treating hPMVEC with cytomix induced Akt activation which was inhibited by siRNAsrc, while siRNAyes had little effect on Akt activity. Inhibiting PI3K using LY294002 prevented cytomix‐induced ERK activation and augmented cytomix‐induced cleaved caspase 3 levels. Taken together our data demonstrate that yes activation blunts the ERK cascade in response to cytomix resulting in greater apoptosis, while cytomix‐induced src activation induces PI3K pathway which in turn activates Akt and ERK to inhibit apoptosis.
Endothelial dysfunction in pulmonary arterial hypertension (PAH)
Authors: Thambiayya Kalidasan
Published: June 2021

PAH is a disease of the small pulmonary arteries. It is characterized by vascular proliferation and remodeling, vasoconstriction, and increases in blood pressure that often results in right ventricular hypertrophy. Endothelial dysfunction seems to occur early in PAH and play an integral role in mediating the structural changes in the pulmonary vasculature. We characterized the time dependent changes in endothelial‐derived vasoactive mediators (prostacyclin, endothelin‐1, and thromboxane) in blood plasma in hypoxia model PAH. Our results demonstrated that prostacyclin significantly decreased (10 folds relative to normoxic mice) as early as 6hrs in hypoxic mice. Tyrosine receptor kinase inhibitor (SUGEN) which causes endothelial dysfunction exacerbated hypoxia‐mediated prostacyclin decrease. The decrease in prostacyclin correlated to the severity of PAH (as determined by increases in right ventricular systolic pressure and right ventricular hypertrophy). Our observations suggested that a) endothelial dysfunction occurs early in PAH progression and may orchestrate the development of PAH; b) patients with low levels of circulating prostacyclin as a result of other lung diseases may be potential candidates for PAH; c) drugs that restore the altered balance of endothelium‐derived vasoactive mediators can be used to treat PAH with some success; and d) a greater understanding of the role of the endothelium in PAH will facilitate the evolution of newer, targeted therapies
Role of FK506 Binding Protein 51 in Pulmonary Arterial Hypertension
Authors: Vasauskas Audrey , Cioffi Donna
Published: June 2021

Pulmonary arterial hypertension (PAH), a disease of the small pulmonary arteries, is characterized by pulmonary vasoconstriction, vascular cell proliferation, and remodeling leading to right ventricular hypertrophy and failure. Despite advances in treatment, disease management and prognosis remain grim. Thus, there is a critical need to better understand the mechanisms underlying cellular remodeling and subsequent reduction of luminal area in pulmonary arteries. In cases of heritable disease, mutations in the bone morphogenetic protein receptor 2 (BMPR2) predispose individuals to development of PAH. Recent studies show an increase in TGFβ‐1 levels and signaling in PAH, and BMPR2 mutations may lead to increased TGFβ signaling. Increases in TGFβ signaling associated with decreased BMPR2 expression can promote cellular reprogramming and vascular remodeling. Recent evidence implicates FK506‐binding protein 51 (FKBP51) as a regulator of cellular remodeling in certain cancers. Additionally, TGFβ‐1stimulates cellular reprogramming in pulmonary artery endothelial cells (PAECs), and a cooperative relationship between TGFβ and FKBP51 exists in malignant cell progression. Our preliminary work indicates increased expression of FKBP51 in PAECs in a rat model of PAH, and treatment of PAECs with TGFβ‐1 increased FKBP51 levels compared to control cells. Overexpression of FKBP51 in PAECs led to an increase in TGFβ. Finally, in BMPR2‐deficient PAECs, FKBP51 and α‐smooth muscle actin protein levels were elevated. Thus, we hypothesize that in PAECs of PAH, and notably in cases of BMPR2 deficiency, FKBP51 is upregulated, increasing TGFβ in a positive‐feedback loop, resulting in the pro‐proliferative, anti‐apoptotic phenotype of PAH. Supported by R01HL107778
Proteomic Study Reveals Altered Carbamyl‐phosphate Synthetase I in Congenital Heart Disease‐associated Pulmonary Arterial Hypertension
Authors: Zhang Xi , Yang Qin , Wang Jun , Liu Zhi‐Gang , Liu Xiao‐Cheng , He Guo‐Wei
Published: June 2021

Pulmonary arterial hypertension (PAH) associated with congenital heart disease and left‐to‐right shunt is characterized by increased pulmonary vascular resistance and remodeling. Endogenous nitric oxide (NO) derived from arginine supplied by the urea cycle is critical for the maintenance of normal pulmonary arterial pressure. The rate‐limiting step in the urea cycle is catalyzed by a mitochondrial enzyme, carbamoyl‐phosphate synthetase I (CPSI). We examined the hypothesis that CPSI may be associated with PAH by an proteomic method using iTRAQ to compare protein profiles in pooled plasma samples of ventricular septal defect (VSD) patients with PAH (VSD‐PAH), VSD, and normal controls (n=20 for each). Among more than 150 differential plasma proteins, CPSI was 2‐fold down‐regulated in VSD‐PAH. We further validated CPSI in new VSD‐PAH and VSD patients as well as controls by ELISA. The plasma CPSI level in VSD‐PAH (109.2±7.718 pg/ml, N=40) was significantly lower (P<0.01) than that in VSD (196.5±35.68 pg/ml, N=21) and normal controls (431.8±41.42 pg/ml, N=37, p<0.0001). Thus we have for the first time identified alterations of the mitochondrial enzyme CPSI in the plasma of VSD‐PAH patients, suggesting important role of the urea cycle‐arginine‐NO pathway in PAH and providing important information for biomarkers in PAH and possible future interventional strategy.

Supported by NSFC 81170148, China Postdoc Sci Foundation 2014M561181, National S & T Major Project 2013ZX09303004‐005.
Identification and Investigation of Mucin 1‐Mediated Kidney Disease
Authors: Schuler Bryce , Tsaih Shring‐Wern , Worthey Elizabeth , Kirby Andrew , Stevens Christine , Daly Mark , Jacob Howard
Published: June 2021

Establishing a causal link between genomic variation and pathology is the foundation of the successful application of genomics to clinical medicine. Whole genome sequencing (WGS) increases the resolution with which we can investigate the genome, but this increased resolution leads to an increase in the number of potentially damaging or disease causing variants identified. Consequently, WGS in the analysis of disease requires both bioinformatics approaches for efficient prioritization of variants as well as experimental evidence that demonstrates that the variant alters the function of the protein which directly contributes to the development of the disease phenotype(s). We have identified a family with an autosomal dominant chronic tubulointerstitial nephritis that presents insidiously between 20 and 60 years of age, leads to end stage renal disease, and subsequently requires dialysis and transplantation. By utilizing a WGS approach in affected members of this family, literature queries, and targeted gene analyses, we were able to identify a single base insertion in mucin 1 (MUC1) that segregated with the disease in this family, was absent in the general population, and was bioinformatically interesting. We are utilizing a mutant Muc1 rat model in an effort to confirm that MUC1‐dysfunction is causative in this phenotype, to more completely characterize this disease process, and to investigate potential treatment strategies with the hope that subsequent findings can, at least in part, be directly applied to patient care.

Funding was provided by the NIH (award 5T32HL007852‐17) and an extramural donation.
Klotho Deficiency Causes Kidney Fibrosis via Activation of the Notch2 Signaling pathway
Authors: Xu Yuechi , Sun Zhongjie
Published: June 2021

Background

Klotho was originally identified as an aging‐suppressor gene. Mutation of Klotho gene causes severe kidney damage. In humans, chronic kidney disease is associated with diminished Klotho protein expression. The purpose of this study is to investigate molecular mechanism that mediates Klotho deficiency‐induced renal fibrosis.

Methods & Results

Trichrome staining showed that Klotho gene mutation enhanced tubular collagen deposition, indicating that Klotho deficiency caused kidney fibrosis. Interestingly, the Notch2 activity was increased significantly in kidneys of Klotho mutant mice, suggesting that Klotho may regulate Notch2 activity. Overexpression of Klotho suppressed Notch2 activity and decreased expression of fibrosis markers (smooth muscle actin and collagen I) in mouse kidney tubule cells. Knockdown of Notch2 also attenuated fibrosis markers. Therefore, Notch2 activation may mediate Klotho deficiency‐induced fibrosis formation. The functional interaction between Klotho and Notch2 was assessed by the lectin precipitate assay. Interestingly, the result showed that Klotho exhibited neuraminidase activity which inhibited Notch2 activity through removing α 2, 6 Neu5Ac from Notch2. A neuraminidase inhibitor (DANA) blocked the inhibitory effect Klotho on activation of Notch2.

Conclusion

Klotho deficiency‐induced renal fibrosis formation may be, at least in part, attributed to activation of Notch2. Klotho regulates Notch2 activity via its neuraminidase activity.
Twist2 is a Novel Regulator of Renal Fibrosis
Authors: Nichols LaNita , Grunz‐Borgmann Elizabeth , Parrish Alan
Published: June 2021

Morphological and functional changes occur in the aging kidney, including the loss of kidney mass and an increase in fibrosis, an abnormal accumulation of the extracellular matrix (ECM). Functional changes include a decrease in glomerular filtration rate (GFR), proteinuria and reduced ability to concentrate urine. Age is a major risk factor for chronic kidney disease (CKD) while kidney dysfunction and fibrosis are hallmarks of CKD. Thus, aging and CKD have many common elements. Male Fischer 344 rats are an excellent animal model as they develop severe renal disease and fibrosis similar to CKD with aging. However, a soy diet started at 16‐months, the age dysfunction is first detected, has been demonstrated to reverse age‐related damage and fibrosis to the kidneys. Sequencing of kidney RNA purified from aged soy‐fed rats, compared to control, found decreased expression of Twist2, a basic Helix‐Loop‐Helix (bHLH) transcription factor. However, rats fed a normal diet had increasing Twist2 expression with age, as compared to young. A stable cell line of Twist2 overexpression was generated in NRK‐52E cells. Again, RNA sequencing revealed the expression of several genes significantly changed, including up‐regulation of Serpine1. Serpine 1 encodes for the serine protease inhibitor, plasminogen activator inhibitor‐1 (PAI‐1). Interestingly, PAI‐1 expression increased with age and parallels increasing Twist2 expression. Increased PAI‐1 expression is associated with increased fibrosis, while PAI‐1‐/‐ mice attenuates this affect. Twist2 recognizes the E‐box element, nCAnnTGn, of which the Serpine‐1 promoter has several. Thus, Twist2 is a novel regulator of Serpine1/PAI‐1 and fibrosis in the kidney.
Analysis of PTEN and ARID1A expression in Endometriosis disease using RT‐PCR assay
Authors: Rosado‐Alicea Jamie , Lopez Pablo , Flores Idhaliz , Ortiz Carmen , Arias‐Delfi Yvette , Monteiro Janice
Published: June 2021

Endometriosis is defined as the presence of endometrial glands and stroma outside of the uterine musculature and endometrial lining. Regulation of gene expression is a flexible, yet stable, mechanism that maintains a cellular memory that could result in a disease state.The aim of this study was to analyse mRNA levels expression in endometrial tissue and endometrium from women with and without endometriosis. We will analyze mRNA transcription activity in PTEN (phosphatase and tensin homolog), and ARID1A (AT rich interactive domain 1A) in fresh tissue from patients with endometriosis using RT‐PCR assay. In this study, we used NCBI database for PTEN and ARID1A transcript. The RNA extraction was performed using commercial kit Qiagen in endometrium and endometriosis fresh tissues. RNA integrity, quality, and purity were stringently analyzed by Experion technology. RT‐PCR analysis showed no consider PTEN and ARID1A mRNA expression in eutopic and ectopic endometrium. The expression levels of PTEN transcript (Exon5 and9) and ARID1A transcript (Exon 9 and 20) by RT‐PCR analysis were observed of mRNA expression of the PTEN and ARID1A in endometriotic tissues vs. controls. We observed that no differences statically between in eutopic and ectopic endometrium patterns of PTEN transcript P value (0.6499) and ARID1A transcript P value (0.9789) in this transcription of target genes. Our study will continue to increase more sample to improve the power analysis of the samples and provide further evidence to different patterns of PTEN and ARID1A transcription of target of these genes in endometriosis to transition to cancer.
The Effects of Genetic Background on Psychiatric Behaviors in CACNA1C +/‐ Mice
Authors: Stephens Phillip , Sittig Laura , Palmer Abraham
Published: June 2021

Bipolar disorder and schizophrenia are genetic‐based psychiatric disorders. The heritability of bipolar disorder and schizophrenia, around 70% ‐ 90% and 83%, respectively, shows that the genetic variation in certain genes help leads to the development of these psychiatric disorders and that studying these genetic variations will help us understand the physiological mechanisms that lead to these psychiatric disorders. Among other genes, CACNA1C was identified as a gene, via large‐scale genome‐wide association analyses, that contributes to a person's risk for these disorders. However, the effects of the genes found only account for a small percentage of the heritable risk of these psychiatric diseases. There has been evidence that the interactions between genes, or epistasis, may account for a significant amount of the heritability. We identified different genetic interactions with CACNA1C by utilizing mouse genetics. The behavioral phenotypes of both wild‐type and CACNA1C +/‐ mice of 19 different genetic backgrounds were observed via a prepulse inhibition test. Each test measures specific behaviors that correlate to neurobiological characteristics of bipolar disorder and schizophrenia. Our data suggests that the CACNA1C +/‐ mutation has different effects on the mouse's prepulse inhibition, depending on the genetic background. The data also suggests that the CACNA1C +/‐ mutation trends toward a decreased startle response in females, while not in males. This allows us to suggest that the CACNA1C gene does undergo epistasis. In the future, we will conduct a genome‐wide association study for both trait markers and modifier markers. We will then identify high‐priority candidate modifier genes that we may be able to confirm in human data sets.
IDENTIFICATION OF A GENOMIC DRUG TARGET FOR KIDNEY INJURY AND THERAPEUTIC SCREENING OF NATURAL PRODUCTS DERIVED SMALL MOLECULES
Authors: Jia Zhen , Pasco David , Johnson Ashley , Garrett Michael
Published: June 2021

Through positional cloning, Arhgef11, a Rho guanine nucleotide exchange factor was implicated in kidney injury exhibited in the Dahl salt‐sensitive (S) rat, a model of chronic kidney disease (CKD). Previous work found that genetic knockdown of Arhgef11 in HEK293 results in reduced RhoA activity (~20%), decreased activation of Rho‐ROCK pathway (~50%), and less stress fibers (vs. control). Primary proximal tubule cells (p‐PTC) cultured from the S rat exhibit increased expression of Arhgef11, activation of Rho‐ROCK, and are more prone (vs. control) to epithelial‐mesenchymal transition (EMT), a driver of fibrosis. S rat primary vascular smooth muscle cells (VSMC) cultured from kidney microvessels (k‐VSMC) also demonstrate activation of Rho‐ROCK that corresponds with increased proliferation, migration, and apoptosis. With knowledge of the role of Arhgef11 and Rho‐ROCK pathway in CKD, we sought to evaluate natural product derived small molecules to identify those that could inhibit Arhgef11‐RhoA interaction or lead to decreased expression of Arhgef11. Using an in vitro cell culture system, several hundred natural product compounds were screened for ability to inhibit RhoA activity and expression of Arhgef11, which identified 3 compounds that are currently being characterized on ability to improve downstream EMT or VSMC phenotypes. In summary, genetic analysis of a model of CKD identified a gene/pathway involved in kidney injury that served as a genomic target to screen natural product derived small molecules that could ultimately lead to new treatment for CKD.
Familial Hyperkalemic Hypertension (FHHt) KLHL3 Mutations Disrupt Binding of ROMK Endocytic Regulatory Protein ARH
Authors: Woodward Owen , Kim Boyoung , Fang Liang , Welling Paul
Published: June 2021

Fammilial Hyperkalemic Hypertension (FHHt) is caused by mutations in CUL3 and KLHL3, components of an E3 ligase complex, and is characterized by a marked reduction in distal potassium secretion and reduction in ROMK (KCNJ1) expression, but the mechanism is not well understood. Here, we explore the possibility that CUL3/ KLHL3 regulate ROMK through direct targeting and ubiquitination of ARH, the key ROMK endocystic regulatory protein, independent of Na+ delivery to the distal nephron. We found endogenous KLHL3 can co‐immunoprecipitate ARH from mouse kidney cortex and that GST‐KLHL3 can directly bind HIS‐ARH, an interaction requiring the propeller domain of KLHL3 and the N‐terminal portion of ARH, and results in increased ARH ubiquitination and degradation. Significantly, we found FHHt mutations in KLHL3 are disrupting. Many of FHHt mutants in the KLHL3 propeller are not efficiently expressed and the loss‐of–function leads to increased ARH abundance when co‐expressed in COS7 cells. One KLHL3 mutant, S433G, had no significant expression defect but blocked the ARH interaction. We conclude that FHHt KLHL3 mutants either through defects in expression or binding result in increased ARH abundance and ROMK endocytosis, contributing to the hyperkalemia of FHHt patients.
Novel pH‐dependent Astrocyte‐Neuron Crosstalk in Hippocampal CA1 Region, Not Observed After the Knockout of the Anion Exchanger 3 (AE3)
Authors: Salameh Ahlam , Boron Walter
Published: June 2021

The anion exchanger 3 (AE3) is highly expressed in CA1 hippocampal neurons but not astrocytes. It acid loads the cytoplasm by facilitating the exchange of intracellular HCO3− for extracellular Cl‐. In the present study we investigate the role of AE3 activity in astrocyte‐neuron crosstalk in hippocampal CA1 region during metabolic acidosis (MAc). We bathed our cells in a control (Ctrl) solution that has [HCO3−] of 22 mM at 5% CO2, which sets pH at 7.4. We used a MAc solution with pH 7.2, lowering [HCO3−] from 22 to 14 mM at a fixed 5% CO2. We used ratiometric fluorescence imaging of the pH sensitive dye BCECF to monitor pHi changes during a 7‐min MAc exposure on: [1] a mixed culture of neurons and astrocytes from the CA1 regions from wild‐type (WT) and AE3 knock‐out (AE3‐/‐) mice, or [2] a culture of astrocytes only from the CA1 regions from WT and AE3‐/‐mice. In the mixed culture, astrocytes' steady‐state pHi is 6.99 ± 0.01 in WT and 6.92 ± 0.01 in AE3‐/‐. The MAc‐induced acidification rate is ‐3.3 × 10‐3 ± 9.1 × 10‐4 in the WT and ‐1.8 × 10‐3 ± 6.7 × 10‐4 in the AE3‐/‐. In the astrocytes‐only culture, steady‐state pHi and MAc‐induced acidification rate in the AE3‐/‐ remain the same as in the mixed AE3‐/‐ culture. However, in the WT astrocytes‐only culture steady‐state pHi (6.91 ± 0.00) and MAc‐induced acidification rate (‐1.7 × 10‐3 ± 6.0 × 10‐5) are similar to the values for AE3‐/‐ astrocytes. The higher steady‐state pHi in the WT mixed culture compared to the WT astrocytes‐only culture presumably occurs because neurons send a message to the astrocytes that [1] increases the astrocytes' steady‐state pHi and [2] speeds the MAc‐induced acidification rate. This crosstalk is absent in AE3‐/‐ cells, which may explain the well‐known hyper‐excitability of AE3‐/‐neurons.