bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective Small Molecule Inhibitors Of Heterotrimeric G‐Protein Signaling For The Treatment Of Ovarian Cancer
Authors: Bigham Kevin J , Maher Ellen , Hazard Starr E , Blumer Joe B , Peterson Yuri K
Published: June 2021

Overactive signaling at the level of hormone, receptor, or G‐protein can initiate and potentiate cellular transformation and other diseases. We hypothesize that direct inhibition of overactive G‐protein signaling is cytotoxic to ovarian cancers. We describe the development of selective small molecule inhibitors which bind to and stabilize the GDP‐bound form of the Galphai1. Initial inhibitors were identified using computational high throughput docking of a 280,000 small molecule database to Galphai1‐GDP and counter‐screened against Galphai1‐GTP and Galphaq‐GDP. Nucleotide exchange assays have verified selective G‐protein inhibition. This inhibition was supported by live cell bioluminescence resonance energy transfer (BRET) experiments measuring the interaction of Galphai1 with the regulatory protein AGS4 in HEK293 cells. Inhibition of Galphai1 but not Galphaq decreased cell viability in SKOV3 ovarian cancer cells. The mechanism of Gi pathway inhibition is also explored. Pharmacologic regulation of both receptor dependent and independent signaling through specific heterotrimeric subunits will provide a unique window into a major signaling axis while providing data and compounds for the treatment of a refractory disease like ovarian cancer.
D2L dopamine receptor‐mediated heterologous sensitization of adenylyl cyclase 2 is Gβγ subunit‐dependent
Authors: Conley Jason Michael , Watts Val
Published: June 2021

The focus of the present study was to identify components of the signaling pathways underlying the enhanced responsiveness of adenylyl cyclase 2 (AC2) to phorbol ester stimulation following persistent activation of the D2L dopamine receptor. The heightened sensitivity of adenylyl cyclases following persistent Gi‐coupled receptor activation is known as heterologous sensitization and is thought to be linked to dependence associated with drugs of abuse. Our studies utilized HEK293 cells expressing AC2 and the D2L dopamine receptor to examine sensitization of AC2. Co‐expression of either the G protein regulator AGS3 or the Gβγ sequestering protein βARKct‐CD8 resulted in a blockade of AC2 sensitization following persistent D2L receptor activation, suggesting that Gβγ subunits are important mediators of AC2 sensitization. Gβγ subunits modulate a variety of effector proteins that are potentially embedded in the signaling pathways that support AC sensitization. To broadly study these signaling pathways, steps have been taken to explore cAMP biosensor technologies and miniaturize our sensitization experiments to make them amenable for siRNA library screening. Taken together, we have identified Gβγ subunits as components of AC2 sensitization and have devoloped assay formats to identify downstream signaling pathways underlying this adaptive response. Our work was supported by grant MH060397 to VJW.
Endothelial CaMKIIs and their alteration in mesenteric arteries from hypertensive mice
Authors: Charbel Chimène , Toussaint Fanny , Blanchette Alexandre , Villeneuve Louis Robert , Ledoux Jonathan
Published: June 2021

Modulation of local calcium levels appears to be an important regulatory mechanism for endothelial function. Localized spontaneous endothelial calcium oscillations have recently been described within the myoendothelial projection. Interestingly, calmodulin‐dependent protein kinase II (CaMKII) is involved in endothelial function and is characterized by its ability to decode oscillatory calcium signals into specific outcomes. The aim of this study was then to determine the activation of CaMKII by calcium pulsars in endothelial cells from mesenteric arteries and their alteration in hypertension. qPCR experiments suggest that all four CaMKII isoforms (α, β, δ and γ) are expressed in mesenteric arteries but their expression was significantly decreased (27–57%) in hypertensive animals. Immunohistochemistry confirmed that all isoforms are expressed in mesenteric endothelium and were homogeneously distributed in normotensive animals. However, hypertension is correlated with a distribution of endothelial CaMKII in clusters. Ca2+ pulsars agonists appear to stimulate clustering of CaMKII as evidenced by immunohistochemistry. This data suggest that endothelial CaMKII is activated by Ca2+ pulsars and that this mechanism is altered in hypertension.

Supported by CIHR, FRSQ, CFI and HSFC
Farnesoid X Receptor and Hepatocyte Nuclear Factor 4 alpha Interact to Regulate Gene Transcription in the Liver
Authors: Thomas Ann M. , Hart Steven N. , Zhong Xiao‐bo , Guo Grace L.
Published: June 2021

Farnesoid X receptor (FXR) is a ligand‐activated nuclear receptor critical for liver function. Studies show mice deficient in FXR develop cholestasis, hyperlipidemia and liver tumors. Hepatocyte nuclear factor 4α (HNF4α) is also a nuclear receptor critical for regulating liver development, differentiation and function. The old paradigm suggested linear activation of target gene transcription following direct binding of FXR to gene regulatory regions. However, we show that FXR activates gene transcription by cooperating with HNF4α to regulate gene transcriptional activation in the liver. Data obtained from chromatin immunoprecipitation followed by massive parallel sequencing (ChIP‐seq) of mouse liver, shows that about 40% of FXR binding sites in liver overlapped with HNF4α binding sites. Genes that are bound by both FXR and HNF4α showed more active transcription, revealed by higher levels of H3K4 mono‐methylation and mRNA expression, than for genes that were bound by either factor alone. In addition, the majority of FXR and HNF4α binding sites are close in proximity, about 25–30 base pairs apart. Luciferase assays suggest that HNF4α may have an additive effect on FXR transcriptional activation. Co‐immunoprecipitation assays imply FXR may directly interact with HNF4α. In conclusion, this study shows evidence of a cooperative interaction between FXR and HNF4α in regulating liver gene transcription.
Sphingosine‐1‐phosphate mediate vasoconstriction in femoral and mesenteric arteries of mice via S1P3 receptors
Authors: Soydan Guray , Waeber Christian
Published: June 2021

We have previously shown that Sphingosine‐1‐phosphate (S1P) induces potent vasoconstriction in cerebral arteries by stimulating S1P(3) receptors, but little is known about the effect of S1P on femoral and mesenteric arteries. We therefore aimed to investigate the functional effect of S1P on femoral and mesenteric arteries of mice. Femoral and second or third branches of mesenteric arteries of C57BL/6 and S1P(3) receptor null male mice were isolated, mounted into a wire myograph. Constriction and relaxation responses were evaluated. S1P concentration‐dependently constricted both femoral and mesenteric arteries with intact endothelium in C57 mice but did not affect the vascular tone in arteries of S1P(3) null mice. Vasoconstriction to KCl and phenylephrine did not differ between C57 and S1P(3) null mice. After a preconstricton with submaximal concentration of phenylephrine, S1P and its vehicle induced a weak vasorelaxation in femoral and mesenteric arteries of both C57 and receptor null mice and these relaxations did not change in the presence of a nitric oxide synthase inhibitor, L‐NAME. Vasodilation to acethylcholine and sodium nitroprusside did not differ between C57 and S1P(3) null mice. Our findings suggest that S1P constricts femoral and mesenteric arteries of mice with intact endothelium through S1P(3) receptors and the vehicle of S1P causes a endothelium‐independent vasorelaxation.
The vasodilatory peptide maxadilan increases plasma leakage in postcapillary venules due to activation of leukocytes via CXCL2‐release
Authors: Svensjö Erik , Saraiva Elvira M , Barja‐Fidalgo Christina , Bozza Marcelo T , Lerner Ethan A. , Teixeira Mauro M , Scharfstein Julio
Published: June 2021

Maxadilan (MAX) isolated from the sand fly Lutzomyia longipalpis induce plasma leakage in postcapillary venules of the hamster cheek pouch (HCP) via PAC‐1‐receptor stimulation. Arteriolar dilation, plasma leakage (FITC‐dextran 150) and accumulation of rhodamine‐labeled leukocytes in the HCP was measured after topical applications of MAX 134 nM alone or combined with 8 μM M‐65, a PAC1‐receptor antagonist or after dextranSO4 (500 kDa, 2–4 mg/kg i.v.) or after repertaxin (CXCR2‐antagonist, 30–60 mg/kg i.v.) in order to study role of leukocyte activation by MAX. Measurements were made in images of a digital camera. Five groups of hamsters were studied, saline control, MAX 134 nM, MAX + M‐65, MAX + DxSO4 and MAX + repertaxin. Topical applications of MAX and M‐65 were made on the HCPs in 250 μl of saline and images were recorded for 60 minutes. There was no plasma leakage increase in the saline control group and a slight increase in leukocyte accumulation but there was a lasting increase in plasma leakage (180%, P < 0.05) and leukocyte accumulation (150%, P < 0.05) in the MAX‐group with peak responses at 40 min and 60 min, respectively, that was inhibited by M‐65, DxSO4, and repertaxin. Arteriolar dilation was inhibited by M‐65 and repertaxin but not by dextranSO4.

Conclusions

PAC1 receptor activation by maxadilan caused plasma leakage and leukocyte accumulation via chemokine (CXCL2) release independent of its vasodilatory effect.
Cell‐permeable Rnd3 protein reduces hemorrhagic shock/resuscitation‐induced microvascular leakage in the rat mesentery
Authors: Doggett Travis Matthew , Kurtz Kristine M , Sulzer Jesse K , Whitaker Annie M , Belaidi Marisa B , Souza‐Smith Flavia M , Molina Patricia E , Breslin Jerome W
Published: June 2021

Microvascular hyperpermeability is a significant clinical problem in trauma medicine. The underlying mechanisms involve formation of actin stress fibers in endothelial cells, leading to increased stress on intercellular junctions. We tested the hypothesis that Rnd3, which inhibits actin stress fiber formation, can inhibit hemorrhagic shock/resuscitation (HS/R)‐induced microvascular leakage. We used a conscious, unrestrained rat fixed‐pressure HS/R model (40 mm Hg for 1 h, followed by Lactated Ringers, 40% total blood removed (TBR) i.v. bolus + 2 X TBR over 1 h) or shams, after which the rats were anesthetized for intravital microscopy of the mesenteric microcirculation. FITC‐albumin (i.v.) served as a tracer of extravasation. Integrated optical intensity (IOI) of extravasated FITC‐albumin was measured. The mesentery was treated with either a cell permeable (CP)‐Rnd3 protein or vehicle. The results show that HS/R caused a marked increase in IOI over time (mean IOI 110 ± 34 at 60 min. vs. 20 ± 17 at BL, P<0.05) which was not observed in sham animals. CP‐Rnd3 treatment attenuated the rise in IOI (mean IOI 35 ± 14 at 60 min. vs. 21 ± 9 at BL, no difference). These data suggest that Rnd3 can resolve HS/R‐induced microvascular hyperpermeability in vivo. This novel strategy could potentially improve clinical management of trauma patients. Supported by NIH P20RR018766 and a grant from the American Heart Association.
Epac1/Rap1 and Rnd3 signaling pathways inhibit hemorrhagic shock‐induced venular hyperpermeability
Authors: Souza‐Smith Flavia Moreira , Doggett Travis M , Breslin Jerome W
Published: June 2021

Hemorrhagic shock (HS) causes significant microvascular leakage in the gut, which complicates resuscitation in shock patients. Our goal is to target signaling pathways that will improve microvascular barrier integrity following HS. We tested the hypothesis that activation of the Epac1/Rap1 pathway, or introducing excess Rnd3 protein to inhibit Rho/ROCK signaling, can reduce HS‐induced hyperpermeability in mesenteric venules. We utilized a model of fixed‐pressure HS in conscious, unrestrained rats. Rats were then anesthetized and the intestinal mesentery was excised to isolate venules for study. The permeability coefficient for FITC‐albumin (Ps) was determined in isolated, perfused, mesenteric venules (70–120 μm) from HS and control rats. The selective Epac1 activator 8‐pCPT‐2′‐O‐Me‐cAMP (8CPT), cell‐permeable (CP)‐Rnd3 protein, or no treatment was then applied to the venules from HS rats. The results show that venules from the HS rats had 5‐fold higher Ps than control. 8‐CPT and CP‐Rnd3 both caused a gradual decrease in Ps in HS venules, that was significantly different from untreated HS venules by the 30 min. time point. Our data suggest that Epac1 and Rnd3 both promote microvascular barrier integrity. In addition, 8CPT and CP‐Rnd3 can be used to reduce HS‐induced venular hyperpermeability. Supported by NIH P20RR018766 and a grant from the American Heart Association.
Coupling of Coronary Blood Flow to Cardiac Metabolism: Role of TRPV1 Channels and Local Changes in pH
Authors: Guarini Giacinta , Ohanyan Vahagn A. , Talasila Phani K. , Bratz Ian N.
Published: June 2021

TRPV1 channels, known to be activated by noxious stimuli such as pH changes, are thought to play a role in the regulation of the vascular tone. Accordingly, we hypothesized that TRPV1 activation by local pH changes during increased metabolic demand and/or ischemia would lead to modulation of myocardial blood flow (MBF). Further we will examine if TRPV1 channel dysfunction could contribute to the microvascular impairment in diabetic cardiomyopathy. To test our hypothesis, control (C57/B6), db/db and TRPV1 null mice (TRPV1−/−) were subjected to contrast stress echocardiography for quantification of MBF at baseline and during the infusion of the TRPV1 agonist capsaicin (CAP; 1–100 μg/kg). Arterial pressures (AP) and heart rate (HR) were simultaneously recordedfor double product (DP) measurement as a surrogate of myocardial oxygen consumption. In controls, CAP produced a dose‐dependent increase in MBF, which was significantly blunted in db/db mice, despite db/db having a higher DP. Low pH changes induced vasodilation in isolated mesenteric microvessels from controls, which was blunted in microvessels from db/db, TRPV1−/− and control after TRPV1 inhibition (SB366791), suggesting a portion of low pH induced vasodilation may be due to TRPV1 activation. We conclude that TRPV1 channels may be involved in the metabolic regulation of myocardial blood flow and this mechanism appears to be corrupted in diabetic mice.
Glucagon‐like peptide‐1 (GLP‐1) improves coronary microvascular endothelial function after cardiac arrest and resuscitation and prevents anoxia‐induced coronary microvascular endothelial cell death
Authors: Dokken Betsy B , Piermarini Charles V , Asghar Mostafa Aeen , Teachey Mary K , Gura Michael , Dameff Christian J , Heller Brian D , Hilwig Ronald W , Kern Karl B
Published: June 2021

Cardiac arrest and resuscitation cause dysfunction of the coronary microcirculation. The aims of this study were to determine: 1) effects of GLP‐1 on coronary endothelial microvascular function after cardiac arrest and resuscitation, and 2) effects of GLP‐1 on coronary endothelial cell viability after anoxia and reoxygenation in vitro. Ventricular fibrillation (VF) was induced electrically in 20 anesthetized domestic swine (30–35 Kg). Following 8 min of untreated VF, animals were resuscitated and randomized to receive either GLP‐1 (10 pM/Kg/min) or saline as placebo (PBO) for 4 hr. Coronary flow reserve (CFR) was measured before and after intracoronary administration of Substance‐P (0.2 nG/2 min) pre‐arrest, 1 and 4 hr post‐resuscitation. In addition, human microvascular coronary endothelial cells (HCMVECs) were subjected to 1 hr of anoxia after pre‐incubation with GLP‐1 (0.3 nM) followed by reoxygenation for 72 hr.

Results

CFR was increased in those treated with GLP‐1 (1 Hr: PBO group 0.8 ± 0.1, GLP‐1 group 1.3 ± 0.1; 4 Hr: PBO group 0.8 ± 0.1, GLP‐1 group 1.1 ± 0.1, p< 0.05). Viability of ECs decreased by 77% in untreated cells; GLP‐1 prevented anoxia‐induced cell death (untreated group 23% viable/total, GLP‐1 group 94% viable/total, p< 0.01).

Conclusions

GLP‐1 attenuates post‐resuscitation coronary microvascular endothelial dysfunction in vivo, and prevents anoxia‐induced HCMVEC cell death in vitro.
Challenging O2 delivery: metabolism coupling in small muscle mass exercise
Authors: Bentley Robert Francis , Hong Terence , Poitras Veronica , Kellawan Jeremy Mikhail , Pak Melissa , Jazuli Farah , Chung Beryl , Tschakovsky Michael E
Published: June 2021

PURPOSE

To understand how O2del: metabolism matching is defended in the face of increased duration of contraction‐induced mechanical impedance to exercising muscle blood flow.

METHODS

7 healthy young males, (21.8 ±1.8 yrs) performed a handgrip ramp exercise protocol (increase contraction force 2.5 kg every 3 min) under Control (CON; 2 s isometric contraction + forearm compression cuff inflation: 4 s relaxation duty cycle) and Impedance (IMP; 2 s contraction + forearm compression + extra 2 s of forearm compression: 2 s relaxation duty cycle). Forearm blood flow ((FBF (ml/min); brachial artery Doppler and Echo ultrasound), mean arterial blood pressure (MAP (mmHg); finger photoplethysmography) were measured. Forearm vascular conductance during relaxation (FVCrelax ml/min/100 mmHg)) was calculated (FBFrelax/MAP x 100).

RESULTS

Data are mean ± SD. Up to 7.5 kg workload, FBF (therefore O2del) was not different between conditions due to compensatory vasodilation in IMP (eg. 7.5 kg FVCrelax IMP 596 ±140 vs. CON 396 ±60, P<0.05). Above 7.5 kg workload a small pressor response in IMP was added (MAP IMP 103.5 ±7.8 vs. CON 98.8 ±8.6, P<0.05), but O2del: metabolism matching was not achieved.

CONCLUSIONS

Compensatory vasodilation successfully maintains O2del: metabolism matching at lower workloads. When it fails, a pressor response is evoked, but cannot maintain O2del: metabolism matching at higher workloads. NSERC.
Inter‐individual differences in coupling oxygen delivery to demand during a progressive exercise test
Authors: Bravo Michael , Kellawan Jeremy Mikhail , Moynes Jaclyn , Bentley Robert , Shantz Rebecca , Tschakovsky Michael E
Published: June 2021

Oxygen delivery (O2del) to exercising muscle is thought to be tightly coupled to metabolic demand, as evidenced by proportional increases in O2del with increasing exercise intensity. Little consideration has been given to investigation of inter‐individual differences in this coupling.

PURPOSE

To characterize inter‐individual differences in O2del:metabolism coupling during progressive increases in exercise intensity.

METHODS

8 healthy males (24.4 ±6.4 yrs) performed rhythmic isometric handgrip contractions (1 s:2 s contraction:relaxation duty cycle, contraction force increased by 2.5 kg every 3 min. until failure). Forearm O2del was calculated as arterial oxygen content (O2 saturation via pulse oximeter; [hemoglobin] via venous blood sample) x brachial artery blood flow (BABF; Doppler and Echo ultrasound).

RESULTS

Data are mean ±SD. From rest to end of the 17.5 kg stage the 1) total blood delivered to the forearm was 3565.282 ±1013.584 mL, coefficient of variation (CV) 28%, range 2321.132 to 5384.500 mL and 2) total O2 delivered to the forearm was 533.306 ±153.192 mL, CV 29%, range 312.520 to 786.140 mL. Mean blood flow at 17.5 kg was 345.510 ±89.782 mL.

CONCLUSIONS

Inter‐individual differences in blood flow and O2del:metabolism coupling during small muscle mass ramp exercise protocol can be substantial. Implications for understanding O2del:metabolism coupling need to be considered.
Voluntary exercise prevents cerebrovascular dysfunction and remodelling associated with atherosclerosis in mice
Authors: Bolduc Virginie , Duquette Natacha , Gillis Marc‐Antoine , Thorin Eric
Published: June 2021

Physical exercise (PEx) has beneficial vascular effects. In mouse cerebral arteries atherosclerosis is associated endothelial dysfunction and changes in the elastic properties. We hypothesized that PEx prevents these damages. Three‐month old (m/o) male LDLr−/−hApoB+/+ mice (ATX, n=9) and wild type (WT, n=9) controls were exposed, or not, to 3 months of voluntary PEx (≈6 km/day). At 6‐m/o, endothelium‐dependent flow‐mediated dilations (FMD), compliance and incremental distensibility were assessed. Brain vessels matrix metalloproteinase 9 (MMP‐9) activity was evaluated by zymography. Versus WT, FMD was reduced in ATX by 76% at 11.5±0.5 dynes/cm2 (p<0.05) but PEx prevented this decline. At 80 mm Hg, cerebrovascular compliance and incremental distensibility rise in ATX by 26 and 38% versus WT, respectively. PEx normalized these parameters (p<0.05). Pro‐MMP‐9 activity was higher in ATX (1.4±0.1 au) versus WT (1.1±0.1), but normalized by PEx (1.2±0.2) (p<0.05). Our data suggest that ATX affects both the endothelial function and wall elasticity in association with a rise in MMP‐9 activity. PEx prevented these changes demonstrating that it is beneficial to the cerebral circulation. Supported by CIHR MOP 89733
Reduced role of large conductance KCa channels in the response to noradrenaline after ischemia‐reperfusion in goat cerebral arteries
Authors: Garcia‐Villalon Angel Luis , Carreño Gonzalo , Fernandez Nuria , Monge Luis , Dieguez Godofredo
Published: June 2021

Vascular calcium‐activated potassium (KCa) channels seem to play a role in the regulation of cerebral blood flow. To analyze their functional state after cerebral ischemia‐reperfusion, the left middle cerebral artery (MCA) of anesthetized goats was subjected to 120‐min occlusion followed by 60‐min reperfusion. Isolated segments from the left (ischemic) and right (control) MCA exhibited constriction to noradrenaline (10−8‐10−4 M, in the presence of β‐adrenoceptor blockade) that was greater in ischemic arteries. The large‐conductance KCa channel inhibitor Iberiotoxin (10−7 M) enhanced the contraction to noradrenaline in both control and ischemic arteries, but this effect was significantly lower in ischemic (21±12 %) than in control vessels (72±18 %, p<0.05). TRAM‐34 (2×10−8 M, intermediate‐conductance KCa channel inhibitor) or Apamin (10−6 M, small‐conductance KCa channel inhibitor) did not modify the noradrenaline effect in both types of arteries. The NO synthesis inhibitor L‐NAME (10−4 M) potentiated the response to noradrenaline similarly in control and ischemic arteries. These results suggest that the response to noradrenaline is enhanced after ischemia‐reperfusion due, in part, to impaired activation of large‐conductance KCa channels. NO might not be accounting for the adrenergic activation of K channels. (Supported by Ministerio de Ciencia e Innovación grant Nº SAF2008‐01410).
Beneficial effects of melatonin on improvement of neurological function in focal cerebral ischemic rats
Authors: Lee Seunghoon , Lee Minkyung , Shin Jinhee , Hong Yonggeun
Published: June 2021

The antioxidative effects of melatonin are known for its protective role against ischemic/reperfusion injury. In this view point the present study was carried out to investigate the effect of long term administration of melatonin on improvement of neurological functions after focal cerebral ischemia in rats. Following the MCAo, the 8 week old Sprague‐Dawley (SD) rats were treated twice daily with melatonin (10 mg/kg) at 7 and 19 hrs and control group animals were treated with autoclaved distilled water or physiological saline instead of melatonin. After 8 weeks of MCAo, the rat brains were collected for gene analysis and histological measurement. Histological data showed the significant reduction in the loss of neural cells on the primary motor cortex in melatonin treated rats. The gene expression data showed the downregulation of iNOS and nNOS whereas the neurotrophic factor, BDNF was significantly upregulated in melatonin treated MCAo rats brain (p<.01). In addition the behavioral test showed the rapid restoration of neurological dysfunction in melatonin treated rats at 4 weeks. However, only the trivial recovery with mild injury score was noticed in non‐treated rats after 8 weeks. Based on the present study, the improvement in the neurological dysfunction following the melatonin treatment in MCAo animals strongly suggest the possible use of melatonin as a therapeutic agent for the focal cerebral ischemia.
Interactions of PPARα and Acid Sensing Ion Channels on Cerebral Perfusion in Mice
Authors: Ndingwan Neh , Oyekan Adebayo O , Yakubu Momoh A.
Published: June 2021

Acid sensing ion channels (ASICs) are expressed in the brain and are made up of various proton sensitive channels which play critical roles in cellular physiology. Different genes have been shown to code for these channels. PPARα, a nuclear transcription factor that regulates many genes is also expressed in the brain and may have a regulatory role in the functions of ASICs. We have investigated the effects of interactions of PPARα and ASICs on the regulation of cerebral perfusion (CP) in mice. Anesthetized wild type (WT) or PPARα knockout (KO) mice received Amiloride (A), ENaC blocker, Benzamil (B), inhibitor of ENaC and Na+/Ca2+ exchange (NCX) blocker; Furosemide (F), inhibitor of Na‐K‐2Cl symporter, or Nigericin (N), ionophore and antiporter of H+ and K+(0.020, 0.35, 6.25, 0.025 mg/kg, respectively). Effects of the treatments were determined by Laser Doppler Scanner (Moor LDI 5152). CP was increased in the WT by 23, 38, or 26% and was attenuated in the KO to −6, 13, or 11% (A, p<0.05), B increased CP in the WT to 24, 38, or 28% and was attenuated to −1, 17, 28% (p>0.05) at 10, 30, or 60 min, respectively (n=3–5, ANOVA). Treatment with F or N reduced CP in the WT and the KO to similar extent. These data indicate that ASICs selectively sensitive to Amiloride and Benzamil are involved in maintaining basal brain perfusion and that PPARα gene is involved. The mechanism for such regulation needs further investigation.
Pressure‐dependent Reversal of Cerebral Vasoconstriction by a KCNQ4/5 (Kv7.4/7.5) Potassium Channel Activator
Authors: Zhong Xi Zoe , Greenwood Iain A. , William Cole C.
Published: June 2021

Appropriate regulation of cerebral arterial diameter is crucial for ensuring adequate blood flow to the brain. Enhanced cerebral vasoconstriction is a hallmark of cardiovascular diseases such as hypertension and subarachnoid hemorrhagic stroke(SAH). Pharmacological enhancement of K+ channel gating may be a viable approach to reverse abnormal vasoconstriction. Here, we report that S‐1 ((S)‐N‐[1‐(3‐morpholin‐4‐yl‐phenyl)‐ethyl]‐3‐phenyl‐acrylamide)), a selective Kv7.4/7.5 (KCNQ4/5) activator, reversed vasoconstriction of pressurized rat middle cerebral arteries due to: 1) inhibition of Kv2.1/9.3 or activation of voltage‐gated Ca2+ channels, and 2) treatment with angiotensin II, 5‐HT, or oxyhemoglobin that contribute to abnormal vasoconstriction in hypertension or SAH. The extent of vasodilatation evoked by S‐1 was pressure‐dependent, increasing in magnitude between 10 and 120 mmHg. The ability of S‐1 to evoke vasodilation, but preserve the myogenic response differs from the effect of ATP‐sensitive K+ channel activators that evoke pressure‐independent dilation. Our findings indicate that Kv7.4/7.5 channels may be a viable therapeutic target to suppress abnormal cerebral arterial vasoconstriction in disease while maintaining the myogenic response, a crucial physiological mechanism of cerebral blood flow autoregulation. (Support CIHR MOP‐13505; Andrew Family Professorship to WCC)
Effect of Treatment with Dipeptidyl Peptidase IV Inhibitor on Vascular Response in Arteries from High Fat Fed Rats
Authors: Oltman Christine Lee , Dake Brian L. , Holmes Amey J. , Yorek Mark A.
Published: June 2021

The purpose of this study was to examine vascular function in coronary, mesenteric and renal arteries from high fat fed rats and rats treated with the dipeptidyl peptidase IV inhibitor, Alogliptin. Rats were fed standard or high fat (45% kcal as fat) diet for 4 weeks then treated with Alogliptin (300mg/kg) in the diet for 12 weeks. Reactivity of small coronary and mesenteric vessels and tertiary branches of the renal artery were studied. Ach responses were attenuated in coronary and mesenteric responses in high fat fed rats. Alogliptin improved Ach responses in mesenteric, but not coronary arteries. Calcitonin‐gene related peptide (CGRP) was attenuated in coronary, but not mesenteric or renal, arteries from high fat fed rats and Alogliptin treatment did not improve the impaired dilation.

% Relaxation

Control
High Fat
High Fat + Alogliptin

Coronary
Ach (1 μM)
29.1 ± 7.3
19.5 ± 4.7
18.3 ± 4.4

CGRP (10 nM)
48.1 ± 9.4
33.6 ± 7.1
29.5 ± 6.7

Mesenteric
Ach (1 μM)
66.2 ± 7.1
39.0 ± 7.5
56.5 ± 6.3

CGRP (10 nM)
70.9 ± 7.7
86.6 ± 2.8
87.3 ± 3.7

Renal
Ach (0.1μM)
38.8 ± 1.5
44.4 ± 1.2
27.4 ± 0.8

CGRP (10 nM)
21.4 ± 1.7
20.1 ± 0.5
24.5 ± 0.6

ANP (10 nM)
65.7 ± 1.9
64.3 ± 1.7
72.0 ± 1.1

SNP (0.1 μM)
51.1 ± 1.7
47.7 ± 1.8
57.4 ± 2.0

Thus, responses to endothelial dependent vasodilation were attenuated in arteries from high fat fed rats and treatment with Alogliptin improved some (mesenteric) but not other (coronary) vessels. Supported by VA merits to Drs. Oltman and Yorek.
PARP‐1 inhibition improves coronary arteriole function in type 2 diabetic mice
Authors: Choi Soo‐Kyoung , Kassan Modar , Galan Maria , Partyka Megan , Matrougui Khalid
Published: June 2021

Objective

Type 2 diabetes is associated with microvascular dysfunction. We hypothesized that increased Poly‐(ADP‐ribose) polymerase 1 (PARP‐1) activity contributes to coronary arteriolar dysfunction.

Methods and Results

Type 2 diabetic db−/db− (diabetic) and non‐diabetic db−/db+ (control) mice were treated with PARP‐1 inhibitors (INO‐1001, 5mg/Kg/day and ABT‐888, 15mg/Kg/day) for two weeks. Isolated coronary arterioles were mounted in an arteriograph. Pressure‐induced myogenic tone (MT) was significantly potentiated, while endothelium‐dependent relaxation was significantly attenuated in diabetic mice compared to control. These results are associated with increased PARP‐1 activity in coronary arterioles from diabetic mice compared to control. Interestingly, diabetic mice treated by PARP‐1 inhibitors significantly reduced the potentiation of myogenic tone and improved the endothelium‐dependent relaxation. Down regulation of PARP‐1 in coronary arterioles using lentivirus‐shRNA‐PARP‐1 significantly improved coronary arteriole function in diabetic mice compared to control. The inhibition of nitric oxide synthesis by L‐NAME significantly reduced the endothelium‐dependent relaxation in coronary arterioles from sham and diabetic mice treated with PARP‐1 inhibitors and lentivirus‐shRNA‐PARP‐1.

Conclusions

We provide evidence of a link between the increased PARP‐1 activity and coronary arteriole dysfunction in type 2 diabetic mice. Thus, PARP‐1 may be a potential target for overcoming diabetic microvessels complications.
Altered Vascular Reactivity Following Exposure to Multi‐walled Carbon Nanotubes Increases Risk for Cardiac Injury
Authors: Thompson Leslie Charles , Frasier Chad R. , Sloan Ruben C. , Mann Erin E. , Harrison Ben , Brown David A. , Brown Jared M. , Wingard Christopher J.
Published: June 2021

The unique physicochemical properties of multi‐walled carbon nanotubes (MWCNT) present potential risks to physiological systems. We report here that an acute pulmonary instillation of MWCNT negatively impacts coronary flow (4.9 ± 0.6 ml/min/g vs. 6.8 ± 0.6 ml/min/g in vehicles) during reperfusion of isolated rat hearts subjected to global ischemia/reperfusion (I/R) injury. We hypothesized that MWCNT exposure blunts relaxation responses of vascular tissues. Male Sprague‐Dawley rats were exposed to 100 μg of MWCNT suspended in 10% surfactant/saline (10%SS) or the vehicle only. 24 hours later, segments of the left anterior descending (LAD) coronary and thoracic aorta were mounted on a multi‐myograph system. The segments were subjected to cumulative doses of acetylcholine (ACH), sodium nitroprusside (SNP), serotonin (5‐HT), or endothelin 1 (ET‐1). The LAD constrictor responses of the MWCNT group generated a greater magnitude of active stress (mN/mm2) to ET‐1 (4.03 ± 0.41 vs. 2.62 ± 0.23) and 5‐HT (3.76 ± 1.20 vs. 0.88 ± 0.34) compared to vehicle. Hillslope comparisons between groups revealed depressed aortic responses to ACH (−0.69 ± 0.23 vs. −1.95 ± 0.23) and ET‐1 (1.08 ± 0.09 vs. 1.74 ± 0.21) in the MWCNT group. From these data we conclude that an acute exposure to MWCNT can be deleterious to vascular reactivity and increases risks for cardiac injury. Supported by NIH ES019311 (JMB) & ES016246 (CJW).
KCa3.1 upregulated in familial hypercholesterolemic (FH) coronary smooth muscle
Authors: Gole Hope , Bowles Douglas
Published: June 2021

Familial hypercholesterolemia (FH) is a key risk factor for development of atherosclerosis. Vascular smooth muscle (VSMC) cell phenotypic modulation plays a key role in the development and stability of atherosclerotic plaques. We have previously shown upregulation of KCa3.1 channels mediates phenotypic modulation of coronary smooth muscle (CSM) cells and inhibition of these channels reduces restenosis following angioplasty. It remains unclear whether KCa3.1 channels are altered in atherosclerotic CSM of individuals with FH. Our objective was to determine whether KCa3.1 channels are upregulated in CSM isolated from FH swine. RCA sections from 2 year old FH swine showed a ~15 fold increase in artery stenosis, accompanied by elevated triglyceride and cholesterol values. In the media of FH coronaries, there was a trend for increased KCa3.1mRNA expression and KCa3.1 protein expression was elevated ~20%. KCa3.1 channel activity was also increased ~2‐fold in CSMCs isolated from FH swine. Our findings demonstrate that KCa3.1 is upregulated in CSM of FH swine and support previous research indicating KCa3.1 plays a key role in the development and progression of atherosclerosis.
cAMP mediated Ca2+‐desensitization of mouse tail arteries involves disinhibition of myosin light chain phosphatase (MLCP) in a PKA‐dependent and independent manner
Authors: Pfitzer Gabriele , Lubomirov Lubomir Todorov , Drinhaus Hendrik , Neulen Axel
Published: June 2021

Here, we investigated whether the non‐canonical cAMP sensor, Epac decreases Ca2+‐sensitivity by activating MLCP. Submaximally contracted (pCa 6.1) α‐toxin permeabilized arteries were relaxed by the specific activator of PKA, 6‐Bnz‐cAMP (Bnz) or Epacs, 8‐pCPT‐O‐Me‐cAMP (pCPT). Phosphorylation of MLC20, the regulatory subunit of MLCP, MYPT1, and VASP was determined by Western blots using phosphospecific antibodies.

Results

Both cAMP analogues relaxed the vessels at constant pCa=6.1 but with different potencies (pIC50 4.6 for 8‐pCPT‐cAMP; 6.6 for Bnz). At equipotent concentrations both compounds increased the rate of dephosphorylation of MLC20 while phosphorylation of MYPT1‐T696/T853 decreased by a similar extent suggesting activation of MLCP. Bnz but not pCPT increased phosphorylation of the PKA sites on VASP (S157) and MYPT1 (S695) the latter being known to prevent subsequent phosphorylation of MYPT1‐T696. The effect of Bnz on MYPT1 phosphorylation was inhibited by the PKA inhibitor, H‐89. Ca2+‐desensitization by pCPT but not by Bnz was partially blocked by NSC 23766, a Rac1 inhibitor. In conclusion, MLCP is activated by PKA and Epac. Both pathways converge at the level of MYPT1 and decrease phosphorylation of T696 and T853, but the upstream mechanisms only partially overlap.

Supported by the German Research Foundation.
Pre‐junctional potentiation of ATP overflow is age‐dependent
Authors: Kluess Heidi A , Stone Audrey J , Evanson Kirk W
Published: June 2021

The purpose was to evaluate the role of α2‐pre‐junctional receptors in ATP overflow in the resistance arterioles from adult and middle aged rats. Using F344 rats (6 months old: n=5 female; n=6 males) and (12 months old: n=5 females; n=5 males), the 1A arterioles from the red gastrocnemius muscle were dissected and cannulated with glass pipettes in a tissue bath. The ATP and null probes were positioned such that the tips of the probes were touching the arteriole in similar anatomical locations and a normal ATP curve was generated (0.78μM to 25 μM ATP). Field stimulation resulted in an increase in ATP overflow in arterioles from 6‐month old rats (1.88±6.14 μM ATP) and 12‐month old rats (34.95±91.30 μM ATP). Adding the α2‐agonist, UK14,304, resulted in no change in 6‐month olds (3.34±3.10 μM ATP), but increased ATP overflow in some 12‐month olds (119.09±256.71 μM). Blocking α2‐receptors using idozoxan, had little effect on 6‐month olds (1.81±1.59 μM ATP), but blocked the action of UK14,304 in 12‐month olds (11.74±23.04 μM ATP). There were no sex‐related differences in ATP overflow. These data indicate that there are significant age‐related changes in mechanisms modulating ATP overflow in skeletal muscle resistance vessels and that α2‐pre‐junctional activation may not act in the way previously described in the heart, brain and tail. This research was supported by NIA (R03AG033245) and the Arkansas Biosciences Institute.
Endothelin‐1 increases the frequency of smooth muscle calcium waves in vena cava but not aorta
Authors: Tykocki Nathan R. , Jackson William F. , Watts Stephanie W.
Published: June 2021

Vascular contraction is associated with a global increase in smooth muscle Ca2+. The frequency and amplitude of localized intracellular Ca2+ release events, such as IP3 receptor‐mediated Ca2+ waves, can also regulate excitation‐contraction coupling in smooth muscle. While the vasoconstrictor ET‐1 does increase IP3 formation, it is unclear if IP3 receptor‐mediated Ca2+ waves are a regulator of ET‐1‐induced contraction in arteries and veins. We hypothesized that ET‐1 increases the frequency and amplitude of Ca2+ waves in rat aorta (RA) and vena cava (RVC). RA and RVC were mounted en face in an imaging superfusion chamber and incubated with the intensiometric fluorescent Ca2+ indicator Fluo 4‐AM (10 μM). Using spinning‐disc confocal microscopy, image sequences were first recorded with buffer alone, and then maximal contractile concentrations of norepinephrine (NE, 10 μM) (RVC) or phenylephrine (PE, 10 μM) (RA), and finally ET‐1 (100 nM). ET‐1 (100 nM) increased Ca2+ wave frequency significantly in RVC versus control (0.72±0.16 Hz vs. 0.45±0.13 Hz), but not in RA. Neither NE (10 μM) nor PE (10 μM) altered Ca2+ wave frequency or amplitude in RA and RVC. These data indicate that intracellular Ca2+ mobilization by ET‐1 is different in arteries and veins. These data also provide indirect evidence that ET receptors are coupled to IP3‐mediated Ca2+ release mechanisms in the vena cava but not aorta. Supported by NIH P01HL70687.
Kv7 channels modulate adrenoceptor‐mediated activity in rat renal artery
Authors: Chadha Preet S , Zunke Friederike , Greenwood Iain A
Published: June 2021

KCNQ‐encoded voltage‐dependent potassium channels (Kv7) regulate vascular smooth muscle contractility. This study examines the contribution of Kv7 channels to physiological vascular reactivity in the rat renal artery. Recordings of isometric tension were made in response to various vasoconstrictors and dilators in the presence and absence of the Kv7 channel blocker linopirdine. Contractions in response to the α‐adrenoceptor agonist methoxamine were enhanced in the presence of linopirdine (1 μM; pEC50, 6.2±0.1 vs 5.6±0.03 vehicle control; n=5–7). However, linopirdine had no effect on contractions to the thromboxane‐mimetic U46619. In pre‐constricted renal arteries, β‐adrenoceptor‐mediated vasodilation to isoproterenol was attenuated in the presence of linopirdine (10 μM; Emax, 50.2±18.7% vs 95.7±5.8% vehicle control; n=4), but was unaffected by the non‐selective Kv1 channel blocker 4‐aminopyridine (1 mM). Dilation to the nitric oxide donor sodium nitroprusside was unaltered by linopirdine. The present data suggest that Kv7 channels may play a physiological role in α and β‐adrenoceptor‐mediated vascular activity. Supported by the British Heart Foundation.
AMP kinase/cyclic AMP/PK‐A crosstalk in vascular smooth muscle
Authors: Stone Joshua Daniel , Narine Avinash , Tulis David Anthony
Published: June 2021

Aberrant vascular smooth muscle cell (VSMC) growth is pivotal in the pathophysiology of vessel disease. We previously reported that the cyclic AMP/PK‐A system has capacity to attenuate growth of serum‐stimulated rat A7R5 VSMCs. Considering the common utilization of a 5′‐AMP substrate, in the current study we hypothesize that regulatory crosstalk occurs between adenosine monophosphate‐activated protein kinase (AMPK) and the cyclic AMP/PK‐A signaling cascades in VSM. Incubation of rat primary VSMCs with the AMPK activator AICAR significantly increased phosphorylation of both a catalytic Thr172 site and a proposed inhibitory Ser485 residue on AMPK. Concomitantly, we observed increases in AMPK activity shown by both phosphorylation of the downstream target Acetyl CoA Carboxylase (ACC) at Ser80 and an AMPK activity assay. These events were completely abridged by both the AMPK and PKA inhibitors Compound C (CC) and PKI, respectively. AICAR also induced CC‐ and PKI‐inhibitable PK‐A activation measured by vasodilator‐stimulated phosphoprotein (VASP) phosphorylation at Thr157 and a PK‐A activity assay. Lastly, preliminary flow cytometry results suggest that AMPK activity inhibits cell cycle progression from G0/G1. These provocative findings reveal a discrete nexus between AMPK and cyclic AMP/PK‐A in VSMCs and lend credence for a potential metabolic target for quelling abnormal VSMC growth.
Nuclear factor of activated T‐cells 5 (NFAT5) regulates vascular smooth muscle cell phenotypic modulation and enhances atherosclerotic plaque development
Authors: Halterman Julia Ann , Kwon H Moo , Wamhoff Brian R
Published: June 2021

Nuclear factor of activated T‐cells 5 (NFAT5) is a tonicity‐responsive transcription factor; however, NFAT5 function in vascular smooth muscle cells (SMCs) is unknown. We have identified NFAT5 as a positive regulator of both the contractile, and paradoxically, the synthetic SMC phenotypes. RNAi‐mediated knockdown of NFAT5 inhibits basal expression levels of classic SMC‐specific contractile genes (i.e. SMαA, Calponin, Smoothelin, SM22α), as well as genes involved in SMC migration and injury (i.e. Cyr61, VEGF‐C, COX2). We have identified Ang II, a contractile agonist, and PDGF‐BB, a growth factor released in vascular injury, as two novel NFAT5‐stimulating factors. Ang II‐induced SMαA promoter activity and RNA expression is attenuated following endogenous NFAT5 knockdown, and unstimulated NFAT5 null mouse embryonic fibroblasts have a 97% reduction in SMαA expression. In addition, both PDGF‐BB and serum‐mediated SMC migration are NFAT5‐dependent. In vivo injury data show NFAT5 is upregulated following rat carotid balloon injury and in the ApoE−/− atherosclerotic plaque. Remarkably, haploinsufficient NFAT5+/−ApoE−/− mice have a 73% reduction in atherosclerotic plaque formation in the descending aorta. In conclusion, we have identified NFAT5 as a novel regulator of both the contractile and synthetic SMC phenotypes and give evidence to its important role in the development of atherosclerosis.
Allometric Scaling Of Muscle Metabolic Enzymes in Mammals
Authors: Moyes Christopher Douglas , Genge Christine E , Kocha Katrinka
Published: June 2021

Are the transcriptional mechanisms that control mitochondrial content in an individual ‐NRF1 and PGC1alpha (PGC1)‐ also responsible for interspecies differences? We explored the origins of allometric scaling of cytochrome oxidase (COX) activities in muscles from 12 rodents differing 1000‐fold in mass. Muscle COX scaling patterns ranged from isometric (soleus) to allometric (tibialis anterior). Consideration of myonuclear domain reduced differences between muscles, but not interspecies differences. In tibialis anterior, there was no significant scaling relationship in mRNA/g for COX4‐1, PGC1 or NRF1 yet COX4‐1 mRNA/g predicted COX activity, PGC1 and NRF1 mRNA correlated with each other, and both predicted COX4‐1 mRNA and COX activity. Multivariate analysis explained 90% of COX variation between species, about equally partitioned between mass effects and mass‐independent effects on PGC1 (or NRF1) mRNA. In mammalian PGC1 proximal promoters, there were no mass‐linked differences in (i) distribution of regulatory elements or (ii) reporter gene activities. Collectively, these studies suggest that not all muscles scale equivalently, but for those that show allometric scaling, transcriptional regulation of PGC1 and NRF1 genes does not account for scaling patterns, though it does explain most of the mass‐independent differences between species. Funded by NSERC Canada.
Obesity/diabetes‐associated gene screening in rhesus monkeys
Authors: Hansen Barbara C , Shamekh Rania , Budagov Temuri , Linden Ellen , Pessin Jeffrey , Atzmon Gil
Published: June 2021

We examined the genetic basis of naturally occurring obesity and type 2 diabetes mellitus (T2DM), in rhesus monkeys (Macaca mulatta, N=81) longitudinally studied under constant environmental and dietary conditions and fully phenotyped. An initial genome wide association study (GWAS) was conducted using Affymetrix 6.0 gene chip arrays. The successful call rate on all chip assays averaged 91.9% with high heterozygosity (60%), demonstrating the applicability of genotyping the monkey using the human platform. Principal component analysis classified the groups accurately. Five SNPs passed the test for multiple comparison correction and were of complete homology between the rhesus and human. We resequenced 4 of those SNPs, revealing one variant that clearly defined the obese/T2DM animals. Nine out of 18 SNPs selected on the basis of candidate human genome sequence polymorphisms associated with obesity/T2DM resulted in a measurable genotype. Muscle (vastus lateralis) expression levels of the four candidate genes with the most significant associated SNPs demonstrated differences in expression compared to normal animals. These data demonstrate the successful use of human SNP platforms to identify genetic variants associated with obesity/T2DM in rhesus monkeys. This research was supported by NIH/NIA HHSN263200800022C and NIA NO1AG3 1012 and by the Einstein Diabetes Research Training Center award DK020541.
Oral rapamycin does not mimic caloric restriction in mice
Authors: Manning‐Geist Beryl , Flagg Bethany , Swoap Steven
Published: June 2021

Rapamycin, an inhibitor of target of rapamycin (TOR), is the only known dietary additive that increases lifespan to the same extent as caloric restriction (CR). While mechanisms for lifespan extension remain unknown, both rapamycin and CR treatments influence signaling pathways that converge on TOR, an important regulator of growth and energy balance. We tested the hypothesis that similarities in lifespan extension of oral rapamycin and a 70% CR diet in mice would extend to physiological variables potentially important for the extension of life, including resting metabolic rate (RMR), heart rate (HR) and body temperature (Tb). C57BL/6J mice housed at 22°C were implanted with EKG telemeters, fed a control, rapamycin, or CR diet for 8 weeks, and then assessed for RMR via indirect calorimetry as well as HR and Tb via telemetry. CR mice, as compared to controls, showed dramatic reductions in minimal RMR (0.14±0.01 vs. 0.92±0.05 mL O2/min), HR (135±11 vs. 420±16 bpm) and Tb (25.7±0.2°C vs. 35.2 vs. 0.3°C). Rapamycin, however, had no significant impact on minimal RMR (0.85±0.07 mL O2/min), HR (405±17 bpm) or Tb (35.0±0.2°C) when compared to controls. These findings suggest that lifespan extension in rapamycin‐fed mice is not due to reductions in RMR, HR or Tb. Instead, it is likely that CR and rapamycin extend life though different mechanisms or that the treatments extend life via a mechanism that does not influence RMR, HR or Tb.