bluuebunny/crossref_metadata_2025 huggingface dataset:
Dopamine β‐Hydroxylase Inhibitor Etamicastat Prevents High Blood Pressure in Mice Lacking Salt‐Inducible Kinase 1 (SIK1)
Authors: Pires Nuno , Igreja Bruno , Moura Eduardo , Bonifácio Maria João , Serrão Paula , Soares‐da‐Silva Patrício
Published: November 2023

In human genetic studies a single nucleotide polymorphism within the salt‐inducible kinase 1 (SIK1) gene was associated with hypertension (1). Moreover, it was recently shown that loss of SIK1 triggers an increase in blood pressure (BP) upon a chronic high‐salt intake in mice (2). Here, we addressed possible acute mechanisms that may relate to the observed high BP in mice lacking SIK1. SIK1 knockout (sik1−/−) and C57BL/6 wild‐type (sik1+/+) littermate mice were challenged for seven days with a normal‐ (0.3% NaCl) or high‐salt (8% NaCl) diet. Systolic BP (SBP) was significantly increased in sik1−/− mice (137.0±17.2 mmHg, n=6) after seven days of high‐salt intake, as compared to sik1+/+ mice counterparts (120.6±4.5 mmHg, n=6). The renin‐angiotensin system (RAS) and the sympathetic nervous system (SNS) were assayed in order to investigate the possible causes for the increase in SBP in sik1−/− mice fed a high‐salt diet. No differences in renin (normal‐salt: 463.4±17.9, high‐salt: 462.9±28.9 pg/ml) and angiotensin II (normal‐salt: 45.8±10.0, high‐salt: 39.0±8.5 pg/ml) serum levels were observed (n=6/group). The activity of dopamine β‐hydroxylase (DβH), the enzyme that converts dopamine (DA) to norepinephrine (NE), was significantly increased in adrenal glands of sik1−/− mice fed a high‐salt diet (356.7±32.8 nmol/mg protein, n=5) as compared to sik1−/− mice on a normal‐salt diet (184.4±14.4 nmol/mg protein, n=5). Similarly, urinary catecholamines (DA, NE and adrenaline) and L‐DOPA were significantly increased (3‐ to 7‐fold increase) in sik1−/− mice fed a high‐salt diet as compared to sik1−/− mice on a normal‐salt intake. Altogether, this data supports the view that sik1−/− mice fed a high‐salt diet develop SNS overactivity. Next, we addressed the question if reducing SNS activity in sik1−/− mice fed a high‐salt diet would ameliorate hypertension. For that purpose, the effect of the peripheral reversible DβH inhibitor etamicastat [also known as BIA 5–453; (R)‐5‐(2‐aminoethyl)‐1‐(6,8‐difluorochroman‐3‐yl)‐1,3‐dihydroimidazole‐2‐thione hydrochloride] was evaluated on the development of high BP upon high‐salt diet (3). Etamicastat treatment (50 mg/kg/day), started prior to high‐salt feeding, completely prevented SBP increase in sik1−/− mice fed a high‐salt diet (116.8±4.7 mmHg, n=5). In summary, it is concluded that the SNS is involved in the development of salt‐induced hypertension in sik1−/− mice and that the DβH inhibitor etamicastat is able to reduce SNS overactivity and high BP in this mouse model of hypertension.

Support or Funding Information

This work was supported by BIAL ‐ Portela & Ca, S.A.
Multi‐Gene Pharmacogenetics and Blood Pressure Control in Patients with Hypertension
Authors: Johnson Micah William , Sprissler Ryan , Olson Thomas P , Beenken Greg William , Snyder Eric M
Published: November 2023

Introduction

Several common and functional genes are known to contribute to renal, blood vessel, and cardiac functional characteristics associated with sodium (Na+) handling, vascular reactivity, and cardiac output, respectively. Many of these same genes are suggested to contribute to differential responsiveness to blood pressure (BP) therapy. Few studies have explored the simultaneous phenotypic consequence of multiple genes critical for BP control. Despite a significant impulse of the medical community towards personalized medicine, BP therapy is typically guided by algorithms that do not include a patient's genetic information.

Purpose

The purpose of this study was to determine the impact of 14 genes on time to BP control in response to common BP therapy, with a specific focus on genes associated with drug metabolism and protein function in the kidney, the blood vessels, and the heart.

Methods

Eighty‐six patients completed one study visit consisting of a buccal swab for DNA extraction and genotyping, measurement of ambulatory BP, and a medical chart review for objective BP history. Genes in the analysis included those that encode for one drug metabolizing enzyme (CYP2D6), renal Na+ handling (SCNN1A, WNK1, SLC12A3, and Alpha Adducin), vascular function (angiotensin, ACE, angiotensin‐II receptor type‐I, and renin), and cardiac function (the β1 and β2 adrenergic receptors ADRB1 and ADRB2, respectively).

Results

Genetic variation of WNK1 (rs1159744) demonstrated differential responses to diuretic therapy (determined as those with blood pressure under 140/90 vs. those above 140/90 according to genotype)(WNK1=GG>CG/CC, P<0.05). Genes that encode ADRB1 (rs1801252), ADRB2 (rs1042713), and CYP2D6 (rs3892097) demonstrated differential response to β‐blocker therapy (ADRB1=Ser/Ser>SerThr/ThrThr, ADRB2=GG>AG/AA, CYP2D6=CC>CT/TT, P<0.05). Genes that encode angiotensin (rs699), ACE (rs1799752), and renin (rs12750834) demonstrated differential response to ACE‐inhibition and angiotensin receptor blockade (angiotensin=CT/TT>CC, ACE=Ins/Ins>InsDel/DelDel, renin=AG/AA>GG, p<0.05).

Conclusion

These results demonstrate that genetic variants encoding proteins involved in renal Na+ handling, vascular function, and cardiac output influence response to BP therapy for their respective drug classes.

Support or Funding Information

Funded by Geneticure
Toll‐like receptor 9 contributes to increased vascular tone via small GTPase activation
Authors: McCarthy Cameron G. , Wenceslau Camilla F. , Ogbi Safia , Szasz Theodora , Webb R. Clinton
Published: November 2023

Toll‐like receptor (TLR)9 is a pattern recognition receptor of the innate immune system. We have previously reported that TLR9 stimulation causes resistance artery dysfunction via increased sensitivity to norepinephrine, and this contributed to the pathogenesis of hypertension. However, the mechanisms underlying this increased vascular tone after TLR9 activation need to be further clarified. We hypothesized that TLR9 activation would increase expression of small GTPases, indicative of actin cytoskeleton remodeling. Isolated mesenteric resistance arteries from Sprague‐Dawley rats (n=4–6) were divided into three sections for Western blot analysis of Rho family small GTPases. Each section was incubated for 20 minutes in Krebs solution with either vehicle (endotoxin free water), TLR9 agonist ODN2395 (2 μM), or ODN2395 after a 5‐minute pre‐incubation with TLR9 antagonist ODN2088 (20 μM). ODN2395 alone increased protein expression of cdc42 (1.7 fold vs. Veh, p<0.05) and RhoA (1.4 fold vs. Veh, p<0.05), and this increase in expression was inhibited by pre‐incubation with ODN2088 (p>0.05). On the other hand, Rac1 was unchanged after incubation with ODN2395 (p>0.05). These results show that TLR9 stimulation leads to activation of some Rho family small GTPases and suggests another mechanism by which TLR9 causes increased vascular tone is actin cytoskeleton remodeling.

Support or Funding Information

American Heart Association (AHA) and National Institutes of Health (NIH)
cFos changes in the hindbrain following central microinjection of nociceptin/orphanin FQ in conscious hypertensive rats
Authors: Conner Caroline , Drzymala Bethannie , Koshy Ligil , Fortepiani Lourdes , Franklin Cynthia , Gottlieb Helmut
Published: November 2023

Intracerebroventricular (ICV) of the opioid like peptide, nociceptin/Orphanin (N/OFQ), produces marked effects on cardiovascular function and decreases in renal sympathetic nerve activity. The present study utilized changes in cFos immunocytochemistry expression to examine the hindbrain sites activated or inhibited, respectively, by ICV injection of N/OFQ in angiotensin II/high salt hypertensive rats (ANGII) as compared to ANGII rats injected with isotonic saline (control). The hindbrain was processed for cFos using a commercially available antibody (Oncogene AB‐5). ICV N/OFQ produced significant decreases in c‐Fos staining in the Area Postrema (C, 100.1 ± 26, ANGII; 69 ±21 P<.001) and anterior nucleus of the solitary tract (NTS) (C, 44 ± 14; ANGII 27 ± 10; P < .01). There were no statistical significant changes to the rostral ventrolateral medulla (RVLM), medium NTS, or locus coeruleus. Together, central ORL‐1 receptor activation in these forebrain regions may contribute directly to or indirectly participate in the neuropathways involved in the cardiovascular effects of N/OFQ
The First Intracellular Loop Regulates The Platelet Thromboxane A2 Receptor‐Gq‐Dependent Signaling And Function
Authors: Karim Zubair A , Lin Olivia , Khasawneh Fadi T
Published: November 2023

While the molecular signaling pathways for the thromboxane A2 receptor (TPR) have been elucidated from multiple organ systems, much less research efforts have focused on the TPR‐ G‐protein (GP) coupling domains (particularly in platelets). In this connection, studies conducted thus far (mostly mutagenesis in nature) have implicated regions from all of the intracellular domains of TPR in coupling to GPs. However, since alterations in the amino acid sequence can lead to effects on protein structure, at least some of these receptor alterations may not be at the critical GP coupling sites. Based on these considerations, we attempted to map the GP coupling domains and investigate human platelet signaling through TPR by employing site‐specific peptides targeted against regions potentially‐involved in the receptor‐Gα subunits interaction process. Our findings revealed that a myristoylated peptide that mimics the first intracellular loop (IL1) of human TPR (i.e., amino acids A52 ‐ F63; abbreviated as Myr‐IL1pep) blocked TPR‐mediated human and mouse platelet aggregation, but (interestingly) it had no detectable effects on platelet shape change (SC). This finding suggests that the IL1 domain of TPR participates in receptor coupling to Gαq but not Gα13. Furthermore, these data are consistent with previous findings that while platelet aggregation is a Gαq‐dependent event, SC is Gα13 dependent. The specificity of this inhibition was shown by the findings that: 1. the control random sequence for IL1 (Myr‐IL1random) did not produce any detectable effect on platelet aggregation induced by TPR activation; and 2. the Myr‐IL1pep itself did not interfere with ADP‐triggered aggregation. Regarding a potential mechanism, immunoaffinity co‐purification experiments revealed that Myr‐IL1pep uncoupled Gαq but not Gα13 from TPR, whereas the Myr‐IL1random did not produce any apparent effects. Finally, the Myr‐IL1pep was found to protect against thrombogenesis, but it does so while also prolonging the tail bleeding time, in mice; whereas control peptides were without any effect on thrombosis or bleeding time. Collectively, our results provide novel information concerning TPR's structural biology and signaling, and may identify new and novel therapeutic agents for the prevention and/or treatment of thrombotic disorders.

Support or Funding Information

This work was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R15HL115567 (to F.T.K). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Inhibition of vWF Secretion with Novel Gα12 N‐terminal α‐SNAP Binding Domain Peptide Increases Survival in Septic Mice
Authors: Rusu Luiza , Christman John W , Du Xiaoping , Minshall Richard
Published: November 2023

Severe sepsis is associated with disseminated intravascular coagulation (DIC) as a result of interdependent mechanisms of systemic inflammation, microvascular thrombosis, and thrombocytopenia. Currently, no drug is available to concomitantly treat these events in sepsis. A poorly understood mechanism and yet critical determinant of sepsis‐induced microvascular thrombosis is von Willebrand Factor (vWF) secretion by activated endothelial cells. We recently discovered that heterotrimeric G protein alpha subunit G12 plays a critical role in basal and evoked vWF secretion by endothelial cells by promoting Weibel‐Palade body (WPB) exocytosis. Based on the observed interaction of Gα12 with αSNAP, a critical member of the exocyst complex required for plasma membrane fusion and exocytosis of WPB contents, we generated a myristoylated Gα12 N‐terminal αSNAP binding domain blocking peptide (Myr‐SBD) and tested the hypothesis that this would selectively and potently inhibit vWF secretion, limit platelet adhesion, and prevent microvascular thrombosis associated with cecal ligation and puncture (CLP) induced sepsis. CLP‐induced fulminant sepsis in mice was associated with a 2–3‐fold increase in plasma vWF within 24 hrs. Importantly, we observed reduced plasma vWF levels 24 hrs after CLP surgery in mice given a one‐time i.v. bolus (80 μg/30 g mouse) of micellar Myr‐SBD (lipid to peptide molar ratio of 90:4) at the time of surgery as compared to Myr‐scrambled peptide or vehicle only group. Strikingly, this was associated with increased survival without adversely inducing hemorrhage and vascular leakage. Furthermore, and consistent with the hypothesis that Gα12‐dependent increase in vWF secretion during sepsis leads to poor outcome, control WT mice succumbed to sepsis in less than 96 hrs whereas 80% of Gα12−/− mice shown previously to have significantly reduced plasma vWF levels survived. Inhibition of Gα12/αSNAP dependent vWF secretion may therefore be an effective strategy for blocking microvascular thrombosis, disseminated intravascular coagulation, and death due to sepsis.

Support or Funding Information

AHA Postdoctoral Fellowship for LR
Angiotensin II Inhibits T‐type Ca2+ Channels in Cerebral Arterial Smooth Muscle Cells
Authors: Hashad Ahmed Mohamed , Sancho Maria , Welsh Donald
Published: November 2023

T‐type Ca2+ channels (CaV3.1 and CaV3.2) are expressed in cerebral arterial smooth muscle cells and they play a key role in regulating arterial tone. In this study, we investigated whether and by what mechanism Angiotensin II (Ang II), a vasoactive peptide produced within the cerebral arterial wall, influences T‐type Ca2+ channels. Using patch clamp electrophysiology and rat cerebral arterial myocytes, Ang II (100 nM) was shown to inhibit T‐type Ca2+ currents in a time‐dependent manner (~ 40% after 15 min); it also shifted the voltage‐dependency of activation by 7 mV. Ang II induced inhibition was mediated through the AT1 receptor, as preincubation with Losartan (1 μM) abolished the effect. Protein kinase C (PKC) blockade by GF 109203X (100 nM) did not eliminate Ang II‐mediated inhibition, demonstrating that this signaling protein was not involved. In contrast, NADPH oxidase inhibitors abolished the inhibitory effect of Ang II revealing a role for reactive oxygen species (ROS) on T‐type Ca2+ channels. To delineate if Ang II is targeting a particular T‐type channel, Ni2+ (50 μM) was added to selectively block CaV3.2. In the presence of Ni2+, Ang II did not induce further inhibition of the residual current, indicating that Ang II was selectively targeting CaV3.2. In summary, Ang II inhibits CaV3.2 channels in cerebral myocytes through the generation of ROS. These findings suggest that T‐type channels are a regulatory target of vasoactive stimuli, known to facilitate the “constrictive” phenotype prevalent with cerebrovascular disease.

Support or Funding Information

Canadian Institutes of Health Research, Alberta Innovates Health Solutions, Vanier Graduate Scholarship (CIHR)
Endogenous dipeptidyl‐peptidase IV modulates skeletal muscle arteriolar diameter in rats
Authors: Neidert Leslie E , Al‐Tarhuni Mohammed , Goldman Daniel , Kluess Heidi A , Jackson Dwayne N
Published: November 2023

Neuropeptide Y1‐36 is a potent vasoconstrictor that plays an important role in resistance of smaller arterioles. The enzyme, dipeptidyl‐peptidase IV (DPP‐IV), is present on the smooth muscle cell membrane and converts NPY1‐36 to its non‐vasoconstricting form, NPY3‐36. New research suggests that DPP‐IV is also released from skeletal muscle as a myokine in response to different stimuli including muscle contractions and whey protein supplementation. The role of DPP‐IV as a myokine is unknown. It was hypothesized that whey protein will release DPP‐IV from the muscle and will attenuate neuropeptide Y Y1‐mediated vasoconstriction in skeletal muscle arterioles.

The left gluteus maximus (GM) muscle of anesthetized male Sprague‐Dawley rats (n=8) was exposed for intravital video microscopy and superfused with physiological salt solution (5 ml/min; pH 7.4, 35°C). Once a stable baseline was established, the GM arteriolar network was scanned and imaged from 1st to 6th order arterioles (1A to 6A). Following baseline imaging, superfusion solution was switched to 100mM whey protein isolate (WPI) dissolved in PSS, to stimulate release of DPP‐IV from the muscle. Imaging of the network was repeated after arteriolar responses stabilized. Next, a DPP‐IV inhibitor (K579; 4μm) was added to the PSS + WPI superfusion and imaging of the GM arteriolar network was repeated. One‐Way ANOVA for each branch of the network was used to determine statistical significance with alpha set at 0.05.

When the WPI solution was suffused over the muscle the entire arteriolar network vasodilated. The magnitude of change in arteriolar diameter increased with progressing arteriolar order, where WPI promoted a 14% increase in arteriolar diameter in 1A (p=0.0002), 17% in 3A (p<0.0001), and 23% in 6A (p<0.0001). However, when the DPP‐IV inhibitor was added to the WPI superfusion vasodilation was lost and arteriolar diameters across the network returned to baseline. These findings show that DPP‐IV is released from skeletal muscle in response to topically applied WPI, which has potential to vasodilate skeletal muscle arteriolar networks, likely via conversion of NPY1‐36 to NPY3‐36. Future research should address if muscle contractions increase DPP‐IV secretion from skeletal muscle and affect local blood flow control.

Support or Funding Information

Funded by NSERC and Western Visiting Scholars Program
Bulk Flow of Interstitial Fluid: a Watershed in the Brain Parenchyma
Authors: Bedussi Beatrice , van Lier Monique GJTB , Bartstra Jonas W , de Vos Judith , Siebes Maria , van Bavel Ed , Bakker Erik NTP
Published: November 2023

Brain interstitial fluid (ISF) and cerebrospinal fluid are involved in the clearance of waste products from the brain. So far, different pathways of fluid drainage have been hypothesized. In particular, previous work pointed towards the perivascular space along cerebral vessels as a major drainage path. However, the pattern of fluid flow in the brain is still controversial. The aim of the current study was to visualize extravascular fluid transport in the normal mouse brain. Therefore, we injected FITC‐dextran (500 kD ) and Texas Red‐dextran (3 kD) in the striatum and in the cisterna magna and allowed the dyes to spread for 30 minutes. Horizontal and coronal brain sections were imaged using confocal microscopy and a 3D imaging cryomicrotome to reveal the pattern of distribution. We observed the tracers to distribute from the striatum towards the ventricular system, where they mixed with the cerebrospinal fluid and moved to the subarachnoid spaces. From there, tracers drained from the brain via olfactory nerves via the cribriform plate. Occasionally we observed that tracer close to the cortical surface found its way to the brain surface along blood vessels. This last observation suggests that there might be a point of highest interstitial pressure, close to the cortex, where ISF flows to either the ventricles or the cortex. After injection in the cisterna magna, tracers distributed over the subarachnoid space and followed the olfactory route out of the brain. Tracers were observed around blood vessels in the SAS and cisterns, but penetration into the brain parenchyma along perivascular spaces was limited. In conclusion, ISF, possibly originating from the capillaries, may generate a cumulating flow, small in regions far from the ventricles and much larger when approaching the ventricles. Disturbances in this transport pathway could influence the drainage of amyloid β and other waste products, which may be relevant for the pathophysiology of Alzheimer's disease.

Support or Funding Information

This work received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 606998.
IL1β Disrupts Barrier Integrity and Elicits Formation of Focal Adhesions in Lymphatic Endothelial Cells
Authors: Adderley Shaquria P , Zhang Xun E , Breslin Jerome W
Published: November 2023

A functional role of changes in lymphatic vessel permeability for the optimization of immune and inflammatory responses has recently been reported. The precise mechanisms within lymphatic endothelial cells that promote changes in barrier integrity remain incompletely understood. In the current study we investigated the effects of two different inflammatory mediators on lymphatic endothelial barrier function: histamine, which produces local inflammatory responses; and IL1β, an inflammatory cytokine commonly elevated in a variety of diseases. Human adult lymphatic endothelial cell (HALEC) and human juvenile lymphatic lymphatic endothelial cell (HJLEC) monolayers were used in order to examine endothelial‐specific mechanisms. Transendothelial electrical resistance (TER) was used as an index of barrier function. Immunofluorescence microscopy was used to examine localization of vinculin and VE‐cadherin. The roles of MLCK and p38 MAPK kinase, both previously reported to be involved in inflammation‐induced elevations in microvascular permeability, were tested using the inhibitors ML7 (1 μM) and SB203580 (10 μM), respectively. The results showed that histamine (10 μM) produces a rapid decrease in resistance, which recovers within 5 mins, whereas IL1β (25 ng/ml) causes an initial increase in resistance followed by a decrease starting at 3 hours and continuing until approximately 4 hours after treatment. The recovery of the IL1β‐induced decrease in barrier function begins approximately 7 hours into the time course. Treatment with IL1β or histamine also increased the number of focal adhesions containing vinculin, 5.5 h after treatment. Surprisingly, inhibition of p38 MAP kinase with SB203580 potentiated the response of IL1β. Inhibition of MLCK with ML7 had no effect on the IL1β response. The data suggest that the effect of IL1β on endothelial barrier function has different dynamics than that of histamine. However, in both cases the cells appear to be responding to stress, with elevated focal adhesions 5.5 h after treatment. The p38 MAPK kinase appears to have a protective role for barrier function in lymphatic endothelium.

Support or Funding Information

Supported by NIH grant R01HL098215.
Disruption of interleukin 12 stimulates and enhances angiogenesis in type 2 diabetes
Authors: Ali Maha Ali Essameldeen , Mali Vishal , Haddox Samuel , Abdelghani Soad , Eldeek Sahar , Abulfadl Atif , Belmadani Souad
Published: November 2023

Introduction

Previous studies reported the involvement of interleukin (IL)‐12 in cardiovascular complications in type 2 diabetes. We previously showed that ischemia‐induced angiogenesis was compromised in type 2 diabetic mice. In this study, we determined whether IL‐12 disruption would rescue angiogenesis in type 2 diabetic mice.

Methods

Type 2 diabetes was induced by feeding control and IL‐12 deficient mice with high fat diet (60% high fat diet “HFD”) for 12 weeks. Body weight, glucose test tolerance and insulin test tolerance were assessed. After 12 weeks of HFD, femoral artery was ligated and then blood flow recovery was assessed once a week for 4 weeks.

Results

Control (C57/Bl6) and IL12−/− mice fed with HFD became markedly obese after 12 weeks and exhibit glucose intolerance and insulin resistance. Using laser Doppler technique, data revealed that blood flow recovery was fully restored in IL12 −/− and in C57/Bl6 mice without HFD 3 weeks after femoral ligation. After 12 weeks of HFD, blood flow recovery was compromised and only reached 50% in control mice. However, in IL‐12−/− fed with HFD, the blood flow recovery was fully restored and reached 100% indicating that the disruption of IL‐12 in type 2 diabetes protects angiogenesis in response to ischemia. The recovery was associated with an enhancement in eNOS/Akt/VEGFR2 phosphorylation, and the reduction in oxidative stress.

Conclusion

The disruption of IL‐12 increases angiogenesis and blood flow recovery after hind limb femoral ligation in obese type 2 diabetic mice independently of obesity and type 2 diabetes.

Support or Funding Information

Embassy of Arab Republic of Egypt Culture and Educational bureau
Smooth Muscle Specific Expression of a Dominant Negative Cullin 3 Mutant (Cul3Δ9) Causes Vascular Dysfunction in Mice Mediated by RhoA/Rho‐kinase
Authors: Agbor Larry N , Ibeawuchi Stella‐Rita C , Hu Chunyan , Quelle Frederick W , Sigmund Curt D
Published: November 2023

Dominant de novo mutations in Cullin 3 (Cul3) resulting in deletion of exon 9 (Cul3Δ9) cause severe early onset human hypertension mediated in part by impairing the degradation of WNK4 in the kidneys. However, the extra renal mechanisms remain uninvestigated. Previously, we showed that knockout of Cul3 in HEK293T cells induced by clustered regularly interspersed short palindromic repeats (CRISPR)‐Cas9 resulted in accumulation of the Cul3 substrate RhoA. We hypothesized that smooth muscle (SMC) expression of Cul3Δ9 protein in mice impairs endogenous Cul3 wildtype (WT) function and causes vascular dysfunction via elevated RhoA/Rho‐kinase signaling. We generated a novel transgenic mouse model inducibly expressing Cul3Δ9 protein specifically in smooth muscle (termed S‐Cul3Δ9) and assessed vascular responses in the cerebral basilar artery and aorta using a pressurized and a wire myograph respectively. Basilar artery from S‐Cul3Δ9 transgenic mice exhibited significantly impaired relaxation responses to acetylcholine (ACh) (at 100 μmol/L: 15±4% S‐Cul3Δ9 vs 65±5% WT, p<0.0001) and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μmol/L: 59±2% S‐Cul3Δ9 vs 90±5% WT, p<0.05). Vasoconstriction responses to angiotensin II (Ang II) and to phenylephrine (PE) were significantly elevated in S‐Cul3Δ9 transgenic mice compared to WT. Consistent with data from basilar artery, aorta from S‐Cul3Δ9 transgenic mice exhibited impaired ACh‐mediated relaxation (at 100 μmol/L: 55±2% S‐Cul3Δ9 vs 71±7% WT, p<0.0001). RhoA protein expression was significant elevated in aorta of S‐Cul3Δ9 transgenic mice (1.6±0.2 S‐Cul3Δ9 vs 1.0±0.1 WT, P<0.05). Interestingly, incubation with the Rho‐kinase inhibitor (Y27632) completely restores ACh relaxation responses to WT levels in both the basilar artery and aorta of S‐Cul3Δ9 mice. Taken together, these findings suggest a novel vascular specific mechanism whereby Cul3Δ9 protein interferes with endogenous Cul3 and impairs the degradation of Cul3 substrates such as RhoA, contributing, at least in part, to vascular dysfunction in mice.

Support or Funding Information

This work was supported by National Institutes of Health Grants HL048058, HL062984, and HL084207 (to C. D. S.).
Impaired myogenic response of MCA elevates transmission of pressure to penetrating arterioles and contributes to cerebral vascular disease in aging hypertensive FHH rats
Authors: Fan Fan , Pabbidi Mallikarjuna , Lin Rick C.S. , Ge Ying , Gomez‐Sanchez Elise P , Rajkowska Grazyna K , Moulana Mohadetheh , Gonzalez‐fernandez Ezekiel , Sims Jerrell , Elliott Matthew R , Paul Ian A , Alexander Alexander P , Mosley Thomas H , Harder David R , Roman Richard J
Published: November 2023

We recently identified a K572Q mutation in Add3 in FHH rats. The present study examined whether it contributes to cerebral vascular dysfunction in aging and after the development of hypertension. The expression of Add3 is lower in FHH than in FHH.1BN rats. The inner diameter of MCA decreased by 80 ± 2 % and 71 ± 4% in FHH.1BN and FHH.Add3 transgenic rats, respectively, when perfusion pressure was increased from 40 to 140 mmHg. The pressure in terminal pial arteries was greatly increased from 37 ± 1 to 63 ± 4 mmHg in FHH rats when MAP was increased from 100 to 160 mmHg but was unchanged (34 ± 1 to 36 ± 1 mmHg) in FHH.1BN rats. The inner diameters of capillaries in the deep cortex were markedly larger (8 ± 2 vs. 22 ± 1 μm) and capillary density was reduced (15 ± 0.4% vs. 8 ± 2%) in FHH than in FHH.1BN rats. The expression of phosphorylated‐ERK1/2 and phosphorylated‐PKC was reduced in the brain of FHH than FHH.1BN rats. CBF rose by 99 ± 7%, 64 ± 5% and 42 ± 4% in FHH, FHH.1BN and FHH.Add3 rats, respectively, when MAP was increased from 100 to 190 mmHg, demonstrating that the impaired autoregulation of CBF in FHH rats was rescued by introduction of wildtype Add3. BBB leakage was greater in FHH rats than FHH.1BN and FHH.Add3 rats after induction of DOCA/salt hypertension. Hypertensive FHH rats exhibited marked neurodegeneration and vascular remodeling of the neocortex and hippocampus with a 33% reduction in the density and size of neurons relative to FHH.1BN and FHH.Add3 rats. Gap junctions between endothelial cells were larger in hypertensive FHH rats with neuronal, mitochondrial and myelin degeneration in the vacuolated area near leaky capillaries. The hypertensive FHH rats took 2.5 times longer to escape from an eight‐arm water maze than hypertensive FHH.1BN rats suggesting spatial learning and memory dysfunction. These findings suggest that the K572Q mutation in FHH rats decreases the expression and activity of Add3 which is involved in phosphorylation of PKC and ERK1/2. This contributes to the impaired myogenic response and autoregulation of CBF, therefore elevating the transmission of pressure in penetrating arterioles that ultimately promotes loss of cerebral capillaries, BBB leakage and neurodegeneration, which may contribute to vascular cognitive impairments with hypertension and age.

Support or Funding Information

This study was supported by grants HL36279 and DK104184 (RJR) from the National Institutes of Health.
Hookah Smoking Does Not Constrict Human Coronary Microvessels
Authors: Rezk‐Hanna Mary , Nelson Michael D , Rader Florian , Mason O'Neil , Tang Xiu , Shidban Sarah , Rosenberry Ryan , Benowitz Neal L , Elashoff Robert M , Lindner Jonathan R , Victor Ronald G
Published: November 2023

Hookah (water pipe) smoking has reached epidemic proportions due to unregulated social media marketing to young adults as a safer avant‐garde alternative to cigarettes. In contrast, much is suspected by tobacco researchers—yet little is proven—about hookah's putative health risks. What distinguishes hookah from all other tobacco products is that burning charcoal is used to heat the tobacco. As a result, hookah smoke delivers a large acute exposure not only to carbon monoxide (CO) but also to fine and ultrafine particles, charcoal combustion products that have been suggested—but unproven—by the air pollution and tobacco literature to constitute putative coronary vasoconstrictor stimuli. To test if hookah smoking constricts human coronary microvessels, in 28 young adult hookah smokers (age 27±5 years, mean±SD, 8 women, 20 men) we used myocardial contrast echocardiography (MCE) perfusion imaging with intravenous lipid shelled microbubbles to measure myocardial blood flow and its two components, microvascular flux rate (β) and functional capillary blood volume (A), and measured myocardial oxygen consumption (MVO2 = (7.2 × 10−4) x (heart rate x systolic blood pressure) + 1.42) as well as exhaled CO and plasma nicotine before and immediately after 30 minutes of ad lib hookah smoking in a custom‐built smoking chamber. With hookah smoking, exhaled CO increased from 3.0±0.4 to 25±2.2 ppm (mean ±SE, p<0.001 pre‐vs. post‐hookah), which approximates the CO boost of a typical ad lib hookah smoking session in hookah bars and indicates an exposure to fine and ultrafine particles that is 10 times greater (and to total particles that is 250 times greater) than that with cigarette smoking. On MCE, myocardial blood flow (A x β) did not decrease but rather increased acutely with hookah smoking (88±10 to 120±19 a.u.·s−1, p=0.02). The increased perfusion matched the mild hookah‐induced increase in MVO2 (6.5±0.3 to 7.6±0.4 ml·min−1, p<0.001), indicating physiologic vasodilation, and was manifested primarily by increased microvascular flux rate (0.7±0.1 to 0.9±0.1 s−1, p=0.01) with unchanged capillary blood volume (133±7 to 137±7 a.u., p=ns), a pattern of coronary microvascular response seen previously with a mild b‐adrenergic agonist. Moreover, the increased MVO2 and accompanying decrease in heart rate variability were accompanied by increased plasma nicotine (0.9±0.1 to 7.8±1.3 mg/mL, p<0.001) and almost completely eliminated by intravenous propranolol, documenting b‐adrenergic mediation. Thus, under these experimental conditions, mainstream hookah smoke either is not an acute coronary vasoconstrictor stimulus or one that is too weak to overcome the physiologic dilation of coronary microvessels caused by mild nicotine‐induced cardiac b‐adrenergic stimulation and/or pharmacologic dilation caused by carbon monoxide.
Influence of Acute Lower Limb Heating on Clinical Measures and Functional Capacity in Peripheral Artery Disease
Authors: Pellinger Thomas K. , Pearce Catherine B. , Simmons Grant H.
Published: November 2023

Acute local heating of human limbs produces increases in conduit artery blood flow and shear stress, and these increases are comparable in magnitude to those achieved during both whole body and single limb exercise. Numerous studies have shown that acute exposure to periods of high shear stress; whether by exercise or heating, results in increased conduit artery flow‐mediated vasodilation immediately following exposure. However, it is unknown if these transient increases in conduit artery vascular function are sufficient to improve the matching of oxygen supply and demand distal to the site of stenosis in patients with peripheral artery disease (PAD). Thus, the purpose of this study was to test the hypothesis that a single bout of lower limb heating (for 15 or 45 minutes) increases resting popliteal artery blood flow and 6 minute walk distance (6MWD) in patients with PAD. One 71 year old male patient with PAD (Intermittent Claudication, Fontaine stage II) attended the laboratory for a screening visit and two experimental visits during which bilateral lower limb heating was carried out via water bath immersion (42C; 40 cm depth) for either 15 or 45 minutes. Clinical and cardiovascular measures including resting single leg popliteal artery blood flow (Doppler ultrasound), ABI, and toe oxygen saturation (pulse oximetry) were carried out prior to and beginning 10 minutes following limb heating. Popliteal vascular conductance (PVC) was calculated as flow/mean arterial pressure. 6MWD and time/distance to pain onset were measured on the screening day and each experimental day 45 minutes post‐leg immersion. The 15 minute leg heating trial increased popliteal artery blood flow from 113.7 to 169.1 mL min−1 (48.6%), PVC from 1.41 to 2.15 mL min−1 mmHg−1 (52.4%), and 6MWD from 660 to 800 feet (21.2%). The 45 minute leg heating trial increased popliteal artery blood flow from 91.8 to 159.3 mL min−1 (73.5%), PVC from 1.17 to 1.99 mL min−1 mmHg−1 (70.6%), and 6MWD from 660 to 770 feet (16.7%). Furthermore, the subject reported onset of leg pain at 2:45 and 325 feet of the screening visit 6MW, which was delayed until 3:00 and 500 feet after the 15 minute leg heating trial and until 4:00 and 550 feet after the 45 minute leg heating trial. Neither of the heating trials evoked changes in ABI or toe oxygen saturation. Although data collection is ongoing, these initial findings suggest that acute lower limb heating in patients with PAD may increase exercise tolerance which, if undertaken routinely, could increase either patient adherence to or efficacy of exercise prescription in this population.

Support or Funding Information

Supported by the Salisbury University Faculty Mini‐Grant
Endothelial calreticulin and its role in vascular function
Authors: Biwer Lauren , Good Miranda , Sonkusare Swapnil , Isakson Brant
Published: November 2023

Myoendothelial junctions (MEJ) are projections from endothelial cells (EC) that make contact with neighboring smooth muscle cells (SMC) and are the anatomical hallmarks of resistance arteries. MEJ also exhibit presence of endoplasmic reticulum (ER), as evidenced from the ER retention signal –KDEL immunostaining and the live ER tracker (glibenclamide) in holes of the internal elastic lamina. Within the ER, calreticulin is a calcium binding protein that can bind approximately 50% of ER calcium and we found it was significantly enriched in the non lipid‐raft fraction of the MEJ in vitro. Due to the importance of ER calcium signals at the MEJ in controlling vascular tone, we investigated the in vivo role of calreticulin in the endothelium and the MEJ. We generated a tamoxifen inducible, endothelial specific calreticulin floxed mouse. To confirm knockdown of calreticulin in the EC, we digested diaphragm skeletal muscle to obtain microvascular EC from mice injected with peanut oil or tamoxifen. Live cells were stained with CD31 antibody (to mark EC), CD45 (to exclude leukocytes), fixed and permeabilized, and stained with calreticulin. We saw a significant decrease in the percentage of CD31+, Calreticulin+ cells after tamoxifen injection, indicating less EC were expressing calreticulin in our knockout mouse. In calreticulin mice with and without tamoxifen, we could not identify any basal change in blood pressure as measured by radiotelemetry. However, after acute injection of 0.1mg/kg PE, the rapid and significant increase in blood pressure seen in control mice was significantly blunted in mice lacking EC calreticulin. Because of this, we used pressure myography on isolated resistance arteries from mice (both third order mesenteric arteries and thoracodorsal arteries) lacking EC calreticulin and found them to have less constriction to PE dose response curves. In order to determine if this blunted response is due to less agonist‐induced ER calcium release, we also measured calcium transients at MEJs of intact resistance arteries. In summary, calreticulin in the EC appears to play an important role in the regulation of vascular tone and blood pressure, possibly through its actions at the ER in MEJs. We hypothesize calreticulin's presence at the MEJ may be to mediate ER calcium release and provide the negative feedback that occurs upon SMC stimulation with PE.

Support or Funding Information

American Heart Association

National Institute of Health
Effects of arginine vasopressin (AVP) deficiency on long term arterial blood pressure in normal and spontaneously hypertensive rats (SHR)
Authors: Villanueva‐Rodriguez Gloria Marcela , Organista‐Esparza Alejandro , Biñuela‐Berni Veronica , Herrera‐Carreon Paulina , Tinajero‐Ruelas Manuel , Rivera‐Cerecero Claudia Veronica , Chavira‐Ramirez Roberto Veronica , Kovacs Kalman , Quintanar‐Stephano Andrés
Published: November 2023

The role of AVP controlling the arterial blood pressure (BP) in basal conditions remains unclear. Previously, we described that neurointermediate pituitary lobectomy (NIL) in rats, induced an early but transient diabetes insipidus and permanent low mean BP (MBP), without changes in aldosterone, Na and K serum levels (1). In order to obtain a deeper insight in the temporal relationship between NIL and MBP, here we evaluated the effects of NIL on AVP and oxytocin (OT) serum levels and MBP from time 0 to 6 hours and at 3, 15, 45 and 90 days post NIL. Water intake and urine output, urine sodium and potassium concentrations and sodium and potassium serum levels, were measured at these same times, whereas the renin‐angiotensin II‐aldosterone system (RAAS) was assessed at 3 and 45 days post NIL. All values were compared with the respective sham operated (SHAM) groups. Values are expressed as mean ± SEM.

Results

NIL caused significant and permanent low AVP and OT serum levels (10.6 ± 0.8 versus (vs) 2.4 ± 0.16 (pg/ml), SHAM vs NIL respectively), transient diabetes insipidus of 15–30 days of duration and a significant and permanent decrease of the MBP in 70% of the NIL animals (106.8 ± 3.15 vs 73.8 ± 3.2 mmHg respectively). Hypotension was apparent 30 minutes after NIL. Indirect systolic blood pressure (SBP) (cuff tail) were measured in SHR and NIL‐SHR rats at 15 and 3 days before and 8, 20, 35 and 45 days after surgeries. In all times SBP was significantly decrease in NIL animals. The last measured was 166.5 ± 16.8 vs 121.4 ± 12.16 mmHg, SHR vs NIL‐SHR). As in previously studies, no significant differences in the RAAS and serum electrolytes were evident, whereas urine electrolytes were lower in NIL rats (Na; 33.08 ± 6.177 vs 58.99 ± 15.061 and K; 67.148 ± 12.515 vs 126.9 ± 26.97 respectively). We concluded that: 1) the permanent deficiency of AVP (NIL animals) is responsible of the long‐term low BP levels. 2) At the present we suppose that in NIL animals, the normal Na and K serum levels are due to the remnant basal concentrations of AVP (2.4 ± 0.16) which well may be enough to maintain the hydro‐electrolytic balance in the body fluids. 3) The decreased Na and K levels in the urine of the NIL animals may be explained partially for the kidney adaptations of the several segments of the nephron to the low levels of AVP. 4) Further studies are needed to explain why 30% of the NIL animals did not develop hypotension, and to explain how heart, blood vessels and kidney mechanisms adapt to these new physiological conditions.

Support or Funding Information

Supported by UAA‐PIFF 14‐1 and CONACYT‐221262 grants (AQS) and CONACYT (GMV) PhD scholarship, México, and the Jarislowsky and Lloyd Carr‐Harris Foundations.
Detrimental Changes in Perivascular Adipose Gene Expression in Metabolic Syndrome
Authors: Seldomridge Ashlee , DeVallance Evan , Lemaster Kent , Branyan Kayla , Frisbee Jefferson , Setola Vincent , Chantler Paul
Published: November 2023

Metabolic syndrome (MetS) is a clustering of major risk factors that increases the risk of cardiovascular mortality. Perivascular adipose tissue (PVAT) is an important regulator of vascular function through release of adipocytokines. Although PVAT is typically beneficial in a healthy individual, an obese individual exhibits greater production of pro‐inflammatory adipocytokines. PVAT has the capability of altering vascular tone through release of nitric oxide (NO) by endothelium nitric oxide synthase (eNOS). Tetrahydrobiopterin (BH4) is a critical cofactor for eNOS, oxidative stress can reduce BH4 production catalyzed by GTP‐cyclohydrolase‐1 (Gch1). This leads to uncoupled eNOS, which can produce superoxide adding to the oxidative burden. The increased oxidative load is additionally problematical as decrease in the antioxidant system is believed to accompany MetS. These suspected alterations in MetS PVAT may further contribute to diminish NO production from endothelium.

PURPOSE

To demonstrate MetS disrupts eNOS and Gch1 expression and therefore decreases PVAT derived NO production.

METHODS

PVAT tissue surrounding the aortic was removed from 17‐week old male obese Zucker (model of MetS) rats and their lean controls (LZR). Levels of eNOS, Gch1, and SOD‐1 expression were measured by qPCR in the PVAT. NO bioavailability was directly assessed using a DAF‐FM diacetate assay of PVAT exudate.

RESULTS

OZR PVAT had reduced NO production by 36% (p≤0.01). Further, OZR caused an up‐regulation of eNOS, specifically a 1.8 fold increase in OZR vs. LZR (p≤0.001). However, Gch1 expression decreased 1.6 fold (p≤0.001) in OZR vs. LZR. SOD‐1 expression showed only a slight decrease in OZR.

CONCLUSIONS

The PVAT is ill equipped to deal with the increased oxidative load of MetS. Consequently, despite increased eNOS expression in MetS, NO levels are decreased, likely due to a decreased Gch1 expression, and less production of BH4. The possible uncoupling of eNOS may further increase the oxidative load. Not only did these changes reduce PVAT derived NO but also reduced endothelium release of NO. Thus alteration to PVAT in MetS greatly reduces the global bioavailability of NO, which likely impairs aortic reactivity.

Support or Funding Information

NIH

AHA
Glutaminase Stimulates the Proliferation of Human Endothelial Cells
Authors: Durante William , Liu Xiao‐ming , Yates Benjamin , Yu Yajie , Peyton Kelly J.
Published: November 2023

Glutaminase (GLS) is a mitochondrial enzyme that metabolizes glutamine to glutamate and ammonia. Two distinct isoforms of GLS, GLS1 and GLS2, have been identified in mammals that exhibit distinct tissue distribution, enzyme kinetics, structural properties and molecular regulation. Although abundant GLS activity has been reported in endothelial cells, its functional role remains largely unknown. In the present study, we characterized the expression of GLS in endothelial cells and investigated the effect of GLS on the proliferation of endothelial cells. Human umbilical vein endothelial cells (HUVEC) expressed high levels of GLS1, but GLS2 was not detected in these cells. Treatment of HUVEC with the non‐selective GLS inhibitor 6‐diazo‐5‐oxo‐L‐ornithine (DON) or the selective GLS1 inhibitor bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide (BPTES) resulted in a concentration‐dependent inhibition of endothelial cell growth. Both GLS inhibitors also blocked HUVEC DNA synthesis and this was paralleled by a significant decrease in the phosphorylation of retinoblastoma protein. In addition, knockdown of GLS1 expression using a siRNA approach inhibited the mitogenic response of HUVEC. Flow cytometry experiments revealed that DON or BPTES arrested HUVEC in the G0/G1 phase of the cell cycle, as demonstrated by an increase in the percentage of cells in G0/G1 with a corresponding decline in the portion of cells in S and G2/M phases. Cell cycle arrest by the GLS inhibitors was associated with a dramatic reduction in cyclin A expression without any change in the expression of the other G1 cyclins, cyclin D1 and E, the cyclin‐dependent kinase inhibitors, p21 or p27, or p53. In conclusion, this study demonstrates that endothelial cells selectively express GLS1, and that GLS1 plays a fundamental role in stimulating human endothelial cell cyclin A expression, cell cycle progression, and proliferation.

Support or Funding Information

Supported by American Heart Association Grants 15GRNT25250015.