bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Acute Responses to Inflammatory Stimuli at Early Stage of Microvessel Remodeling
Authors: Yuan Dong , Zhou Xueping , He Pingnian
Published: June 2021

Vascular remodeling has been implicated in many chronic inflammatory diseases. This study aims to investigate the impact of chronic inflammation‐induced vascular remodeling on the acute venular responses to inflammatory mediators. Experiments were conducted in mesenteric venules using a survival rat model. Microvessel permeability was determined by Lp measurements and changes in VE‐Cadherin and F‐actin were viewed with confocal images. In day 1, basal and platelet activating factor (PAF, 10 nM) stimulated Lp were measured in individually perfused venules under aseptic conditions. Then blood flow was resumed in the perfused vessel and rat was recovered from anesthesia. In day 3, the baseline Lp measured in the same vessel showed no significant changes from day 1, but vessel diameters increased 74 ± 8 % (n = 8). When each vessel was exposed to PAF, the mean peak Lp reached 5.1 ± 1.2 times that of the response in Day 1. VE‐Cadherin staining at Lp peak showed frequent breaks with apparent gaps between endothelial cells (ECs), a pattern different from that observed in day 1. Phalloidin staining showed disorganized and fragmented F‐actin in both ECs and pericytes. The venular intercellular junctions per circumference in day 1 showed no significant difference from the enlarged venules in day 3, indicating no increases in EC number. Our results demonstrated that the observed morphological changes 3 days after the initial stimulation did not change the basal Lp, but manifested augmented acute response to PAF, indicating that the cellular phenotype at early stage of vascular remodeling are capable of mediating acute exacerbated vascular dysfunction upon additional stimulation. Supported by HL56237 and HL084338.
Measurements of isolated glomerular albumin permeability do not change in a variety of proteinuric conditions
Authors: Sage Leslie M , Ferguson Joanne , Neal Christopher R , Harper Steven J , Bates David O , Salmon Andrew HJ
Published: June 2021

Albuminuria is the hallmark of glomerular disease. The oncometric assay of glomerular albumin permeabilityA has been used to measure glomerular reflection coefficient to albumin (σalb) in clinically‐relevant circumstancesA. We have previously reported theoretical limitations of this assayB, and here report practical assay limitations.

Single glomeruli, isolated by graded sieving, were held on an aspiration pipette. Glomerular volume (V) was recorded during incubation in BSA solution (Valb) of known oncotic pressure. The incubating solution was exchanged for an iso‐oncotic solution of dextran molecules (>100kDa MW after dialysis), and glomerular volume was remeasured (Vdex). Vdex/Valb describes σalb.

σalb was measured in healthy mouse glomeruli before and after exposure to pronase and protamine. No changes were observed (pronase: pre 0.99±0.02; post 0.99±0.01 (7); p>0.9) (protamine: pre 0.95±0.01; post 0.95±0.01 (7); p>0.8).

In addition, σalb was not different between glomeruli from mice with (0.95±0.03 (30)) or without (0.95±0.02 (24)) streptozotocin‐induced diabetic albuminuria (p>0.7), nor between glomeruli from rats with (1.00±0.01 (9)) or without (0.99±0.01 (8)) spontaneous proteinuria (p>0.6).

We conclude that this assay is inadequate for glomerular albumin permeability measurements.
Na‐K‐ATPase activity involved in noise induced blood‐labyrinth‐barrier leakage
Authors: Shi Xiaorui , Nuttall Alfred L.
Published: June 2021

Na+/K+‐ATPase plays an important role in mediating barrier integrity in the polarized epithelial cells. In the cochlea, we found that Na+/K+‐ATPase subunit alpha 1 was the most abundant protein of the vascular tissue in the blood‐labyrinth‐barrier (BLB) and was expressed predominantly at the luminal side of the endothelium. Na‐K‐ATPase is known to be required for controlling the tight junctions in epithelial cells. In this study, we investigated the involvement of Na+/K+‐ATPase in noise induced BLB leakage. In mice, the expression and activity of Na+/K+‐ATPase were found to be correlated with the changes of expression of tight junction proteins as well as the change in BLB permeability. Increased expression of Na+/K+‐ATPase, at both transcriptional and protein levels, occurred after noise exposure at 120 dB SPL for 3h per day for two days. However, Na+/K+‐ATPase enzymatic activity was significantly decreased. There was a significant decrease in endothelial tight junction membrane contact points. In vitro inhibition of Na+, K+‐ATPase by ouabain corroborated the in vivo studies and showed swelling of capillary endothelial cells and increased ratio between occludin phosphorylation and dephosphorylation. The results support a functional link between Na+, K+‐ATPase and occludin in the BLB as one of the mechanisms for noise induced in the BLB.

Support: NIDCD DC 00888, NIDCD DC 00105, NIDCD DC 005983 (p30)
Rnd3 modulates the balance of RhoA and Rac1 activity during thrombin‐induced hyperpermeability
Authors: Kurtz Kristine M , Doggett Travis M , Breslin Jerome W
Published: June 2021

We tested the hypothesis that Rnd3, an endogenous inhibitor of Rho, attenuates thrombin‐induced barrier dysfunction not by inhibiting Rho per se, but rather by affecting the collective balance of activity between RhoA, Rac1, and Cdc42. The signaling activity of these small GTPases was assessed in human umbilical vein endothelial cells (HUVEC) treated with thrombin (1U/ml) for 5, 30, 60, and 120 min. using the commercially available G‐LISA kit. HUVEC were mock‐transfected or transfected with pMAT‐FLAG‐Rnd3, Rnd3 siRNA, or siCONTROL RNA. The results show that although Rnd3 overexpression did not inhibit the initial (5 min.) activation of RhoA by thrombin, it did attenuate thrombin‐induced inhibition of Rac1 activity. Rnd3 knockdown extended the time course of RhoA activation by thrombin, but the time course of changes in Rac1 activity was not significantly affected. Thrombin did not significantly alter Cdc42 activity at any of the time points. The data indicate that Rnd3 is not simply an inhibitor of RhoA activity, but rather modulates the overall balance of small family GTPase activities, particularly between RhoA and Rac1. Supported by NIH RR018766 and a grant from the American Heart Association.
Microprojections amplify EC response to SMC stimulation: A theoretical study
Authors: Nagaraja Sridevi , Kapela Adam , Tsoukias Nikolaos
Published: June 2021

Microvascular tone and blood flow are controlled by a wide network of signaling pathways. To increase fundamental understanding of these signaling events we have developed detailed mathematical models of calcium dynamics and membrane electrophysiology in isolated endothelial and smooth muscle cells and validated them against subcellular‐ and cellular‐level data. Single cell models were then integrated into multicellular/whole vessel models to incorporate microprojections in EC with localized IKCa channels and microdomains in SMC with localized NaKα2 isoform sodium potassium pumps and study their effects on agonist induced vasorelaxation and vasoconstriction. Myondothelial IP3 diffusion reduces SMC depolarization during stimulation with Norepinephrine. The effect was increased threefold by the presence of microprojections. Thus, simulations suggest a role for microprojections in amplifying an IP3 mediated negative feedback loop during SM stimulation. Accumulation of K+ ions between the microprojections and SMC seems to play a minor role in inducing hyperpolarization in SMC via activation of NaKα2 pumps. On the contrary, it antagonizes transfer of hyperpolarization via MEGJ by reducing EC hyperpolarization. The proposed integrative computational model allows the analysis of complex microcirculatory function in health and disease and provides a tool for hypotheses testing and for guiding new experimentation in the microcirculation.

(Supported by NIH SC1HL95101).
Sympathetic modulation of baseline hindlimb blood flow and vascular conductance in a model of prediabetes using young Zucker Diabetic Fatty rats
Authors: Novielli Nicole Maria , Al‐Khazraji Baraa K , Ellis Christopher G , Jackson Dwayne N
Published: June 2021

Although associated with vascular disease, few studies have addressed the impact of prediabetes on vascular function. Thus, we sought to identify differences in baseline sympathetic vascular control between male Zucker (pre)Diabetic Fatty rats (PD) and lean controls (CTL) (n = 11/group). Hindlimb blood flow (Qh) and vascular conductance (VC) were assessed under local infusion of BIBP3226 (NPY Y1‐receptor (Y1R) antagonist), prazosin (α1‐receptor antagonist) and dual blockade. With no differences in baseline, BIBP3226 increased Qh 0.3 ± 0.1 ml/min in both groups and VC increased 4.2 ± 0.9 and 4.5 ± 1.1 μl/min/mmHg in PD and CTL respectively (P < 0.05). Prazosin increased Qh 0.2 ± 0.04 and 0.2 ± 0.1 ml/min and VC increased 3.3 ± 0.5 and 4.2 ± 1.0 μl/min/mmHg in PD and CTL respectively (P < 0.05). Dual blockade increased Qh 0.2 ± 0.05 ml/min in both groups and VC increased 4.6 ± 1.2 and 4.4 ± 0.72 μl/min/mmHg in PD and CTL respectively (P < 0.05). Recovery time from maximum after drug infusion to 37% of baseline (1/e) for Qh and VC was shorter in PD vs. CTL (% of CTL, Qh and VC respectively: BIBP3226 68% and 53%, Prazosin 75% and 81%, dual blockade 78% and 70%) (P < 0.05). In support, red and white vastus muscle Y1R, α1R, and tyrosine hydroxylase expression was higher in PD vs. CTL (P < 0.05). These data suggest that the SNS is upregulated in prediabetes leading to persisting effects on skeletal muscle blood flow. NSERC and NIH.
Role of 20‐HETE in Differential Effects of High Salt Diet on Resistance Artery Function in Dahl Salt‐Sensitive (SS) Rats and SS‐5BN Consomic Rats
Authors: Beehner Kathleen , Falck John R. , Roman Richard J. , Lombard Julian H.
Published: June 2021

This study tested the hypothesis that an increased production of 20‐hydroxyeicosatetraenoic acid (20‐HETE) by cytochrome P450‐4A ω‐hydroxylase (CYP4A) enzymes and/or an enhanced sensitivity to 20‐HETE contributes to impaired vascular relaxation in Dahl salt‐sensitive (SS) rats. The role of 20‐HETE in modulating vessel responses was assessed by comparing vascular relaxation to reduced PO2 and acetylcholine (ACh) in pressurized middle cerebral arteries of SS rats vs. consomic SS‐Chr 5BN/Mcwi (SS‐5BN) rats having CYP4A genes from chromosome 5 of the Brown Norway rat introgressed onto the SS genetic background. Compared to SS‐5BN rats, vasodilator responses to ACh and hypoxia were impaired in SS rats fed either low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl) diet. Vascular relaxation in SS rats could be restored to levels comparable to those in the SS‐5BN rats by inhibiting CYP4A enzymes with dibromododecenyl methylsulfimide (DDMS; 50 μM). In wire myograph studies, mesenteric arteries of SS rats fed HS diet for 3 days or 3 weeks also exhibited an enhanced response to the 20‐HETE agonist 20–5,14‐HEDE (WIT003) compared to SS rats fed LS diet and SS‐5BN rats fed either HS or LS diet. These findings suggest that alterations in 20‐HETE synthesis and/or sensitivity contribute to salt‐induced alterations in vascular function in SS rats (NIH #HL65289, #HL72920, #HL92026; #DK‐38226: #T32 HL007852).
Salt‐Induced Increase in 20‐HETE Production Alters Vascular Function of Mesenteric Resistance Arteries of Dahl Salt‐Sensitive Rats
Authors: Raffai Gabor , Wang Jingli , Falck John R. , Roman Richard J. , Lombard Julian H.
Published: June 2021

Cytochrome P450‐4A ω‐hydroxylase (CYP4A) enzymes and their metabolite, 20‐hydroxyeicosatetraenoic acid (20‐HETE) are potential contributors to inflammation and vascular alterations with elevated dietary salt and/or hypertension. This study evaluated the role of the 20‐HETE/CYP4A system in modulating salt‐induced changes in vascular reactivity in mesenteric resistance arteries from Dahl salt‐sensitive (SS) rats. High salt (HS; 4% NaCl) diet increased expression of mRNA for the 4A3 and 4A8 isoforms of CYP4A and increased 20‐HETE production by ~25% in mesenteric arteries of SS rats. In SS rats fed HS diet, CYP4A enzyme inhibition with dibromododecenyl methylsulfimide (DDMS) [30–50 μM]: 1) reduced norepinephrine sensitivity; 2) rescued impaired dilation in response to reduced PO2; 3) switched the mechanism of hypoxic dilation from cyclooxygenase products to NO; and 4) selectively augmented vasodilation to the NO donor DETA NONOate. The 20‐HETE agonist 20‐OH‐5,14 HEDE (WIT003) [1μM] restored norepinephrine sensitivity and attenuated the restoration of hypoxic dilation in DDMS‐treated vessels of salt‐fed rats. These results suggest that the 20‐HETE/CYP4A system modulates both norepinephrine and NO sensitivity, suppresses hypoxic dilation, and could contribute to vascular inflammation and increased vascular resistance during the early stage of salt‐sensitive hypertension. (NIH #HL65289, #HL72920, #HL92026; #DK‐38226).
Functional response to endothelin in macro‐ and microvascular endothelial cells
Authors: Shlyonsky Vadim , Ben Soussia Ismail , Cheriaa Nidhal , Naeije Robert , Mies Frederique
Published: June 2021

Several studies suggest that macro‐ and microvascular endothelial cells differ in their structure and function. We compared human pulmonary microvascular endothelial cells (MvEC) with human macrovascular endothelial cells from pulmonary artery (PAEC) and from aorta (AoEC) with regard to their functional response to endothelin‐1. We used RTQ‐PCR, whole‐cell patch‐clamp technique, Fluo‐4 measurements of intracellular calcium and NO‐release measurements in order to study functional effect of endothelin‐1. RNA expression of endothelin receptor B was detected in AoEC, PAEC and MvEC. Expression of ETB receptor in MvEC was 3–5 times higher compared to AoEC and PAEC. Endothelin‐1 transiently increased membrane permeability of all three types of endothelial cells. There was no difference in peak ion current response between these different cells, however integral response (duration and current increase) of MvEC was bigger compared to PAEC and AoEC. By the contrast, measurements of intracellular calcium increase by endothelin‐1 did not reveal any difference in this functional response between cells tested. Finally, NO‐release in response to endothelin‐1 was not detectable in PAEC and AoEC, while in MvEC endotheline‐1 stimulated significant NO‐production. These results suggest, that microvascular endothelial cells present more pronounced ability to respond to endothelin‐1, thus balancing its vasoconstrictive action in microvasculature. Supported by FNRS and ULB funds.
Effect of juvenile growth on arteriolar eNOS phosphorylation
Authors: Kang Lori S. , Nurkiewicz Timothy R. , Wu Guoyao , Boegehold Matthew A.
Published: June 2021

Nitric oxide (NO) often contributes little to endothelium‐dependent dilation of arterioles in the young but mediates a large portion of these responses in adults. The aim of this study was to identify the mechanism by which NO emerges as an important endothelial factor in arterioles during juvenile growth. NO release in response to endothelium‐dependent agonists (ACh, A23187) was 5 to 6‐fold higher in gracilis muscle arterioles from 42–46 day‐old rats than in those from 25–28 day‐old rats. However, there were no differences between age groups in arteriolar wall eNOS expression or levels of the eNOS co‐factor BH4, and L‐Arg levels were actually lower in the older vessels than in the younger vessels (104 ± 6 vs.126 ± 3pmol/mg, p<0.05). In contrast, agonist‐induced phosphorylation of eNOS at its Ser1177 residue was almost 75% higher in arterioles from 42–46 day‐old rats than in those from 25–28 day‐old rats. Arterioles from the older rats did not show increased expression of Akt/PKB or PKA, kinases that mediate eNOS Ser phosphorylation, or heat shock protein 90 (Hsp90), which facilitates this process. These findings indicate that eNOS becomes more susceptible to activation during growth due to a change in the regulation of its phosphorylation status, possibly reflecting increased kinase or Hsp90 activity rather than expression. Support: AHA 0755264B, 09GRNT2250298 (MAB); AHA 0755024Y (GW); NIH ES015022, ES018274 (TRN).
Chronic treatment with propranolol does not alter sympathetic and cardiovagal baroreflex sensitivity in the Postural Orthostatic Tachycardia Syndrome
Authors: Fu Qi , VanGundy Tiffany B , Galbreath M Melyn , Shibata Shigeki , Jarvis Sara S , Levine Benjamin D
Published: June 2021

Patients with Postural Orthostatic Tachycardia Syndrome (POTS) have marked increases in heart rate (HR) during orthostasis, while β‐blocker is commonly used to reduce tachycardia. We tested the hypothesis that chronic β‐blocker treatment alters baroreflex sensitivity in POTS. Eighteen POTS patients (age 16–44) were treated with placebo (n=10) or propranolol (n=8, 80 mg qd) for 1 mo. Sympathetic baroreflex sensitivity was quantified by 1) linear correlation between muscle sympathetic nerve activity (MSNA) and stroke volume supine and during a graded upright tilt, and 2) relating sympathetic bursts to the fall of diastolic blood pressure (BP) during Valsalva (VAL). Cardiovagal baroreflex sensitivity was assessed during early phase II (decreasing BP) and phase IV (increasing BP) of VAL. Patients’ quality of life was evaluated using SF‐36 form. Propranolol decreased upright HR but had limited impacts on MSNA. Sympathetic baroreflex sensitivity remained unchanged after propranolol treatment [−1.04±0.40 bursts/min/mL pre vs −0.99±0.33 post, P=0.78; −0.97±0.30 bursts/20s/mmHg pre vs −1.01±0.33 post, P=0.69). Cardiovagal baroreflex sensitivity during decreasing BP (5.05±3.58 ms/mmHg pre vs 5.75±2.03 post, P=0.44) and during increasing BP (14.23±7.59 ms/mmHg vs 13.05±4.43; P=0.92) of VAL was comparable before and after propranolol treatment. Patients’ quality of life remained unchanged after treatment. Placebo had no effects on any variables measured. These results suggest that chronic propranolol treatment decreased tachycardia but did not alter baroreflex sensitivity in POTS patients. Patients’ quality of life was not improved by propranolol. More effective therapies to improve not only symptoms but also patients’ overall well‐being are needed.
Carotid Baroreflex Function in African Americans at Rest
Authors: Fulton Diana , Hodge T. Glenn , Vespoli Kalen , Keller David M.
Published: June 2021

African Americans (AA) are at relatively high risk for the development of hypertension. However, little is known regarding dynamic blood pressure control (e.g., arterial baroreflex function) in this racial population prior to the development of hypertension. The purpose of this study was to test the hypothesis that normotensive AA would exhibit reduced carotid baroreflex control of heart rate (HR) and mean arterial pressure (MAP) at rest compared to age, gender and fitness‐matched Caucasian Americans (CA). Eight subjects (4 in each racial group) underwent multiple 5‐sec trials of Neck Pressure (NP, +40 Torr: simulated carotid hypotension) and Neck Suction (NS, −60 Torr: simulated carotid hypertension). The peak HR and MAP responses to NP and NS were determined relative to pre‐stimulus values and used to calculate the Response Range (i.e., the difference between averaged peak responses to NP and NS) for the respective variable for each subject. Although somewhat lower in AA (14.5±3.2 mmHg), the MAP Response Range was not statistically different compared to CA (20.1±1.7 mmHg, P = 0.19). However, the HR Response Range approached statistical significance in AA (17.2±4.3 bpm) compared to CA (30.8±3.5 bpm, P = 0.051). These findings indicate possible inherent differences in carotid baroreflex function between racial populations at different risk for the development of hypertension.

Supported by NIH Grant R15HL096103
Salt induced vascular response through adenosine A2A receptor: role of CYP epoxygenase and soluble epoxide hydrolase
Authors: Nayeem Mohammed A , Zeldin Darryl C. , Boegehold Mathew A , Ponnoth Dovenia S , Roush Kevin P , Falck John R
Published: June 2021

High salt intake can change the effect of adenosine on arterial tone in mice. The aim of this study was to clarify the mechanism by which this occurs. Using aortas from mice fed a 4% (HS) or 0.45% (NS) NaCl diet for 4–5 wks, concentration response curves for NECA (adenosine analog) and CGS 21680 (A2A AR agonist) were obtained with glibenclamide (Glib, KATP channel blocker; 10−5M), 5‐hydroxydecanoate (5‐HD, mito‐KATP channel blocker; 10−4M), MS‐PPOH (CYP2J2 blocker; 10−5M), AUDA (sEH blocker; 10−5M) and DDMS (CYP4A blocker; 10−5M). Relaxation to 10−6 M NECA was greater in the HS group (28.4 ± 3.9%) than in the NS group (4.1 ± 2.3%). Relaxation to 10−6 M CGS 21680 was also greater in HS (27.9 ± 4.5%) than in NS (4.9 ± 2.2%). In HS, the CGS 21680 response was greatly reduced by MS‐PPOH (to 4.7 ± 2.0%) and 5‐HD (to 8.9 ± 2.2%), and abolished by Glib (−1.0 ± 5.9%). In NS, the CGS 21680 response was increased by AUDA (to 29.2 ± 3.1%) and DDMS (to 27.2 ± 3.0%). Compared to NS, HS vessels showed increased CYP2J2 expression (46% higher) but decreased sEH and CYP4A expression (60% and 30% lower, respectively). These data suggest that in mice fed normal diet, activation of arterial A2A receptors causes constriction via increased sEH activity and formation of CYP4A metabolites. However, in mice fed high salt, activation of these receptors triggers relaxation via formation of CYP2J2 metabolites. HL027339, HL094447, z01‐ES‐025034, AHA2250298, GM‐31278
Differences in the cardiac proteome in rats selected for either low or high running capacity
Authors: Burniston Jatin , Kenyani Jenna , Wastling Jonathan , Koch Lauren , Britton Steven
Published: June 2021

Selection on running capacity has created rat phenotypes of high capacity runners (HCR) that have enhanced cardiac function and low capacity runners (LCR) that exhibit features of metabolic syndrome. Proteins that regress with differences in running capacity may be mechanistically responsible for this divergence in disease risk. Hearts of 6 HCR and 6 LCR from generation 23 were analysed using difference in‐gel electrophoresis (DIGE) and protein spots were identified from database searches of tryptic peptide mass fingerprints and fragment ion mass spectra. The running capacity of HCR was 4‐fold greater than LCR. DIGE resolved 957 gel spots and proteins were identified unambiguously in 369 spots. Protein expression profiling detected 68 statistically significant (P<0.05; false discovery rate <10 %, calculated using q‐values) differences between HCR and LCR hearts. In HCR, there was robust up‐regulation of each enzyme of the beta‐oxidation pathway, whereas, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCR also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of alpha B‐crystallin at serine 59. Our data show that selection on running capacity is associated with changes in cardiac metabolism and oxidative stress pathways.
Involuntary breathing movements improve cerebral oxygenation during apnea struggle phase in elite divers
Authors: Dujic Zeljko , Uglesic Lovro , Breskovic Toni , Valic Zoran , Heusser Karsten , Marinovic Jasna , Ljubkovic Marko , Palada Ivan
Published: June 2021

We investigated whether the involuntary breathing movements (IBM) during struggle phase of apnea, together with peripheral vasoconstriction and progressive hypercapnia, have a positive effect in maintaining cerebral blood volume. The central hemodynamics, arterial oxygen saturation, brain regional oxyhemoglobin, deoxyhemoglobin and total hemoglobin changes (bO2Hb, bdHb and bTHb, respectively) and IBM were monitored during maximal dry apneas in 8 elite divers. The frequency of IBM increased and their duration decreased towards the end of the struggle phase, whereas the amplitude was unchanged. In all subjects a consistent increase in bdHb and bTHb was also found during struggle phase, whereas bO2Hb changed biphasically. Mean arterial pressure (MAP) was elevated during the struggle phase although there was no further rise in the peripheral resistance, suggesting unchanged peripheral vasoconstriction and implying the beneficial influence of the IBM on the cardiac output recovery. The IBM‐induced short lasting sudden increases in MAP were followed by similar oscillations in bO2Hb. These results suggest that an increase in the cerebral blood volume observed during the struggle phase of dry apnea is most likely caused by the IBM at the time of the hypercapnia‐induced cerebral vasodilatation and peripheral vasoconstriction.

This study was supported by the Croatian Ministry of Science, Education and Sports.
The Role of Nitric Oxide in the Urine Concentration Mechanism in Diabetic Rats
Authors: Cipriani Penelope , Kim Sunhye L. , Klein Janet D. , Denson Chad E. , Sim Jae H. , Sands Jeff M. , Blount Mitsi A.
Published: June 2021

Diabetes mellitus (DM) results in osmotic diuresis and lowered levels of nitric oxide (NO). We examined how absence of NO affects transporters involved in urine concentration in DM. Sprague Dawley rats were injected with streptozotocin to induce DM. Control and DM rats were given L‐NAME, a non‐selective inhibitor of nitric oxide synthase. Urine osmolality was lower in DM rats, however in L‐NAME treated DM rats levels were similar to untreated and L‐NAME treated animals. L‐NAME treatment did not change urine output however L‐NAME treated DM rats produced less urine than untreated DM rats. UT‐A1 was increased in DM animals. L‐NAME treatment alone did not alter UT‐A1 abundance however DM‐treated rats had increased UT‐A1 expression but the abundance was less than untreated‐DM animals. AQP2 abundance was up both DM and DM‐L‐NAME rats compared to the control group. L‐NAME treatment had no effect on control or DM rat AQP2 expression. Similarly, NKCC2 expression was increased in both DM and DM‐L‐NAME rat. L‐NAME did not alter NKCC2 abundance compared to control or the DM untreated animal. Increased protein expression of UT‐A1, AQP2 and NKCC2 observed in DM rats provides a compensatory mechanism however lack of NO prevents the increase of UT‐A1 in DM but reduces urine osmolality and output. Data suggest that alteration of NO levels may be beneficial in treatment of polyuria occurring in DM.
Retinal Reactivity in Type 2 Diabetics
Authors: Lott Mary EJ , Bettermann Kerstin , Slocumb Julia , Smith Bruce , Gardner Thomas , Gabbay Robert
Published: June 2021

Diabetics have impaired vascular reactivity in the microcirculation; however, the time course of this impairment is unclear. Imaging of the retinal blood vessels provides unique insight into the microvasculature.

PURPOSE

To measure retinal diameter responses to vasoconstrictor and vasodilator stimuli in persons with type 2 diabetes compared to healthy controls.

METHODS

Retinal blood vessels were continuously imaged with the Dynamic Vessel Analyzer (Imedos, Jena, Germany) as six type 2 diabetics without retinopathy (57±7 yrs) and four healthy controls (53±10 yrs) inhaled 100% oxygen (5 min) and during flicker light‐induced stimulation (5 min).

RESULTS

The diabetic compared to the control group had attenuated retinal vein vasoconstriction that was apparent within 2 minutes of hyperoxia (diabetics: −0.7±1.5%, −0.6±1.8%, −2.2±2.7%, −1.1±3.5% vs. controls: −2.9±1.2%, −5.4±0.9%, −7.5±1.6%, −8.4±0.8% at 2, 3, 4, and 5 min; P<0.05). Similar responses were observed in the retinal artery. Diabetics compared to controls also exhibited attenuated vasodilator responses (Artery: 1.1±1.0% vs. 8.6±5.3%; Vein: 2.4±3.2% vs. 7.2±2.6%; P<0.05).

CONCLUSION

Type 2 diabetics have both attenuated retinal vasodilator responses and early attenuated vasoconstrictor responses in which the mechanisms are unclear. Funded by Pennsylvania Department of Health using Tobacco CURE Funds.
Increased decorin mRNA expression leads to larger collagen fibril diameter in SHR kidney cortex
Authors: Skogstrand Trude , Leh Sabine , Moen Ingrid , Stuhr Linda Elin Birkhaug , Reed Rolf , Iversen Bjarne Magnus , Hultström Michael
Published: June 2021

It has been shown that the renal hypertensive damage in SHR is accompanied by changes in the synthesis, deposition and breakdown of collagen, in particular in the juxtamedullary cortex (JMC). In the present study, we investigated gene expression using Illumina beadarray microarrays comparing renal cortex from 60 week old SHR with WKY rats.

The results showed increased expression of decorin (2.3 fold increase P<0.01). Decorin is a small leucin rich protein (SLRP) which binds collagen and is important in collagen fibril formation. In addition, fibronectin, another SLRP was also increased (1.8 fold increase P<0.01). Finally several forms of fibrillar collagen were upregulated, collagen Ia1 (1.4 fold increase P<0.01), collagen IIIa1 (2.7 fold increase P<0.01) and collagen Ia2 (2.9 fold increase P<0.01). This indicates a changed collagen fibril composition and assembly process.

Further, collagen fibril diameter measurements were performed using TEM. The fibril diameters were increased in the JMC of SHR compared to the outer cortex (P<0.05), while diameters in superficial and juxtamedullary cortex were not different in WKY.

In conclusion, renal interstitial fibrosis in JMC of SHR is composed of thicker collagen fibrils. This increased fibril diameter may be a result of increased SLRP expression in damaged cortex as well as a changed composition of the collagen fibrils.
Dipeptidyl Peptidase IV Inhibition Attenuates Blood Pressure Rising in Young Spontaneously Hypertensive Rats (SHR)
Authors: Pacheco Bruna Piccolo Muniz , Crajoinas Renato Oliveira , Couto Gisele Kruger , Davel Ana Paula Couto , Rossoni Luciana Venturini , Girardi Adriana Castello Costa
Published: June 2021

The present study aimed to assess the effect of the dipeptidyl peptidase IV inhibitor sitagliptin on renal function and blood pressure in young (5‐wk‐old) pre‐hypertensive and adult (14‐wk‐old) hypertensive SHR. Sitagliptin was given by oral gavage twice daily at a dose of 20 mg/kg for eight days. As a control, same age SHR received vehicle. After eight days of sitaglipitin administration, systolic (121 ± 3 vs 149 ± 9 mmHg, P = 0.01), diastolic (84 ± 4 vs 105 ± 9 mmHg, P = 0.02), and mean arterial pressures (104 ± 3 vs 125 ± 8 mmHg, P = 0.01) were significantly lower in young treated SHR compared to control. The cumulative urinary volume, sodium and lithium excretion were significantly higher in young treated animals than in controls. Sitagliptin also decreased Na+/H+ exchanger NHE3 expression in proximal tubule (PT) microvilli membrane vesicles (32 ± 4%; P < 0.01) and increased NHE3 phosphorylation at serine 552 (38 ± 6; P < 0.001). In the adult hypertensive SHR DPPIV inhibition did not cause any effect on renal fuction. Likewise, no differences in blood pressure were observed between adult SHR treated with sitagliptin and its correspondent control. Our data suggest that DPPIV inhibition can prevent or delay increases in blood pressure in young pre‐hypertensive SHR partially by inhibiting PT NHE3.

Supported by FAPESP and CNPq
Effect of intracerebroventricular (ICV) and subcutaneous (SC) benzmail administration on AngII‐salt hypertension
Authors: Hirsch Dalay Marie , Guzman Pilar A , Rodriguez Myraida , Toney Glenn , Osborn John W.
Published: June 2021

AngII‐salt hypertension in the rat is mediated, in part, by activation of the sympathetic nervous system. In the present study we tested the hypothesis that benzamil (Benz) blockable sodium channels and transporters in the brain contribute to this salt‐sensitive model. Male Sprague Dawley rats consuming a high salt diet (2.0% NaCl) were instrumented with telemeters to measure mean arterial pressure (MAP), ICV cannulae for continuous Benz or vehicle infusion, and placed on one of 6 treatments for the duration of the protocol; Benz‐ICV (4 or 8 nmol/day), vehicle‐ICV (Veh‐ICV; 2 groups), or Benz‐SC (4 or 8 nmol/day). MAP was monitored for 3 days, then AngII was administered (150 ng/kg/min, sc) for 14 days. There were no differences in MAP between the six groups prior to AngII. MAP increased ~30 mmHg in both Veh‐ICV groups by Day 14 of AngII. At the low dose, neither Benz‐ICV, nor Benz‐SC, affected the hypertensive response to AngII. In contrast, AngII‐salt hypertension was abolished by Benz‐ICV at the higher dose. Benz‐SC at the higher dose attenuated AngII‐salt hypertension by 50%. We conclude that benzamil‐sensitive sodium channels/transporters in the brain contribute to AngII‐salt hypertension in the rat. Supported by R01 HL076312
Induction of hypertension and peripheral inflammation by deletion of extracellular superoxide dismutase in the central nervous system
Authors: Lob Heinrich E. , Marvar Paul J. , Guzik Tomasz J. , Weyand Cornelia , Gordon Frank J. , Harrison David G.
Published: June 2021

Superoxide has been implicated in central neural control of blood pressure. The circumventricular organs (CVO) lack a well‐formed blood‐brain barrier and produce superoxide (O2·‐) in response to angiotensin II and other hypertensive stimuli. The extracellular superoxide dismutase (SOD3) is expressed in cells associated with the CVO. To understand the role of SOD3 in the CVO in blood pressure regulation, we injected an adenovirus encoding Cre‐recombinase (AdCre, 5x108 particles/ml) intracerebroventricularly (ICV) in mice with loxP sites flanking the SOD3 coding region. Deletion of CVO SOD3 increased baseline blood pressure modestly and markedly augmented the hypertensive response to low‐dose angiotensin II (140 ng/kg/day). Deletion of CVO SOD3 also increased sympathetic modulation of heart rate and blood pressure variability, increased vascular superoxide production and T cell activation as characterized by increased circulating CD69+/CD3+ cells. Deletion of CVO SOD3 also markedly increased vascular T cell and leukocyte infiltration caused by angiotensin II. We conclude that SOD3 in the CVO plays a critical role in regulation of blood pressure and its loss promotes T cell activation and vascular inflammation. These findings provide insight into how central signals produce vascular inflammation in response to hypertensive stimuli such as angiotensin II. Funded by NIH R01 HL39006 and PPG HL58000.
Novel Mechanisms of Activation of Endothelial Nitric Oxide Synthase Mediated by Endothelial Transient Receptor Potential Vanilloid Type 1
Authors: Ching Li‐Chieh , Kou Yu Ru , Pan Ching‐Chian , Lee Tzong‐Shyuan
Published: June 2021

OBJECTIVE

We investigated the molecular mechanism underlying the role of transient receptor potential vanilloid type 1 (TRPV1) in the activation of endothelial NO synthase (eNOS) in endothelial cells (ECs) and mice.

METHODES AND RESULTS

In bovine aortic ECs, TRPV1 ligands (evodiamine or capsaicin) promoted NO production, eNOS phosphorylation and TRPV1‐eNOS interaction, all of which were abrogated by the TRPV1 antagonist capsazepine and extracellular Ca2+ remove. TRPV1 ligands also enhanced the phosphorylation of Akt, calmodulin‐dependent protein kinase II (CaMKII) and TRPV1, and increased interactions of TRPV1 with Akt and CaMKII. Inhibition of PI3K and CaMKII suppressed ligand‐induced increases in TRPV1 phosphorylation, TRPV1‐eNOS interaction and eNOS activation. In vivo experiemnts revealed that TRPV1 activation increased the phosphorylation of Akt, CaMKII and eNOS in the aortas of wild type mice, whereas it failed to activate eNOS in the aortas of TRPV1 deficient mice.

CONCLUSION

TRPV1 activation in ECs may trigger a Ca2+‐dependant PI3K/Akt/CaMKII signaling leading to enhanced phosphorylation of TRPV1, increased TRPV1‐eNOS interaction and ultimmately eNOS activation. Thus, in addition to its channel function, the TRPV1 may possess a potential role serving as a scaffold for recruitment and interaction with eNOS, Akt and CaMKII, which may be vital for regulating eNOS activation.
Nitric Oxide Enhances NFATc3 Nuclear Import and Decreases Export
Authors: Bettini Layne , Albertoni‐Borghese Florencia , Majowicz Mónica , Vidal Norberto , Bosc Laura Gonzalez
Published: June 2021

We have previously shown that nitric oxide (NO) enhances NFATc‐mediated aquaporin‐2 (AQP2) promoter activation in MDCK cells. Also, we have shown that elevation of Ca2+ and NO are required for NFATc3 nuclear accumulation in cerebral artery smooth muscle. Ca2+‐activated calcineurin dephosphorylates NFATc allowing its nuclear import. GSK3, protein kinase A and JNK2 phosphorylate NFATc. The goal of this study was to determine the mechanism by which NO enhances NFAT activity. MDCK cells were transfected with a GFP‐NFATc3 plasmid and nuclear fluorescence intensity was measured over time. To study nuclear import, cells were treated with a Crm‐1 exportin inhibitor, 40 nM leptomicin B, and the Ca2+ ionophore (NFATc activator), 1 μM ionomycin (Io) ± 0.1 mM NONOate (NO donor). To study export, cells were treated for 1 h with Io and then imaged in the presence of 1 μM cyclosporin A (calcineurin inhibitor) ± 0.1 mM NONOate.

NO significantly enhanced Io‐induced NFATc3 nuclear import increasing the Ymax without affecting the K (Ymax= 3.6±0.5 vs. 2.4±0.3, n=5, p<0.05). Also, NO decreased NFATc3 nuclear export by increasing the plateau without affecting the K (Plateau= 0.35±0.03 vs. 0.14±0.03, n=5, p<0.05). Our results suggest that NO enhances NFATc nuclear import and decreases export. Future studies will address if NO via protein kinase G is inhibiting any of the kinases that phosphorylate and inactivate NFATc. Supported by AHA SDG.
Nitric oxide (NO) released from NO donors has hypotensive effect and it induces rat aorta relaxation due to the activation of K+ channels, guanylyl‐cyclase and SERCA
Authors: de Carvalho Pereira Amanda , Vercesi Juliana Aparecida , Biazzotto Juliana Cristina , da Silva Roberto Santana , Bendhack Lusiane Maria
Published: June 2021

NO reduces cytosolic Ca+2 in the vascular smooth muscle cells by cGMP‐PKG, K+ channels activation and Ca+2 uptake via sarcoplasmic reticulum Ca‐ATPase (SERCA). This study aimed to investigate if NO released from PY and SNP activates K+ channels and the enzymes guanylyl‐cyclase (GC) and SERCA. We also studied its effect on the mean arterial pressure (MAP). Vascular reactivity to PY and SNP were studied in denuded rat aortic rings pre‐contracted with phenylephrine (Phe). Concentration‐effect curves for PY and SNP were constructed with or without the inhibitors of GC (ODQ 1μM), SERCA (Thapsigargin, TG 1μM) and K+ channel (TEA 1mM). MAP was recorded in conscious hypertensive rats after in bolus injection of PY (5, 7mg/Kg) or SNP (35μg/Kg). PY induced hypotensive effect at the doses of 5 mg/Kg (−18.3 ±3.8 mmHg) and 7mg/Kg (−24±4mmHg) which effects were lower but longer (10.4min) than SNP 35μg/Kg (−80±6.1, 1.18min). PY and SNP presented similar efficacy in inducing aortic ring relaxation, but PY (pD2: 6.5±0.1) was less potent (pD2) than SNP (pD2: 8.07±0.1). The relaxation was inhibited by ODQ (PY: 37.9±3.6%, SNP: 29.3±2.6%) and TG (pD2: PY 5.7±0.2, SNP: 7.69±0.1). However, TEA reduced the relaxation to PY (pD2: 5.8±0.1) but it had no effect on SNP. In conclusion, NO released from PY has hypotensive effect and it induces relaxation due to the activation of K+ channels, sGC and SERCA. Supported by Fapesp and CNPq.
Time‐course effects of low salt diet on the regulation of cardiac NO bioactivity: Role of NAD(P)H oxidase
Authors: Skayian Yester S , Coffin Samuel , Huang Harer , Wang Ziping , Hintze Thomas H
Published: June 2021

The time‐course effects of low salt diet (LSD) on ANG II, MVO2 and NO bioactivity were studied in gp91phox (−/−) (KO) mice. A 2 week LSD significantly (P<0.05) increased the plasma ANG II levels in both wild type (WT) and KO mice. An 8 week LSD also increased plasma ANG II levels in WT (355±77 to 508±36pg/ml, P<0.05, n=10) but there was no change in KO mice. On normal diet (ND), stimulation of NO production by bradykinin (BK) induced a concentration dependent reduction in MVO2 (−32±2% and −25±2%) in vitro in both WT and KO mice respectively. The reduction in MVO2 was significantly (P<0.0001) attenuated by L‐NAME in WT and KO mice. BK‐induced reduction in MVO2 was significantly attenuated in WT (−14±1%, P<0.0001) on 8 week LSD but not in KO mice. The BK‐induced reduction in MVO2 was restored in WT by apocynin (−25±1%), by losartan (−25±1%) or by tiron (−28±2%). Similar effects were seen in KO mice on 2 week LSD. Systolic and diastolic blood pressure (BP) was significantly increased after 8 week LSD in WT (123±2/79±1 to 143±4/90±3, P<0.01, n=10). No significant increase in BP was seen in KO mice on 8 week LSD or WT and KO mice on 2 week LSD. Protein expressions of gp91 and p47 increased significantly (P<0.05) in WT on 8 week LSD. Thus LSD increases plasma ANG II and reduces NO bioactivity in the heart. The assembly and activation of NAD(P)H oxidase by ANG II may be the basis of LSD induced cardiovascular dysfunction. Supported by PO‐1 HL 43023 and 50142.
Age‐ and sex‐related changes in purinergic metabolism in the skeletal muscle vasculature
Authors: Stone Audrey J , Evanson Kirk W , Kluess Heidi A
Published: June 2021

The purpose of this study was to determine if vascular ecto‐ATPase was affected by sex or age in skeletal muscle arteries and arterioles. We hypothesized that females would have higher ATPase activity than males, and that old rats would have lower ATPase activity than young rats. The red gastrocnemius 1A (G1A) arterioles and femoral arteries were removed and cleaned from 2mo, 6mo, and 24mo old Fisher 344 rats. Whole vessel segments were incubated in a phosphate reagent for 50min and then analyzed using a spectrophotometer. Males had significantly higher ATPase activity than females at 2mo (106±22, n=8 vs. 39±7pM phosphate, n=8) and 6mo (208±56, n=7 vs. 44±14 pM phosphate, n=8) in the G1A, p<0.05. Endothelial removal in 2mo G1A reduced the variability (males: 86±7, n=5; females: 71±10, n=5 vs. males: 88±18, n=8; females: 60±17 pM phosphate, n=7), but it was not statistically different. Males (1184±175 pM phosphate) had significantly higher ATPase activity than females (489±36 pM phosphate) in the femoral in 6mo and 24mo (1510±70 vs. 908±76 pM phosphate) rats, p<0.05. In the femoral, ATPase activity was 47% higher in 24mo males than in 2mo old males ( p<0.05). ATPase activity was 50% higher in 24mo females than in 2mo and 6mo females(p<0.05). In general, males appear to have higher ATPase activity than females, and ATPase activity increases with age. Supported by NIH and Arkansas Biosciences Institute.
Intrinsic circadian oscillation of myosin light chain phosphorylation in vascular smooth muscle cells
Authors: Saito Toshiro , Hirano Mayumi , Ide Tomomi , Sunagawa Kenji , Hirano Katsuya
Published: June 2021

The onset of coronary artery disease, such as myocardial infarction, shows an apparent circadian variation. However, its molecular mechanism still remains to be elucidated. This study investigated the role of intrinsic vascular clock in the circadian regulation of vascular contractility. The porcine coronary artery smooth muscle cells in culture were exposed to 100 nM dexamethosone for 2 hr (Dex shock) to induce a circadian rhythm. The response of the myosin light chain (MLC) phosphorylation to the contractile stimuli was evaluated as an index of vascular contractility. A real‐time PCR analysis revealed a circadian oscillation of the expression of clock genes, such as bmal1 and per1. The resting level of MLC phosphorylation did not significantly change after DEX shock. In contrast, the level of MLC phosphorylation induced by thrombin exhibited a circadian oscillation with a 25.3‐hr cycle, reaching peaks at 36 hr and 60 hr. MLC phosphorylation changed from 0.85 at a nadir to 1.05 PO4 mol/MLC mol at a peak. However, there was no change in the level of thrombin receptor. The thrombin‐induced contraction of smooth muscle cells also exhibited a circadian oscillation after DEX shock. The results demonstrated the existence of an intrinsic circadian oscillation of the response of MLC phosphorylation. The present study thus suggests the vascular clock to contribute to the circadian regulation of vascular contractility.
Monoamine oxidase activity in skeletal muscle conduit and resistance vessels
Authors: Evanson Kirk W , Stone Audrey J , Kluess Heidi A
Published: June 2021

The purpose of this study was to examine monoamine oxidase (MAO) activity in the vascular smooth muscle of conduit and resistance vessels. Femoral arteries and red (RG1A) and white (WG1A) first‐order gastrocnemius arterioles were excised from 6‐month old male, F344 rats (n=5). The vessels were cut into 2mm segments and homogenized in 120μl of enzyme buffer (100mM HEPES, 5% glycerol). Luminometric (Promega) and spectrophotometric (Thermo Scientific) assays were used to quantify MAO activity and total protein content of the vessel, respectively. Relative luminescence units (RLU) were proportionate to MAO activity. MAO activity was greater in the femoral arteries (18.59±2.26 RLU/μg; p<.05) as compared to RG1A (11.62±1.82 RLU/μg) and WG1A (12.03±1.39 RLU/μg) vessels. These data suggest that MAO activity is greater in the larger conduit vessels than in the resistance vessels irrespective of vessel size, and that there are no differences in MAO activity between the red and white first‐order gastrocnemius arterioles. This research was supported by the Arkansas Biosciences Institute and the National Institute on Aging.
Impact of hypertension and hormonal status on relaxation of the pudendal vasculature in aging female rats
Authors: Hannan Johanna , Allahdadi Kyan , Giachini Fernanda , Dorrance Anne , Webb R. Clinton
Published: June 2021

The prevalence of female sexual dysfunction (FSD) and cardiovascular disease increases with age. We examined the impact of hypertension and hormonal status on pudendal artery function, a key resistance vessel for blood flow to the genital tissue. We hypothesize that pudendal arteries from aged stroke prone hypertensive rats (SHRSP) have impaired endothelium‐ and soluble guanylyl cyclase (sGC)‐mediated relaxation. Ovariectomies (OVX) or sham surgeries were performed in 7 month old SHRSP and Wistar Kyoto (WKY) female rats. At 11 months of age, pudendal arteries were mounted and exposed to increasing concentrations of phenylephrine (PE), acetylcholine (ACh) and sGC agonist BAY 41‐2272 (+/− sGC antagonist, ODQ). Pudendal arteries from SHRSP exhibited greater contraction to PE and impaired ACh‐mediated relaxation. These differences were further exacerbated in SHRSP‐ and WKY‐OVX. All groups demonstrated equal pudendal artery relaxation to BAY 41‐2272. In the presence of ODQ, relaxation to BAY 41‐2272 was abolished in SHRSP+/−OVX; however inhibition was overcome in the WKY and reduced by 50% in WKY‐OVX. In aged hypertensives, pudendal artery relaxation via ACh and sGC‐mediated pathways is impaired and this abnormality may contribute to FSD in hypertension. (Funded by Heart & Stroke Foundation Canada and NIH)