bluuebunny/crossref_metadata_2025 huggingface dataset:
Small skeletal muscle veins exhibit substantial myogenic response, which is mediated by hydrogen peroxide‐induced activation of TP receptors
Authors: Debreczeni Béla Zoltán , Gara Edit , Veresh Zoltán , Tamás Róbert , Hamar János , Koller Ákos
Published: June 2021

We hypothesized that isolated small veins (similarly arterioles) develop an appreciable myogenic tone in response to elevation of intraluminal pressure and aimed to elucidate the constrictor mechanism(s) involved. Thus small veins were isolated from gracilis muscle of male Wistar rats, then cannulated and incubated in the presence of 10 mmHg of intraluminal pressure at T = 37°C in a special pressure myograph chamber. Isolated small veins developed a substantial myogenic tone in response to increases in intraluminal pressure (from 1 to 12 mmHg). Calculated maximum myogenic tone was 70 ( 5 % of PD at 10 mmHg. Presence of indomethacin or catalase or SQ 29,548 reduced significantly the pressure‐induced myogenic tone. Also, H2O2 (10‐9‐10‐5 M) elicited concentration dependent constrictions, which were inhibited by the indomethacin or SQ 29,548. Arachidonic acid (10‐7–10‐4M) elicited concentration dependent constrictions, which were inhibited by indomethacin or SQ 29,548. In conclusion, in isolated small veins, 1) increases in pressure and H2O2 elicit substantial constrictons, both of which are inhibited by catalase, indomethacin or SQ 29,548 suggesting that the myogenic mechanism is mediated by H2O2 ‐induced arachidonic acid derived constrictor metabolite(s) such as prostaglandin H2/thromboxane A2 acting on TP receptors.

Support: AHA Founders Aff. 0855910D and Hungarian Sci. Res. Funds, OTKA‐T48376.
The effect of network pattern alterations on microvascular resistance in hypertension
Authors: Yang Ming , Murfee Walter Lee
Published: June 2021

Microvascular rarefaction, defined by a loss of vessels, is a common characteristic of hypertension and has been linked to elevated microvascular resistance. The objectives of this study were to determine whether rarefaction is associated with other network pattern alterations in hypertensive rat mesenteric networks and to whether these alterations lead to increased resistance. Mesenteric tissues from age‐matched (15–16 weeks) male spontaneously hypertensive rats (SHR) and normotensive Wistar‐ Kyoto (WKY) rats were harvested and labeled for PECAM, an endothelial cell marker. SHR networks displayed decreased microvascular area, arteriolar‐venular (AV) length, the number of AV branches, capillary segments and increased AV connections per network compared to WKY networks. Based on segment lengths, branching locations, and an assumed pressure drop of 65 mmHg, resistance per network was calculated using a computational model. For simulations with uniform vessel diameter and with the diameters based on reported intravital measurements, SHR resistance was not elevated compared to WKY level. Our results suggest that microvascular pattern alterations associated with hypertension are more complex than vessel loss and that rarefaction is not necessarily associated with elevated microvascular resistance. Supported by the Tulane Hypertension and Renal Center of Excellence.

Grant Funding Source: Tulane Hypertension and Renal Center of Excellence.
Computational Analysis of a Microfluidic Device for Measuring Oxygen‐Dependent ATP Release from Erythrocytes
Authors: Sove Richard , Ghonaim Nour W. , Ellis Christopher G. , Goldman Daniel
Published: June 2021

Release of adenosine triphosphate (ATP) from erythrocytes is believed to be a key component of the system for regulating microvascular blood flow. Recently, shear rate‐dependant ATP release from erythrocytes was measured using a microfluidic device (Wan et al, PNAS, 2008). These authors found ATP release occurred within 25–75ms following increased shear. The present work seeks to apply a similar approach to oxygen‐dependant ATP release, the dynamics of which are thought to be important in regulation of microvascular O2 delivery. Our computational model allows for the analysis of such a system to: i. design the optimal device to measure ATP release and ii. quantitatively interpret the resulting output signal. A computational model was constructed based on hemodynamics, convective‐diffusive transport of O2 and ATP, ATP/luciferin‐luciferase reaction kinetics, and optics. The computational model shows erythrocyte O2 levels can be altered rapidly enough to measure a delay in ATP release of 25ms. We show that this computational model is appropriate for analyzing properties associated with erythrocyte ATP release including the relationship between hemoglobin O2 saturation and magnitude of ATP release.
Shear Stress Generated by Different Fluid Compositions Induces Differential Endothelial Signaling in Intact Venules
Authors: Xu Sulei , Yuan Dong , He Pingnian
Published: June 2021

Disturbed shear stress promotes atherosclerosis, thrombosis, and inflammation of the vessel wall. Most shear stress studies have been conducted in large arterial segments or cultured endothelial cells (ECs). Shear stress induced EC signaling and its role in the regulation of microvessel function have not been well explored in intact venules. The objective of this study was to investigate the EC [Ca2+]i and nitric oxide (NO) responses at the onset of shear stress generated by different fluids. Shear stress was quantified by measuring the flow velocity and fluid viscosity using a high speed camera and cone‐plate viscometer. The changes of EC [Ca2+]i and NO were measured in Fura‐2 AM and DAF‐2 DA loaded individually perfused rat mesenteric venules. Transient increases in EC [Ca2+]i occurred only with whole blood perfusion. The peak EC [Ca2+]i of 203 ± 17 and 288 ± 18 nM were observed with shear stress at 11 and 32 dyn/cm2(n=5), respectively. Shear stress generated by the perfusion of albumin–Ringer, plasma, and Dextran 70‐albumin‐Ringer solution caused no changes in EC [Ca2+]i. However, both plasma and whole blood perfusion induced shear magnitude‐dependent NO production. These results indicated that in intact venules shear stress‐induced increases in EC [Ca2+]i require the presences of erythrocytes, while shear stress‐induced NO production can be independent of Ca2+ signaling. Supported by HL56237 and HL084338.
Contribution of Cav1.2 Channels to Coronary Microvascular Dysfunction in Metabolic Syndrome
Authors: Berwick Zachary C , Dick Gregory M , Bender Shawn B , Moberly Steven P , Kohr Meredith C , Goodwill Adam G , Obukhov Alexander G , Tune Johnathan D
Published: June 2021

Previous investigations by our group indicate that diminished functional expression of KV channels impairs control of coronary blood flow in obesity/metabolic syndrome (MetS). The goal of this investigation was to test the hypothesis that coronary microvascular dysfunction in MetS is also related to subsequent increases in CaV1.2 channel activity. Initial studies revealed that inhibition of KV channels with 4‐aminopyridine (4AP, 0.3 mM) increased intracellular [Ca2+], contracted isolated coronary arterioles, and decreased coronary reactive hyperemia, and that these effects were reversed by blockade of CaV1.2 channels. Further studies in chronically instrumented Ossabaw swine showed that inhibition of CaV1.2 channels with nifedipine (10 μg/kg, iv) had no effect on coronary blood flow at rest or during exercise in lean swine. However, blockade of CaV1.2 channels significantly elevated coronary blood flow, conductance, and the balance between flow and metabolism in swine with the MetS (P < 0.05). These changes were associated with a ~50% increase in inward CaV1.2 current and elevations in α1c and α2δ1 channel subunit expression in coronary smooth muscle cells from MetS swine. Taken together, these data indicate that increased functional expression of coronary CaV1.2 channels contributes to coronary microvascular dysfunction in the MetS.
The ratio of C‐peptide to insulin is critical for low oxygen tension‐induced ATP release from human erythrocytes (RBCs)
Authors: Richards Jennifer , Stephenson Alan H. , Ellsworth Mary L. , Sprague Randy S.
Published: June 2021

Exposure to low oxygen tension (pO2 ~ 20mmHg) stimulates ATP release from RBCs, allowing these cells to direct blood flow to areas of increased O2 need. We showed that low pO2‐induced ATP release from RBCs is inhibited by insulin at concentrations present in prediabetes contributing to defective vascular control. Here we investigated the hypothesis that C‐peptide, at physiological concentrations, attenuates the adverse effects of insulin on low pO2‐induced ATP release from human RBCs. Although C‐peptide and insulin are co‐released at equimolar concentrations, due to differing clearance rates, they circulate at different concentrations. When insulin and C‐peptide were administered at a 1:1 ratio (ratio when released), low pO2‐induced ATP release was not decreased (n=5). However, since this ratio is not present in vivo for extended periods, we tested additional ratios of C‐peptide to insulin: 6:1 and 4:1. We determined that a ratio of 6:1 (n=5) inhibits ATP release induced by low pO2. However, at 4:1, a ratio present physiologically in healthy humans, C‐peptide and insulin did not inhibit low pO2‐induced ATP release (n=7). Our results indicate that the ratio of C‐peptide to insulin is important for low pO2‐ induced ATP release from RBCs and suggest that co‐administration of C‐peptide with insulin could attenuate insulin‐induced adverse effects on microvascular perfusion. Supported by grants from the ADA and NIH.
Role of erythrocyte‐ vs. wall‐derived signals in flow regulation in heterogeneous microvascular networks
Authors: Roy Tuhin K , Secomb Timothy W
Published: June 2021

Postulated mechanisms for metabolic flow regulation are examined using a theoretical model to simulate blood flow, mass transport and vascular responses in heterogeneous microvascular networks. The model accounts for myogenic, shear‐dependent, and metabolic flow regulation. Metabolic signals are assumed to be propagated upstream along vessel walls via a conducted response. Arteriolar tone is assumed to depend on the conducted metabolic signal as well as local wall shear stress and wall tension. In heterogeneous networks, unequal partition of hematocrit at diverging bifurcations can lead to low hematocrit in low‐flow vessels. If the metabolic signal is generated solely by an erythrocyte‐dependent mechanism, such vessels then experience a reduced metabolic signal and may constrict further, leading to further decreases in hematocrit and oxygen delivery. Inclusion of a metabolic signal dependent on oxygen levels at the vessel walls can prevent this effect. The different functional ranges of oxygen tension at which these signals operate suggest a role for both types of signals in flow regulation. Supported by NIH grant HL070657.
Assessing the contribution of distinct vascular segments in arterial insufficiency
Authors: Arciero Julia , DiStasi Matthew , Unthank Joseph
Published: June 2021

Clinical studies indicate that vascular adaptations following an acute major arterial occlusion are capable of completely restoring resting perfusion. Similar responses are reported in animals, but the specific role of collaterals and the distal microcirculation remains controversial. In this study, a mathematical model was developed to predict how changes in the diameters of collateral and microvascular segments would contribute to compensation after focal femoral artery occlusion. The model was based upon proximal and distal pressures as well as dimensions of the femoral, saphenous, profunda femoral, and gracilis arteries in mouse hindlimb, and future studies will include microvascular dimensions in adductor and gastrocnemius muscles. The model confirms experimental observations that arterial occlusion shifts the site of primary vascular resistance from arterioles to small arteries which form collateral pathways. The model also predicts that a 60% increase in collateral artery diameter could explain the perfusion data even if distal vessel diameters were reduced as much as 22% due to reduced pressure.
Aging impairs electrical conduction along resistance artery endothelium via enhanced signal dissipation through KCa channels
Authors: Behringer Erik , Segal Steven
Published: June 2021

Aging results in endothelial cell (EC) dysfunction yet little is known of how intercellular transmission of electrical signals is affected. We hypothesized that aging impairs electrical conduction via current dissipation through Ca2+‐activated K+ channels (SKCa/IKCa). EC tubes (width: 60 μm; length: ≥ 2 mm) isolated from Young (3–6 mo, n ≥ 10) and Old (24–26 mo, n ≥ 8) superior epigastric arteries of male C57BL/6 mice were studied using dual simultaneous intracellular microelectrodes placed at separation distances of 50–2000 μm. Membrane potential (Vm) was greater in Old (−35 ± 2) vs. Young (−26 ± 1 mV; P < 0.05). Conduction amplitude (CA; ΔVm at Site 2/nA current injected at Site 1) was less in Old vs. Young (at 500 μm: 6 ± 1 vs. 10 ± 1 mV/nA; P < 0.05). SKCa/IKCa activation (1 μM NS309) increased Vm similarly for Old (Δ = −32 ± 2) and Young (Δ = −35 ± 2 mV) and the reduction in CA was greater in Young vs. Old (−6 ± 1 vs. −4 ± 1 mV/nA; P < 0.05). SKCa/IKCa inhibition (300 nM apamin + 100 nM charybdotoxin) evoked greater depolarization in Old (Δ = 14 ± 2) vs. Young (Δ = 7 ± 1 mV; P < 0.05) and the resulting increase in CA was greater in Old (45 ± 7%) vs. Young (22 ± 6%; P < 0.05) which restored conduction in Old to that of Young (both = 10 ± 1 mV/nA). We conclude that aging impairs electrical conduction along the endothelium by increasing signal dissipation through enhanced SKCa/IKCa activation. (NIH R01HL086483, R37HL041026, F32HL110701)
Age‐associated vascular oxidative stress, Nrf2 dysfunction and NF‐κB activation in the non‐human primate Macaca mulatta
Authors: Ungvari Zoltan , Bailey-Downs Lora C. , Gautam Tripti , Sosnowska Danuta , Mingyi Wang , Monticone Robert E. , Telljohann Richard , de Cabo Rafael , Sonntag William E. , Lakatta Edward , Csiszar Anna
Published: June 2021

Aging promotes oxidative stress in vascular endothelial and smooth muscle cells contributing to the development of cardiovascular diseases. Nrf2, a transcription factor, is activated by reactive oxygen species (ROS) in the vasculature of young animals, leading to up‐regulation of ROS detoxifying and antioxidant genes. The present study was designed to elucidate age‐associated changes of Nrf2‐driven free radical detoxification mechanisms in the vasculature of non‐human primates. We found that carotid arteries of aged rhesus macaques (~20 yrs) exhibit significant oxidative stress (as indicated by the increased 8‐iso‐PGF2α and 4‐HNE content and decreased GSH and ascorbate levels) as compared young macaques vessels (age: ~10 y). Although NF‐κB is activated, age‐related oxidative stress does not activate Nrf2 and does not induce Nrf2 target genes (NQO1, GCLC and HMOX‐1). In cultured vascular smooth muscle cells (VSMCs) derived from young and old M. mulatta, treatment with H2O2 and high glucose significantly increases transcriptional activity of Nrf2 and up‐regulates the expression of Nrf2 target genes in the young but not aged macaques. Taken together, aging is associated with Nrf2 dysfunction in M. mulatta arteries, which likely exacerbates age‐related cellular oxidative stress, promoting NF‐κB activation and vascular inflammation in aging.
Hypoxia/Reoxygenation (H/R) Stress Increases Organic Anion Transporting Polypeptide 1a4 (Oatp1a4)‐mediated Transcellular Transport at the Blood‐Brain Barrier (BBB): Relevance to CNS Drug Delivery
Authors: Ronaldson Patrick Thomas , Slosky Lauren Marie , Sanchez-Covarrubias Lucy
Published: June 2021

Our objective was to investigate effects of H/R stress, a component of ischemic stroke, on Oatp1a4‐mediated transcellular transport at the BBB. Rats (200–250 g) were subjected to normoxia (Nx, 21% O2, 60 min), hypoxia (Hx, 6% O2, 60 min) or H/R (6% O2, 60 min followed by 21% O2, 10 min). In rat brain microvessels, Oatp1a4 expression was increased following H/R but was unchanged by Nx or Hx. Uptake of Oatp1a4 substrates (i.e., taurocholate, atorvastatin) were increased following H/R. Oatp1a4 inhibitors (i.e., fexofenadine, estrone‐3‐sulfate) blocked taurocholate and atorvastatin brain uptake in H/R animals, further implicating Oatp1a4 in BBB transcellular transport of these drugs. Additionally, H/R reduced serum levels of transforming growth factor‐β1 (TGF‐β1) and decreased microvascular expression of TGF‐β receptor activin receptor‐like kinase (ALK)‐5. Pretreatment with SB431542, an ALK5 inhibitor, increased Oatp1a4 functional expression both in H/R and Nx animals. These novel data demonstrate that targeting an endogenous BBB transporter may be a viable approach for delivering drugs to the brain during cerebral ischemia. Our study also identifies a BBB signaling mechanism (i.e., TGF‐β/ALK5 pathway) that can be exploited to control expression of Oatp1a4 during H/R and, therefore, optimize CNS drug delivery. This work was supported by NIH R01 grant DA‐11271 and an AAPS New Investigator Grant.
Novel Role of Local Lamellipodia in Endothelial Barrier Function
Authors: Breslin Jerome W
Published: June 2021

Dynamics of the actin cytoskeleton and VE‐cadherin, both implicated in the control of endothelial permeability, are poorly understood. Using live cell imaging of confluent human umbilical vein endothelial cells (EC) expressing either GFP‐actin or GFP‐VE‐ cadherin, we tested the hypothesis that inflammatory agents disrupt the cortical actin cytoskeleton and VE‐cadherin localization at intercellular junctions. Time‐lapse images were recorded during baseline and after addition of agents that either increase permeability (10 μM histamine, 1 U/ml thrombin) or decrease permeability [0.5–2 μM sphingosine‐1‐phosphate (S1P); 2 μM S1P1 receptor agonist SEW2871]. Transendothelial electrical resistance (TER) served as an index of barrier function. The results revealed an unexpected finding: Actin‐rich local lamellipodia frequently protrude and retract between EC. Histamine and thrombin both significantly decreased local lamellipodia protrusion frequency. Conversely, S1P and SEW2871 rapidly increased protrusion frequency concomitant with an increase in TER. Changes in VE‐cadherin were slower, although normally continuous VE‐cadherin was clearly disrupted by thrombin or histamine. In summary, our results suggest a novel concept that local lamellipodia may help regulate the paracellular passage of fluids and solutes across the endothelium. Supported by NIH R01HL098215 and R21AA020049.
Effects of cigarette smoke extracts on cardiac stem cell Paracellular and Trans‐cellular permeability
Authors: Sumanasekera Wasana K. , Sumanasekera Gamini U. , Rokosh Gregg , Tran Hieu T.
Published: June 2021

Cigarette smoking is a risk factor for several cardiovascular diseases and premature death. The integrity of tight junctions between cardiac cells is important in keeping proper cardiovascular function. The main objective of our study is to investigate the possibility of impairment of tight junction barrier by cigarette smoke extracts (CSE). We hypothesized that CSE cause increase in para‐cellular and trans‐cellular permeability in c‐kit positive rat cardiac stem cells (rCSC). CSE induced development of paracellular gaps has not been reported in cardiac stem cells. This is truly a novel area and there was only one report discussing CSE induced changes in endothelial cell permeability. In our study cell junction permeability was detected using para‐cellular permeability assay. Following establishment of rCSC monolayer on trans‐well polycarbonate inserts, Fluorescein isothiocyanate (FITC)‐labeled dextran was added to the inner chamber and Para‐cellular permeability of FITC‐dextran across the cell monolayer was detected at 490 λ. As an indicator of CSE induced changes in trans‐cellular permeability, trans–cell electrical resistance was also detected. In addition, CSE induced changes in cell membrane damage was assessed using LDH release cytotoxicity assay. CSE caused increase in Para‐cellular permeability, LDH release, and decrease in Trans–cell electrical resistance in a dose dependent manner. The mechanisms underlying CSE induced modulation of cell permeability is currently under investigation. Preliminary data indicates that oxidative stress play a role in CSE induced changes in permeability. Research support was provided by Sullivan University College of Pharmacy faculty development grant awarded to Dr. Wasana Sumanasekera.
T‐type Ca2+ Channels and The Induction of CICR in Vascular Smooth Muscle
Authors: Bigdely-Shamloo Kamran , Vigmond Edward J , Welsh Donald G
Published: June 2021

Recent work has revealed a microdomain in cerebral arterial smooth muscle comprised of caveoli and sarcoplasmic reticulum. Immunolabeling techniques indicate that T‐type Ca2+ channels and ryanodine receptors localize to this microdomain while L‐type Ca2+ channels do not. Given these observations, T‐type Ca2+ channels were hypothesized to initiate a CICR‐like response. To address this hypothesis, a model of smooth muscle Ca2+ handling was developed. Our physical model was derived from direct dimensional measurements and consisted of a large cytosolic domain and a structured subspace of caveoli and SR. T‐type Ca2+ channels and ryanodine receptors were localized to the subspace whereas L‐type Ca2+ channels, Na+/Ca2+ exchanger, PMCA and SERCA pump were placed outside this region. The kinetics of SR Ca2+ uptake/release were derived from previous models. The activity of sacrolemmal pumps, exchanges and channels were obtained from various sources and scaled in a smooth muscle specific manner. A 20 mV depolarizing step from −60 mV induced an initial CICR process that gave way to periodic SR Ca2+ release every 10sec. This spark‐like release pattern was dependent upon continuous Ca2+ influx through T‐type Ca2+ channels and the recovery of ryanodine receptors from Ca2+‐induced inactivation. Varying the depolarizing step from −50 to −30 mV did not affect the frequency of SR release, a finding consist with T‐type Ca2+ channels acting as a two‐state switch. Supported by the Canadian Institute for Health Research.
Interactions between A2A adenosine receptor, hydrogen peroxide, and KATP channel in coronary reactive hyperemia
Authors: Sanjani Maryam Sharifi , Asano Shinichi , Dick Gregory , Teng Bunyen , Tilley Stephen , Ledent Catherine , Mustafa S Jamal
Published: June 2021

Myocardial metabolites such as adenosine mediate reactive hyperemia (RH) partially through glibenclamide‐sensitive KATP channels in coronary vascular smooth muscle (VSM). We tested the hypothesis that A2A receptors link to KATP channel activation via hydrogen peroxide (H2O2). Using A2A knockout (KO), A2BKO, and A2A/2B double KO mice, we performed RH experiments in Langendorff‐perfused hearts and whole‐cell patch clamp experiments on isolated VSM cells. Flow repayment (ml/g) following a 15 sec occlusion in WT hearts was 6.3 ± 0.4 and reduced in A2AKO (4.5 ± 0.2), but not A2BKO (6.1 ± 0.3), mice. Catalase (1250 U/ml), which breaks down H2O2, significantly reduced flow repayment (ml/g) in WT (4.4 ± 0.4) but not A2AKO (4.1 ± 0.4) hearts. Patch clamp experiments demonstrated that adenosine (10 μM) activated a glibenclamide (10 μM)‐sensitive conductance (nS/pF) in VSM cells from WT (0.05 ± 0.01) but not A2A/A2BKO (0.02 ± 0.01) mice. Additionally, in WT VSM cells, H2O2 (1 mM) activated a glibenclamide‐sensitive conductance (0.07 ± 0.02 nS/pF). Our data indicate that A2A receptors are coupled to KATP channels, in part, via the production of H2O2 as a signaling intermediate. HL027339, HL094447, HL071802, T32 HL090610.
IL‐6 Deletion Does Not Alter LV Remodeling and Dysfunction in Pressure Overload
Authors: Lai N. Chin , Tang Eric , Pandey Amit , Gao Meihua , Tang Tong
Published: June 2021

Chronic pressure overload, as seen in persistent hypertension, is a major risk factor for congestive heart failure. Left ventricular (LV) dysfunction in the pressure‐overloaded heart is associated with increased cardiac IL‐6 expression. Chronic IL‐6 infusion also leads to LV dysfunction, indicating a causal effect of IL‐6 on cardiac function deterioration. In this study, we tested the hypothesis that IL‐6 deletion would be beneficial to the pressure‐overloaded heart. Transverse aortic constriction was performed with a 27ga needle on IL‐6‐deleted (IL6KO) mice and C57BL/6J mice (CON) to induce pressure overload. Both the hearts from CON and IL6KO mice showed decreased LV contractile function 2 and 4 weeks after pressure overload, which also induced an increased in LV end‐diastolic diameter and LV wall thickness. No group differences were found in LV hypertrophy and re‐activation of the fetal gene program. There were also no differences between CON and IL6KO mice in LV fibrosis or expression of extracellular matrix proteins after pressure overload. In addition, no group differences in apoptosis or autophagy were observed.

Conclusion

These data indicate that IL‐6 deletion does not affect LV hypertrophy, dysfunction, or fibrosis in the setting of pressure overload. Reduction of molecular signaling elicited by proinflammatory IL‐6 family members, rather than the sole decrease of IL‐6 (such as immunodepletion with IL‐6 antibody), may be required to block deterioration of cardiac function in pressure overload.
Role of caveolin‐3 and mitochondria in protecting the aged myocardium
Authors: Fridolfsson Heidi Nicole , Reichelt Melissa E , Peart Jason N , Ali Sameh S , Finley J Cameron , Niesman Ingrid , Head Brian P , Kidd Michael W , Patel Piyush M , Headrick John P , Roth David M , Patel Hemal H
Published: June 2021

The aged heart is more susceptible to injury with limited understanding of the cause. We have shown that caveolin‐3 (Cav‐ 3, a heart scaffolding protein) is critical to cardiac protection. Cav‐ 3 protein is 50% reduced in 2 year old hearts. Cardiac myocytespecific overexpression of Cav‐3 (Cav‐3 OE) increased Cav‐3 expression through 2 years of age and resulted in augmented ischemic tolerance with age as assessed in Langendorff perfused heart. Mitochondrial morphology and function are disrupted in the aged heart leading to increased cell death during stress. Mitochondria from aged Cav‐3 OE mice maintain structural morphology similar to young mice and display improved function and reduced reactive oxygen species generation relative to aged transgene negative mice. Cav‐3 was shown to localize to mitochondria and result in increased mitochondrial membrane rigidity as assessed by electron paramagnetic resonance, suggesting mitochondrial Cav‐3 as a possible molecular mechanism of mitochondrial stabilization with age. Our data reveal a parallel decline in cardiac ischemic tolerance and Cav‐3 expression, and indicate that overexpression of Cav‐3 can eliminate this decline. Cav‐3 may contribute to maintenance of proper mitochondrial function and be a novel means to limit cardiac aging.
The disruption of the intracellular Ca2+ homeostasis is associated with heart disfunction in chronically malnourished Wistar rats
Authors: Mendes Luiza Villarinho Pereira , Gonsalez Sabrina Ribeiro , Oliveira-Pinto Leonardo Maciel , Nascimento Jose Hamilton Matheus , Einicker-Lamas Marcelo , Vieyra Adalberto , Cunha Valeria do Monti Nascimento , Lara Lucienne Silva
Published: June 2021

It is known the association between malnutrition and hypertension in the adult life. The aim of this study was to determine the consequences of the brazilian regional basic diet (RBD) submitted chronically to Wistar rats in the function and in the intracellular Ca2+ homeostasis of the heart. After weaning, the rats were fed with RBD and conventional diet for 10 weeks (n=10 each). The rats were subjected to echocardiography. A cohort of 5 hearts was isolated, perfused with Krebs buffer in the presence or absence of isoproterenol (ISP, from 0.3 to 1000 nM) by the Langendorff method. The microssomal fractions of the ventricles were obtained to measure the Ca2+‐ATPase activities. Ecocardiographic analysis showed a decrease in the cardiac output (53%), ejection fraction (71%), stroke volume (48%), and left diastolic ventricular diameter (22%) in the RBD. The left ventricular developed pressure was decreased in the RBD (34%). Furthermore, the contractility (+LVdP/dt) and relaxation (−LVdP/dt) in the presence of ISP are disrupted. The lung weight/body weight ratio were increased in 75%. We observed that the total Ca2+‐ATPase activity was two times higher in the RBD; due to a decrease in SERCA and an increase in the PMCA activities. Our data indicate that chronic malnutrition unbalances the SERCA and PMCA activities that could affect the heart hemodynamic.
Understimation of Murine Cardiac Hemodynamics using Invasive Catheters
Authors: Constantinides Christakis , Angeli Stelios , Kossivas Fotis , Ktorides Panayiotis
Published: June 2021

This study quantifies an error in the estimation of hemodynamic indices of global cardiac function from use of commercial catheters in mice. Male C57BL/6 mice (n=5, age=8–12wks, weight=25.5±3.3 g) were imaged on a 7T MRI scanner under 1.5% ISO in 100% O2; computational myocardial and catheter finite element models were constructed. A composite model of the catheter‐myocardium was imported in XFdtd for electric (E) field simulations. Comparison of end‐diastolic (ED) and end‐systolic (ES) volume estimates from such simulations show mean underestimation errors between −8.3±52.9 and −68.9±10.4%. Propagation of such errors in stroke volume, cardiac output, and ejection fraction estimation yields errors of 85.4±29.1, 182.3±117.4, 132.9±171.1%, respectively, given a catheter E‐field detection sensitivity of 5%. Modification of the commercial catheter design is shown to minimize underestimation errors of such indices, based on simulations. Elicited response improvements (peak amplitude and spatial extent) are quantified to be 405% and 52% (3.8 vs. 2.5 mm at 1% E‐field fall‐off) in ED, and 934 and 80% in ES, for the new catheter, compared to the commercially available design. Reductions in absolute hemodynamic error estimates were also computed (4.8–60.8%) for the new catheter in one mouse. Implementation of the newly proposed catheter design can lead to improved accuracy of indices of cardiac function.
Sustained differential effects of a transient antioxidant diet and voluntary exercise training on vascular functions in aging mice
Authors: Nguyen Albert Quyen , Leblond François , Thorin Éric
Published: June 2021

A healthy diet and regular exercise delay cardiovascular diseases in aging, but little is known about the mechanisms. We tested the effects of an early transient antioxidant diet and voluntary exercise on the vascular function of aging mice. One month old C57/Bl6 mice were split into 3 groups: one was supplemented with the antioxidant catechin (30mg/kg/d; CAT), a second was given access to an exercise wheel (EX) and a third remained sedentary (S), all until 9 months (9m). Mice were then aged for another 3 months in sedentary conditions with no treatment (12m). Femoral artery reactivity and compliance were assessed in a pressure myograph (80 mm Hg). At 9m, arterial compliance, represented by the Young's elastic modulus (β), did not differ between S (0.08±0.01) and CAT (0.08±0.01) mice, while it was greater in the EX group (0.05±0.01, p<0.05); 12m mice retained their elastic properties except for the EX group (0.07±0.01, p<0.05). The wall media‐tolumen ratio was similar in all groups. Endothelium‐dependent dilations to acetylcholine were similar in all groups at 9 and 12m. This dilatory response was, however, reduced by 60% (p<0.05) after inhibition of NO production in S mice, but not in the other groups at both ages. In conclusion, both CAT and EX improved vascular endothelial function by preserving NO‐independent vasodilator mechanisms, while only EX improved arterial compliance. Supported by CIHR MOP14496.
Vasomotor responses in facial and finger skin to acute changes in blood pressure
Authors: Kashima Hideaki , Ikemura Tsukasa , Yamaguchi Yuji , Hayashi Naoyuki
Published: June 2021

We examined the effects of acute changes in blood pressure on facial and finger vasomotion in nine healthy males using laser speckle flowgraphy and laser‐dopplar flowmetry, respectively. Skin blood flow was measured during acute changes in the rise of blood pressure (using maximum static handgrip: HG), and in the reduction of blood pressure (using acute release of 2‐min thigh cuff pressure at 250mmHg: CR). Skin vascular conductance (VC) values at baseline and the first four seconds of HG and CR were calculated from the ratio of skin blood flow and mean arterial pressure. During the HG, VC in forehead, eyelid, nose, cheek and upper lip significantly increased, whereas that in finger decreased compared to the baseline. During the CR, VC in forehead, eyelid, nose, cheek, upper lip and finger significantly decreased compared to the baseline. The results obtained in finger skin were similar to previous studies (Durand et al 2004, Saad et al 2001). These results obtained in facial skin suggest that changes in VC do not serve to maintain facial skin blood flow against acute changes in blood pressure.
Influence of Cellular Magnesium Concentration on Fibroblast Phenotype Differentiation
Authors: Battis Christine M. , Kombi Therese , Dubyak George , Romani Andrea
Published: June 2021

Given their common lineage from mesenchymal stem cells, fibroblasts and vascular smooth muscle cells share the ability to differentiate into mineralizing cells similar to osteoblasts and chondrocytes. Recent studies have reported a reduction in NT5E mRNA and CD73 protein expression together with accumulation of Ca*Pi crystals in patients with calcification of lower extremity arteries and joint capsules, suggesting that (myo)fibroblasts modulate vascular calcification via differentiation into an osteogenic phenotype characterized by increased expression of calcification inducers and loss of calcification inhibitors including PPi. PPi level is maintained through production from extracellular ATP via eNPPs, transport via Ank protein, and degradation via TNAP. Magnesium is also influential in that low Mg2+ level promotes calcification. As 3T3‐fibroblasts grown in low [Mg2+]o exhibit a 25% decrease in cellular ATP content, we hypothesized that a decrease in extra‐ and intra‐cellular Mg2+ content decreases ATP availability and extra‐cellular PPi level, which then promotes Ca*Pi precipitation and calcification. Supporting this hypothesis, our results indicate a decrease in extra‐ and intra‐cellular ATP and extracellular PPi in fibroblasts grown in low [Mg2+]o, together with higher intracellular PPi levels. The influence of low Mg2+ content on expression and activity of eNPPs, TNAP and Ank protein is currently being assessed to determine the mechanism responsible for the decrease in extracellular PPi level. Supported by NSF‐REU DBI1004909 to AR and NIH‐GM36387 to GD.
Involvement of CaM kinase II in impairments of endothelial function and eNOS activity in aortas of type 2 diabetic Goto‐Kakizaki rats
Authors: Matsumoto Takayuki , Nemoto Shingo , Taguchi Kumiko , Kamata Katsuo , Kobayashi Tsuneo
Published: June 2021

We examined the relationship between calcium/calmodulin‐dependent protein kinase II (CaMKII) pathway and endothelial dysfunction in aortas from Goto‐Kakizaki type 2 diabetic rats. The ACh‐induced relaxation, NO production and Ser1177‐endothelial NO synthase (eNOS) phosphorylation were attenuated in diabetic aortas (vs. controls). ACh increased the CaMKII phosphorylation within endothelial cells only in control aortas. ACh‐stimulated Thr286‐CaMKII phosphorylation within endothelial cells was decreased in diabetic aortas (vs. controls). ACh‐induced relaxation, NO production, and phosphorylation of eNOS and CaMKII were inhibited by CaMKII inhibitors in control aortas, but not in diabetic ones. Preincubation of aortic strips with a protein phosphatase‐1 inhibitor or with a protein phosphatase‐2A (PP2A) inhibitor corrected the above abnormalities in diabetic aortas. The expression of PP2A type A subunit was increased in diabetic aortas. The ACh‐stimulated Thr320‐phosphorylation level of PP1α was lower in diabetic aortas than in their controls, but the total PP1α protein level was not different. These results suggest that the aortic relaxation responses, NO production, and eNOS activity mediated by CaMKII phosphorylation are decreased in type 2 diabetic model, and that these impairments of CaMKII signaling may be due to enhancements of PP1α activity and PP2A expression.
Effects of long‐term aerobic exercise on vascular function mediated by insulin and insulin‐like growth factor‐1 in hypertension
Authors: Chao Wen-Chia , Yang Ai-Lun , Su Chia-Ting , Lin Yi-Yuan
Published: June 2021

Hypertension induces vascular dysfunction and decreases nitric oxide (NO) bioavailability. However, whether exercise influences insulin‐ and insulin‐like growth factor‐1 (IGF‐1)‐mediated vascular function in hypertension remains unclear. Thus, we aimed to investigate the effects of long‐term aerobic exercise on vascular function mediated by insulin and IGF‐1 in spontaneously hypertensive rats (SHR). Three groups of rats were used: SHR with aerobic exercise (SHR‐EX), SHR, and Wistar‐Kyoto rats (WKY) as the normotensive control group. The exercise group ran on the treadmill at moderate intensity for 1 hr/day, 5 days/week for 8 weeks. Vasorelaxant responses of rat aortas were analyzed in the organ baths. The aortic protein expression, such as insulin and IGF‐1 receptors, was also examined by Western blot. We found that, compared with the WKY, the SHR group had significantly (p<0.05) lower vasorelaxant responses to insulin and IGF‐1. However, the aerobic exercise significantly (p<0.05) improved the adverse effects in the SHR‐EX group. After treating the inhibitors of NO synthase and phosphatidylinositol‐3 kinase (PI3K), the differences of vascular responses among three groups were eliminated. In addition, the protein expression of insulin and IGF‐1 receptors was significantly (p<0.05) increased in the SHR‐EX group. Our results suggested that long‐term aerobic exercise ameliorated hypertension‐induced vascular dysfunction mediated by insulin and IGF‐1.
WNK3 Prevents the Nedd4‐2 Inhibition of the Renal Na‐Cl Cotransporter (NCC)
Authors: Arroyo Juan Pablo , Rojas-Vega Lorena , Lagnaz Dagmara , Castañeda-Bueno María , Ronzaud Caroline , Vázquez Norma , Staub Olivier , Gamba Gerardo
Published: June 2021

NCC and ENaC are inhibited by the E3 ubiquitin ligase Nedd4‐2 (JASN, 2011) and elimination of two canonical SGK1 phosphorylation sites on Nedd4‐2 (S222A and S328A), renders the Nedd4‐2 mediated inhibition of NCC and ENaC irrecoverable by SGK1. In search of new regulatory proteins, we found that the NH2 terminal domain of WNK3 interacts with Nedd4‐2. In Xenopus laevis oocytes inhibition of NCC by Nedd4‐2 is completely reversed in the presence of WNK3. In oocytes injected with NCC alone, WNK3 increases NCC activity by 264 ± 22 %, while in the presence of NCC and Nedd4‐2, the WNK3‐induced increase was of 353 ± 44 %. Additionally, in contrast to SGK1, WNK3 further activates NCC in the presence of double mutant Nedd4‐2 S222A/S328A to 501 ± 48 %. Thus, in the presence of wild type or mutant Nedd4‐2, WNK3 is a more potent stimulator of NCC. In the absence or presence of Nedd4‐2, NCC activation by WNK3 is prevented by elimination of WNK3 histidine 1497 and glycine 1508 that have been shown to be critical for interaction between WNKs (TBiochem J, 2011). In order to test for non‐specific WNK interactions we tested WNK4 and saw no difference in the Nedd4‐2 mediated inhibition of NCC. Thus, we propose that WNK3 is a regulator of the Nedd4‐2‐NCC pathway.
Role of Ecto‐5′–nucleotidase (CD73) in the Development of Renal Fibrosis
Authors: Carota Isabel , Höcherl Klaus , Castrop Hayo
Published: June 2021

Adenosine has been shown to be involved in the progression of fibrotic remodeling in various organs including the kidney. In the present study, ecto‐5′‐nucleotidase (CD73)‐deficient mice were used to address the route of adenosine generation during the development of renal fibrosis.

To induce renal fibrosis, CD73+/+ and −/− mice were subjected to unilateral ureter obstruction (UUO) for 7 days (n=6 each). UUO induced renal fibrosis in both genotypes, and this was accompanied by a marked increase in CD73 protein abundance in WT mice (196±35% of the contralateral kidney, p=.026). Compared to WT mice, UUO kidneys from CD73−/− showed significantly lower mRNA and protein levels of fibrotic markers like alpha‐SMA, fibronectin, TGF‐beta, Ki67, MMP‐2, MMP‐13, MMP‐14, and of the collagens Col 1a1, Col 1a2, Col 3a1 and Col 4a3, as determined by qRT‐PCR, western blotting (p<.01 for WT vs. KO, each), and immunohistochemistry. F4/80 antigen staining revealed a massive infiltration of macrophages/monocytes in the cortex of fibrotic WT kidneys, whereas only scattered cells were observed in kidneys from CD73−/− mice. In summary, CD73 is induced in the UUO model, and CD73‐dependent extracellular generation of adenosine appears to promote the development of renal fibrosis. Consequently, CD73 may constitute a pharmacologic target for the management of renal fibrosis.
Carbamylated LDL: novel potential mechanistic factor for progression of kidney disease
Authors: Apostolov Eugene O. , Fite Todd W. , Gaidar Iurii A. , Shah Sudhir V. , Basnakian Alexei G.
Published: June 2021

Carbamylated LDL (cLDL) is an abundant form of LDL resulting from ApoB modification by blood urea‐ or thiocyanate‐derived cyanate. It has a variety of vasculature‐targeting properties, and was found to be markedly elevated in mice with chronic kidney disease (CKD) and in CKD patients. However, its role in CKD progression has not been determined. We used two mouse models to address this issue. In first, CKD was induced by surgical deprivation of renal function in ApoE−/− mice, and in second, the mice were weekly IV injected with cLDL (32 mg/kg, IV‐cLDL model) for 6 weeks. Both models caused >100‐fold accumulation of cLDL in kidneys compared to controls. ICAM‐1, the mediator of cLDL‐induced leukocyte adhesion, and CD11b‐positive leukocyte count indicative of inflammation were significantly elevated the cLDL‐enriched kidneys. The IV‐cLDL mice developed 2.5‐fold elevation of serum creatinine and 30% decrease of GFR indicating the loss of kidney function. Kidney microcirculation assessed by ultrasound echography showed 40–50% drop in the microcirculation of IV‐cLDL mouse kidneys. Finally, immunohistochemical analysis of kidneys of CKD patients revealed cLDL accumulation, ICAM‐1 overexpression and leukocyte infiltration. Our results indicate that cLDL induces renal injury and identify a novel mechanistic factor for progressive kidney disease. Study was supported by AHA (EOA), NIH R01 and VA grants (AGB, SVS).
Regulation of Annexin A1 expression in renal interstitial fibroblasts during chronic kidney disease
Authors: Neymeyer Hanna , Reverte Virginia , Hohberger Stephan , Saez Fara , Bachmann Sebastian , Salazar Francisco Javier , Paliege Alexander
Published: June 2021

Aim of the present study was to characterize the expression of the anti‐inflammatory and anti‐fibrotic protein annexin A1 (ANXA1) in renal fibroblasts in a rat low nephron number model of chronic kidney disease (CKD). Expression and distribution of ANXA1 was studied by real time PCR and double labeling immunofluorescence with Ecto‐5′‐nucleotidase (5′NU) as a marker for cortical fibroblasts and alpha‐smooth muscle actin (α‐sma) as marker for myofibroblasts. At ten month of age, animals with CKD were hypertensive (162 ± 5 vs. 124 ± 4 mmHg) and demonstrated a significantly increased abundance of ANXA1 immunoreactive cells in the renal cortical interstitium. ANXA1 mRNA levels were increased as well (+190 ± 34%, p<.05). 5′NU positive fibroblasts were identified as the dominant ANXA1 expressing interstitial cell type both, in controls and in animals with CKD whereas myofibroblasts in the CKD animals did not show ANXA1 signal. Treatment of cultured human fibroblasts with the pro‐fibrotic cytokine transforming growth factor beta (7,5 ng/ml for 48h) resulted in an augmented α‐sma and collagen I biosynthesis whereas ANXA1 expression was reduced. In conclusion, our results thus identify renal cortical interstitial fibroblasts as a major source for renal ANXA1 in controls and during CKD. Modulation of fibroblast ANXA1 expression may be an important mechanism for the control of extracellular matrix synthesis.
Endothelin ETA receptor blockade does not attenuate the rise in early markers of acute kidney injury following bilateral renal ischemia
Authors: Boesen Erika I , Zoll Kyle E
Published: June 2021

The role of endothelin‐1 (ET‐1) in renal ischemia/reperfusion (I/R) injury is unclear. We tested whether pretreatment with a selective ETA receptor antagonist (ABT‐627; 10 mg/kg/d p.o., 3 days) would attenuate acute kidney injury in male C57BL/6 mice (n=5–8). At 24 h after 20 min bilateral renal ischemia, I/R significantly increased ET‐1 mRNA levels (qPCR) relative to sham‐operated mice in renal cortex (2−ΔΔCT=6.7+/−2.3 vs 1.0+/−0.1; P=0.06), outer medulla (6.0+/−1.0 vs 1.0+/−0.1; P<0.01) and inner medulla (2.1+/−0.3 vs 1.1+/−0.3; P=0.05). Plasma creatinine concentration was also significantly increased in I/R (2.4+/−0.2mg/dL) compared to sham operated mice (1.0+/−0.1mg/dL); this was not attenuated by ABT‐627 (2.8+/−0.1 mg/dL). Neutrophil gelatinase‐associated lipocalin (NGAL) is produced by renal tubular cells in response to injury and was markedly increased in I/R versus sham‐operated kidneys in the renal cortex (2−ΔΔCT=62+/−13 vs 1.1+/−0.2; P<0.05) and outer medulla (24+/−4 vs 1.3+/−0.4; P<0.05), and this effect was not significantly attenuated by treatment with ABT‐627 (70+/−25 and 34+/−9 for cortex and outer medulla). These data suggest that despite marked ET‐1 upregulation in the kidney following I/R, ETA receptor activation may not play a major role in the initial loss of renal function or in tubular injury within the first 24 h following I/R.
Role of Calcineurin in Polycystin Protein Trafficking to the Primary Cilium in LLCPK Cells
Authors: Gilder Allison Louise , Chapin Hannah C , Caplan Michael J.
Published: June 2021

Pathological mutations in PKD1 and PKD2 cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). PKD1 and PKD2 code for Polycystin 1 (PC1) and Polycystin 2 (PC2), respectively. PC1 and PC2 traffic to the primary cilium in epithelial cells, including LLCPK cells. They assemble with one another to form a complex that may mediate aspects of ciliary signaling. It has been shown that, in both ciliated and non‐ciliated cell lines, expression of PC2 is necessary in order to permit PC1 to traffic to the cell surface membrane. To identify potential regulatory partners of PC1, we conducted a genome wide siRNA knockdown screening study in HEK 293T cells. This analysis suggested that knockdown of the protein phosphatase, calcineurin, decreased PC1 cell surface localization. Interestingly, in C. elegans, dephosphorylation of PC2 by calcineurin has been shown to be necessary for PC2 trafficking to the male sensory neuronal cilia. We find that inhibition of calcineurin through Cyclosporine A treatment in LLCPK cells produced a decrease of PC1 and PC2 accumulation in the primary cilium. In addition, Cyclosporine A‐treated zebrafish embryos develop a curly tail phenotype that is characteristic of zebrafish in which functional expression of PKD1 a/b or PKD2 has been disrupted. We hypothesize that calcineurin may play a role in transport of the PC1/PC2 complex to the primary cilium, perhaps by mediating the dephosorylation of PC2.