bluuebunny/crossref_metadata_2025 huggingface dataset:
The functional role of bestrophins and TMEM16A in rat mesenteric small arteries
Authors: Aalkjaer Christian , Nielsen Vibeke Secher , Matchkov Vladimir
Published: June 2021

Two Ca2+‐activated Cl− currents (ICl(Ca)) are present in vascular smooth muscle (VSMC). One is cGMP dependent, the other is characterized as a classical ICl(Ca). Here we downregulate bestrophin‐3 (best3) and TMEM16A, to assess their importance for the two ICl(Ca) and for the role of ICl(Ca) in vascular function.

Best3 and TMEM16A were downregulated in rat small mesenteric arteries using siRNA. Knockdown was confirmed at mRNA and protein levels 3 days after transfection.

Best3 downregulation suppressed the cGMP‐dependent ICl(Ca) while TMEM16A downregulation suppresed both ICl(Ca)s. Both best3 and TMEM16A downregulation suppressed oscillations in vascular tone i.e. vasomotion. Best3 downregulation was without effect on sensitivity and maximal force production to norepinephrine while TMEM16A downregulation significantly reduced both. Also the force production to 125 mM KCl was reduced by TMEM16A downregulation.

We conclude that TMEM16A is essential for both ICl(Ca)s while best3 may be modifying the TMEM16A current characteristics in a subset of channels. The cGMP‐dependent ICl(Ca) is critical for vasomotion and normal expression of TMEM16 is essential for VSMC conctractility.
Interaction between eNOS translocation and phosphorylation in flow –induced NO production
Authors: Boric Mauricio P , Figueroa Xavier F , González Daniel R , Durán Walter N
Published: June 2021

Flow‐induced eNOS activation may involve the interaction of several signaling processes, such as eNOS phosphorylation, binding to Ca2+‐calmodulin, dissociation from caveolin (cav‐1) and/or translocation from membrane. We studied NO production (chemiluminescence), eNOS subcellular distribution and phosphorylation in intact resistance vessels of the isolated rat mesenteric bed submitted to step changes of flow rate. Flow increase (2 to 10 mL/min) induced a biphasic rise in NO production: a transient phase that peaked at 15‐sec and lasted 3–5‐ min, followed by a sustained phase, which lasted until the end of stimulation. Concomitantly, flow caused a rapid translocation of eNOS from the microsomal compartment to the cytosol and Golgi, which was paralleled by a 50% reduction in the eNOS‐cav‐1 association. In addition, the transient NO production, eNOS translocation, and eNOS dissociation from cav‐1 depended on extracellular Ca2+. In control or Ca2+‐free conditions, flow rise increased eNOS phosphorylation at ser‐1177 in an Akt‐dependent manner. Blockade of the PI3K‐Akt pathway abolished the sustained, but not the transient phase, of flow‐induced NO release. We conclude that flow‐induced NO production involves rapid Ca2+‐dependent eNOS translocation from membrane, while PI3K‐Akt‐ dependent phosphorylation accounts for the sustained eNOS activation by prevailing flow.

Fondecyt 1090757 & Anillos‐Act71
Increased Circulating Microparticles in Diabetic Rats Mediate Leukocyte Adhesion in Intact Venules
Authors: Stork Christian , He Pingnian
Published: June 2021

Microparticles (MPs) are membrane bound vesicles that express phosphatidylserine and released by activated and apoptotic cells. MP amount increases under inflammatory conditions. The objective of this study is to investigate if streptozotocin‐induced diabetic rats had increased circulating MPs and the roles of MPs in development of diabetic microvascular complications. Plasma MPs were quantified by flow cytometry with Annexin‐V staining and isolated by centrifugation. MP interaction with endothelium and blood cells was investigated by perfusing isolated MPs to individually perfused rat mesenteric microvessels and observed by confocal imaging. The diabetic plasma MP count significantly increased from 5.3 × 106 (normal rats) to 33 × 106 per ml. Perfusing isolated diabetic MPs to the normal venule resulted in rapid MP adhesion on the microvessel wall, which induced immediate leukocyte adhesion following resumed blood flow (19 leukocytes/100 μm vessel length). Although MP‐mediated leukocyte adhesion did not affect basal permeability, the application of a chemoattractant fMLP caused a 5‐fold increase in Lp. These results indicate that the increased circulating MPs under diabetic conditions are not only results of vascular cell activation and apoptosis, but also mediators that promote blood cell adhesion and activation resulting in exacerbated microvessel dysfunction. Supported by HL56237 and HL084338.
Increased nitric oxide is necessary but not sufficient to increase permeability in the absence of calcium influx in intact rat venules
Authors: Wang Mingxia , Yuan Dong , He Pingnian
Published: June 2021

Our studies showed that inhibition of nitric oxide synthase (NOS) prevented inflammatory stimuli‐induced permeability increases without affecting the initial increase in endothelial (EC) [Ca2+]i, suggesting that eNOS activation downstream from Ca2+ signal is the key determinant of permeability increases. However, we also found that H2O2‐induced initial NO production does not cause an immediate permeability increase in the absence of an initially increased EC [Ca2+]i. We therefore hypothesize that increased NO alone is not sufficient to increase permeability in the absence of increased EC [Ca2+]i. To test this hypothesis, microvessel permeability was measured by hydraulic conductivity Lp in individually perfused rat venules. Increased NO was initiated by NO donors and confirmed by DAF‐2 measurements. Increased EC [Ca2+]i was induced by platelet activating factor (PAF, 10 nM). Results showed that perfusing Spermine NONOate (100 μM) or S‐Nitroso‐ N‐acetyl‐D, L‐penicillamine (SNAP, 400 μM) alone did not increase Lp, but both NO donors potentiated PAF‐induced Lp peak from 9 ± 1.2 (without NO donor) to 43 ± 13.0 and 27 ± 4.1 times the control value. These results support the hypothesis that increasing NO alone is not sufficient to increase permeability in the absence of Ca2+ influx, but in the presence of stimuli‐induced Ca2+ signal, the higher NO level potentiated the increased permeability. The results suggest that increased NO and EC [Ca2+]i are both necessary and function co‐dependently to increase microvessel permeability. Supported by HL56237 and HL084338.
Pericytes Contribute to Exercise‐Induced Skeletal Muscle Hypertrophy
Authors: Zou Kai , Huntsman Heather D. , Valero Carmen , Skelton John T. , Adams Joseph T. , Mahmassani Ziad , Boppart Marni D.
Published: June 2021

Pericytes are perivascular cells with stem cell potential found in a variety of tissues. We have demonstrated that pericytes accumulate in skeletal muscle overexpressing the α7 integrin (α7Tg) and α7Tg mice exhibit increased muscle growth following exercise.

PURPOSE

The purpose of this study was to determine whether pericytes contribute to muscle growth and whether this effect is enhanced by exercise.

METHOD

Sca‐1+CD45− cells were isolated from α7Tg gastrocnemius (GAS), and labeled with DiI. Recipient C57BL6 mice were exposed to 30 min downhill running 1 hour before cell injection (4 × 104) into GAS. The contralateral leg was injected with saline. Animals were then assigned to either sedentary (SED) or exercise trained (EX) group. EX mice were exposed to downhill running 3 times/wk for 2 wks.

RESULTS

Sca‐1+CD45− cells expressed pericyte markers (CD90, CD140b, NG2, and CD146). DiI‐labeled pericytes did not directly form new fibers and did not fuse with existing fibers. However, Pax7+ satellite cells, mean fiber cross sectional area, percentage of larger caliber fibers (2000 to 3000 μm2) and number of nuclei per fiber were significantly increased in Cell/EX compared to Saline/EX (P<0.05).

CONCLUSIONS

This study demonstrates that exercise enhances the ability for pericytes to indirectly increase muscle growth and may provide the mechanistic basis for exercise‐induced hypertrophy.
GATA‐4 Gene Transfer Increases Mesenchymal Stem Cell Mediated Myocardial Salvage through miRNA Regulation and Bioactive Molecules Migration between Cells
Authors: Xu Meifeng , Yu Bin , Pasha Zeeshan , Wang Yigang
Published: June 2021

We hypothesized that GATA‐4 enhanced mesenchymal stem cell (MSC) mediated ischemic heart repair via regulating miRNA (miR).

Methods and Results

GATA‐4 was transduced into MSC (MSCGATA‐4) using the murine stem cell virus retroviral expression system. Overexpression of GATA‐4 downregulated miR‐15 family (miR‐15b, miR‐16, and miR‐195) and upregulating miR‐221/222 in MSC assayed using miR microarray and quantitative RT‐PCR. GATA‐4 significantly increased MSC survival evaluated by counting Annexin‐V+ cell and by MTT intake. MSCGATA‐4 markedly protected myocytes (CM) against ischemic injury examined through measurements of LDH release, MTT intake, and Annexin‐V staining. Fluorescence in situ hybridization and immunostaining results showed that miR‐221 as well as Bcl‐w and VEGF, two proteins of target genes of miR‐15 family, were transferred from MSC to CM. Gap junctions were observed between CM and MSC in a co‐culture system. The rats transplanted MSCGATA‐4 following left anterior descending coronary artery ligation showed a significantly improved cardiac function by echocardiography. Furthermore, histological studies revealed reduced infarction size and increased angiogenesis.

Conclusions

Overexpression of GATA‐4 regulates miR expression in MSC, which plays an important role in MSCGATA‐4 mediated salutary effects on improving cardiac function and cardioprotection.
Hypothermia Protects Against Myocardial Microvascular Damage (No‐Reflow) Even When Initiated After Coronary Artery Reperfusion
Authors: Hale Sharon Lee , Kloner Robert A
Published: June 2021

We tested the effects of hypothermia (H) on no‐reflow when H is initiated only at reperfusion; a time point that many studies have shown is too late to reduce myocardial infarct size (IS).

Methods

Rabbits received 30 m coronary artery occlusion and 3 h reperfusion. Hearts were treated with topical H (~32ºC) at 5 m before (HI, n = 14) or 5 m after (HR, n = 12) reperfusion. Controls remained normothermic (N, ~38ºC, n=14). Ischemic risk zone, no‐reflow zone, and necrosis were measured.

Results

The ischemic insult was comparable in all groups (26–30% of left ventricle, p = ns). As we and others have observed, H started at this time failed to significantly reduce IS compared with N hearts (27 ± 3% of risk zone, HI; 42± 4%, HR and 34 ± 5%,N p =NS, ANOVA). However, the extent of the no‐reflow defect was significantly reduced in hearts treated with H, even when started after reperfusion (46±9% of the necrotic area, HI; 50±5%, HR; versus 80±8%, N; p <0.05 HI and HR vs. N). There was a significant treatment effect of H on the regression between the extent of the no‐reflow zone and that of the necrotic zone (ANCOVA, p = 0.003).

Conclusions

This study shows that myocardial temperature reduction dramatically reduces no‐reflow injury even when H is initiated too late to reduce overall IS. These data suggest that the microvasculature is especially receptive to the salvaging properties of hypothermia.
Quercetin glucuronide preserves cardiac function in porcine hearts during perfusion with human whole blood by inhibiting platelet/neutrophil activation and preserving venular barrier function
Authors: Juchem Gerd , Bauer Andreas , Postrach Johannes , Reichart Bruno , Nees Stephan
Published: June 2021

Quercetin glucuronide (QG) reportedly inhibited the activation of blood cells of the acute immune system and stabilized venular wall models (cells of human origin) in presence of inflammatory mediators. We hypothesized that QG would protect isolated pig hearts during acute activation of the immune system (xenogenic perfusion with human whole blood). 12 hearts of non‐transgenic landrace pigs were explanted (organ preservation solution supplemented with 50 μM QG) and immediately cirumfused for 180 min in an ex vivo working‐mode with human blood in the absence (control group, n=6) or presence of 50 μM QG in the blood (n=6). Hemodynamic function was evaluated and inflammatory interactions were histologically investigated. All hearts maintained mandatory perfusion pressure of >55 mmHg throughout the whole study period. Cardiac function was significantly improved in the QG‐group and very similar to that of pig hearts perfused with autologous blood. Stroke work index (mean ± SEM) was 4272±434 erg/g in the QG‐group vs. 2790±558 erg/g in controls without QG (P<0.05) after 30 min and was still significantly higher at the end of the perfusion period (QG: 3145 ± 160 vs. control: 1570 ± 300 erg/g; p<0.05). Venulothrombosis, venulitis, and excessive accumulation of leucocytes could not be detected in the QG‐group after specific staining and microscopic examination of ventricular myocardium. Supported by a grant of the DFG.
Loss of flow mediated dilation accompanies endothelial dysfunction in the preconditioned state
Authors: Frame Mary D , Dewar Anthony M
Published: June 2021

To examine flow mediate dilation in the preconditioned state, terminal arteriolar junctions were observed in the hamster cheek pouch using intravital microscopy (pentobarbital 70mg/kg, N=28). Repeated occlusion of one downstream arteriolar branch induced dilation in both the unoccluded downstream branch B (where shear increased) and in the upstream feed F (where shear decreased). To test whether occlusion induced dilation was NO‐mediated, we applied nitro‐arginine (LNNA, 10‐5M) to either B or F (micropipette). LNNA prevented occlusion induced dilation in B (where shear increased) and not F. Thus dilation where shear increased is interpreted as flow mediated. To test whether occlusion induced dilation in F was propagated via gap junction (GJ) signals arising from B, 18‐alpha‐GA (10‐4M) was applied to either B or F. GJ inhibition blocked all dilation in F and none in B. Finally preconditioning induced pharmacologically with nitroprusside or b2‐adrenergic agonism (isoproterenol, ISO) caused endothelial dysfunction (bradykinin, acetylcholine). Occlusion in the preconditioned state caused the same flow changes in B or F, but no dilation. Local elevations in shear induce a local dilation and initiate a GJ mediated propagated dilation. Preconditioning inducing endothelial dysfunction includes absence of flow mediated dilation, and lack of GJ mediated propagated dilation. (NIH HL55492; AHA 0655908T)
Early MRI findings of targeting the NG2 proteoglycan in GBM
Authors: Rygh Cecilie Brekke , Thuen Marte , Poli Aurélie , Wang Jian , Løkka Guro , Huuse Else Marie , Thorsen Frits , Enger Per Øyvind , Chekenya Martha
Published: June 2021

Glioblastoma multiforme (GBM) is an aggressive brain tumor where patients have median survival of only 14.6 months. High NG2 expression in GBMs correlates with increased angiogenesis, drug resistance and shorter overall survival. We aimed to target NG2 with monoclonal antibodies (mAb) combined with natural killer (NK) cells in an adoptive immunotherapy approach in athymic rats bearing human GBM. The treatment response was monitored longitudinally using multiparametric MRI including morphological, dynamic contrast enhanced (DCE) and diffusion weighted imaging. The animals were divided into no treatment (n=8), mAb (n=9), NK cells (n=12) and mAb+NK cells (n=8). The overall survival, histological analyses and DCE‐MRI showed that the combined mAb+NK had therapeutic efficacy, where the leakage space (ve) was significantly increased indicating increased cell death. Changes in the microvascular parameters transfer constant (Ktrans) and area under the curve (AUC) were transiently detected in the mAb and NK cell groups. Diffusion values showed no significant change over time. Multiparametric MRI may facilitate individualized therapy by early detection of treatment responses.

Support: The Norwegian Cancer Society, Western Norway Regional Health Authority and National Functional Genomics Programme (FUGE) of the Research Council of Norway
Simvastatin and GGTI‐2133, a gernaylgeranyl transferase inhibitor, increase erythrocyte (RBC) deformability, but inhibit low oxygen tension‐induced ATP release
Authors: Clapp Kelly Marie , Ellsworth Mary L , Sprague Randy S , Stephenson Alan H
Published: June 2021

Endogenous cholesterol synthesis is reduced by statin drugs that inhibit HMG‐CoA reductase. This pathway also synthesizes isoprenoids (Geranylgeranyl‐pyrophosphate and Farnesyl‐pyrophosphate), lipid molecules responsible for posttranslational modification of small GTP‐binding proteins such as Rho. Some potential effects of statins, beyond lowering blood cholesterol levels are thought to occur by inhibiting the Rho/Rho kinase pathway. We reported the Rho kinase inhibitor, Y27632, increases RBC deformability and low O2 tension‐induced ATP release from RBCs. Here we investigated the hypothesis that treatment with simvastatin or the geranylgeranyl transferase inhibitor, GGTI‐2133 would increase both RBC deformability and low O2 tension‐induced ATP release. Sprague‐Dawley rats were fed simvastatin supplemented chow (20mg/kg/day) for 30 days. RBCs obtained from rats fed statin supplemented chow were more deformable than RBCs from control rats (p< 0.05, n=6). Similar results were seen with healthy human RBCs treated with GGTI‐2133 (10 μM, p < 0.01, n=8). Statin treatment, however inhibited low O2 tension‐induced ATP release from rat RBCs (n=6) and the same result was seen after treatment with GGTI‐2133 (p<0.05, n=7). These results suggest that inhibiting geranylgeranylation impairs low O2 tension‐induced ATP release, despite increased RBC deformability. HL‐89094 and HL‐64180
Characteristics of cell‐free layer variation in a 25‐μm tube
Authors: NAMGUNG BUMSEOK , CHO SEUNGKWAN , ONG PENG KAI , SAKAI HIROMI , KIM SANGHO
Published: June 2021

To examine the effect of aggregation on the cell‐free layer variation, in vitro experiments were performed by perfusing human red cells through a 25‐μm tube. The level of aggregation was adjusted to physioloigcal and pathological levels seen in humans. In addtion to qunatifing the layer width with its mean, additional information on its periodicity and variation frequency was studied by using the auto‐correlation and power spectral density analysis. Interstingly, the contact frequency between red cells and inner tube wall was significantly reduced in hyper‐aggregating conditions as compared to that in non‐aggregating conditions. This result might be due mainly to the enhancement of the layer formation which impaired the opportunity for red cells to become in contact with the tube wall. In the anaysis of the layer periodicity, relatively small magnitude of the auto‐correlation length (< 6.6 μm) as compared to the diameter of red cell (~8 μm) was found, which may imply that the layer variation exhibits non‐periodicity regardless of red cell aggregation conditions. The power spectral density analysis revealed that the temporal variation of the layer was significantly decreased in hyper‐aggregating conditions than that in non‐aggregating conditions.
The Risks of Chronic Internal Diseases Among the Workers of Uranium Processed Enterprise in Kazakhstan
Authors: Bekenova Farida , Abduldayeva Aigul
Published: June 2021

Data on the prevalence rate of chronic internal diseases (CID) among the nuclear industry workers are scarce and contradictory. The aim of the study was to investigate the prevalence rate and relative CID risks among the workers at uranium processed company who are affected by occupational radiation‐chemical effects of low intensity. It was studied the prevalence (Pr), relatively rough (RR) CID risks among 912 workers at Hydrometallurgical Plant ‐ plants for the production of nitrous oxide ‐ uranium oxide. Comparative group was consisted of 788 workers at bearing plant (BP). The most common CID were arterial hypertension (AH)‐24.7%, chronic gastritis (CG)‐17.4%, chronic bronchitis (CB)‐19.3%. These diseases have excess RR gmzvspz (p<0,0001):AH‐2.4(95%CL1,9–3,0); CG‐2.1(95%CL1,6–2,7), CB‐2.0(95%CL 1,6–2,6;). A more accurate judgments about excess Pr disease is possible on the basis of SRR assessment (the method of stratification), which allows to monitor the confounding effects. Specific confounding for hypertension are heredity, obesity, metabolic syndrome, alcohol abuse, high anxiety, addiction to salty foods, and for CG‐H.pylori; for HB ‐ smoking. Thus, standardized for sex, age, seniority and the main predictors, relative risks were higher than the rough risks, which confirms the increased risk of hypertension, CG, CB in terms of combined radiation and long‐term chemical exposure.
The influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation
Authors: Ilderbayev Oralbek , Dalenov Erbolat
Published: June 2021

The purpose of this experimental research is to study the influence of gamma‐radiation on metabolic processes of white rats undergone to ionizing radiation. The is a conclusion of the ethical committee (protocol N5, 2010, State medical university, Semey). Animal experiments were carried out in accordance with the order of the Helsinki Declaration on the humane treatment of animals. The levels of LPO, diene conjugates (DC) and MDA in thymus, spleen and lymph nodes of small intestine were tested.

In the II series of experiment 30 days after irradiation by common gamma‐rays in the dose equal to 6 Gray, the reliable increase of DC contents was observed, that was 2.75 times higher in liver, 1.37 times higher in lymph nodes of small intestine, 2.5 times higher in thymus and 2.38 times higher in spleen as compared to control animals. The MDA amounts significantly increased in thymus (in 1.68 times) and in spleen (in 1.7 times, ð<0.05). The increase of MDA concentration in liver was equal to 1.63 times (ð<0.05). No changes were observed in lymph nodes of small intestine. Finally, obtained research results show that in case of exposure to common gamma‐rays in a dose of 6 Gray, the concentrations of DC and MDA increase in liver, thymus and spleen.
Vasoactive agents result in coordinated changes in vascular smooth muscle cell elasticity and extracellular matrix adhesion
Authors: Hong Zhongkui , Sun Zhe , Li Zhaohui , Meininger Gerald A.
Published: June 2021

Coordination of mechanotransduction and force transmission within the blood vessel wall requires a physical adhesion of vascular smooth muscle cells (VSMCs) to extracellular matrix proteins (ECM) and surrounding VSMCs. Our hypothesis was that integrin adhesion to the ECM is dynamically up‐regulated in VSMC during contractile activation and likewise down‐regulated during relaxation to support changes in vessel diameter and wall tension. Real‐time measurements of cell adhesion and cell elasticity were obtained with atomic force microscopy (AFM) in response to angiotensin II (Ang II) and adenosine (Ado). ECM adhesion behavior was evaluated with fibronectin (FN) coated microbead‐tipped (5 μm dia) AFM probes. Single VSMCs were isolated from rat cremaster skeletal muscle arterioles and cultured 3–8 days before AFM experiment. The results show that the administration of Ang II (10−6M) in cell bath immediately increased the strength of adhesion between the AFM probe and cell as well as probability of binding between FN and the cell. Cell stiffness also increased in parallel with adhesion upon Ang II stimulation. In contrast, Ado (10−4M) decreased cell elasticity and the adhesion of integrins to FN. These findings support the hypothesis that a vasoconstrictor can activate through inside‐out mechanisms both the contractile process and cell adhesion to the ECM in a coordinated fashion, while a vasodilator has the opposite effects. The role of the changes in adhesion may be related to altered mechanics or cell signaling involving the focal adhesion sites. (1P01HL095486 to G.A.M. and AHA 0835676N to Z.S.)
Erythrocyte shunting patterns are identical for endothelial dysfunction and for loss of connexin 43
Authors: Frame Mary D , Rivers Richard J , Yang Jay , Secomb Timothy W
Published: June 2021

To study shunting (preferential flow pathways), we observed arterioles in the hamster cheek pouch by intravital microscopy (pentobarbital 70mg/kg, N=48). Erythrocyte (RBC) fluxes into sequential branches (4–8) from a central feed were measured (fluorescently labeled RBC). In controls, each branch received 10–25% of total inflow. Preconditioning, either with nitroprusside or controlled thermal injury (50°C, 20s) by microcautery (300μm), induced shunting, with 80–90% of RBCs passing along the central feed to one distal branch. Endothelial dysfunction induced with tissue bath nitro‐arginine (10‐5M) led to the same shunting pattern, as did tissue blockade of gap junctions (18‐alpha‐GA, Gap‐27(43), sucrose). Cx43 was targeted for knock‐down using RNAi applied to the cheek pouch 3 days in advance, decreasing Cx43/GAPDH protein by 60%. Shunting was seen in all RNAi(Cx43) animals, but not with RNAi(Cx45) treatment. Dilations to bradykinin (10‐7M), acetylcholine (10‐6M), isoproterenol (10‐5M) and adenosine (10‐4M) were attenuated in preconditioning, indicating endothelial dysfunction. Thus, a common maldistribution of RBC fluxes is seen both in the absence of gap junctional activity and in the preconditioned state, and may occur in many pro‐inflammatory states associated with endothelial dysfunction. (NIH HL55492; AHA 0655908T)
Inter‐individual differences in rapid vasodilation in older males with and without type 2 diabetes
Authors: Kellawan J. Mikhail , Bravo Michael F. , Moynes Jackie S. , Walsh Jeremy J. , Bentley Robert F. , Tschakovsky Michael E.
Published: June 2021

PURPOSE

To identify if rapid vasodilatory responses differ between individuals with or without type 2 diabetes.

METHODS

13 males (7 type II diabetics (T2D), 6 healthy controls (CON)) lay supine with the arm at heart level and completed 6 trials of single handgripping contractions (3 trials/day/contraction intensity, on 2 separate days, 1 s contraction duration) at 10, 20, and 40 % of their maximal voluntary contraction (randomly ordered). Forearm brachial artery blood flow (FBF, echo and Doppler ultrasound), mean arterial pressure (MAP, finger photoplethysmography) were measured continuously.

RESULTS

tension‐time integral of single contraction force production (kg·s) vs. FBF relationships were constructed for each individual. There was no difference in the slope (4.7 ± 2.9 vs. 4.8 ± 2.5) or y‐intercept (y‐int) (35.2 ± 32.5 vs. 23.3 ± 15.2) between T2D and CON. However, there was considerable variation in both the slope and y‐int between subjects in each group. T2D coefficient of variation (COV) of slope was 64% and y‐int was 93%. COV for the slope in CON was 52% and y‐int 65%. Despite the variation, the r2 value for each individual was strong, ranging from 0.34–0.7 (σ = 0.51 ± 0.14) in T2D and 0.44–0.97 (σ = 0.76 ± 0.22) in CON.

CONCLUSION

Older males exhibit substantial inter‐individual rapid vasodilatory responses to muscle contraction. These individual differences do not appear to be influenced by type 2 diabetes.
Nitric oxide availability is reduced in sickle cell disease even in presence of low amounts of intravascular cell‐free hemoglobin
Authors: Deonikar Prabhakar , Kavdia Mahendra
Published: June 2021

Cell‐free Hb is released by intravascular hemolysis in sickle cell disease and can affect the nitric oxide availability. Role of small amounts of cell‐free Hb in lowering NO availability and thereby the development of pulmonary hypertension seen in sickle cell disease has been questioned. To analyze the effect of intravascular cell‐free Hb on NO availability under sickle cell disease conditions, we developed a mathematical model describing NO transport in a 50 μm arteriole. Simulations were performed to assess the significance of cell free layer thickness, blood velocity and NO‐RBC reaction rate constant on NO bioavailability. We observed that NO bioavailability was lower under sickle cell conditions as compared to physiological conditions. In presence of 0.5 μM cell‐free Hb, NO bioavailability reduced ~3 to 7 fold. Increasing cell free layer thickness did not increase NO availability significantly for cell‐free Hb concentrations ≥ 1 μM. Our results indicate that intravascular cell‐free Hb concentrations as low as 0.5 μM can reduce the NO availability significantly and in presence of cell‐free Hb ≥ 1 μM, diffusional resistance for NO transport from the cell free layer become less significant. Thus, a low amount of cell‐free Hb reduces NO availability and may contribute to the development of sickle cell complications of pulmonary hypertension. Supported by AHA 0530050N & R15 HL087287 grants.
Oxygen dependence of respiratory rate (VO2) in rat spinotrapezius muscle in situ measured with phosphorescence quenching microscopy (PQM)
Authors: Golub Aleksander S , Pittman Roland N
Published: June 2021

Objective

The sensitivity of isolated cells to PO2 is described by Hill's equation with a low Km (< 5 torr). Cells in intact tissues are more sensitive to O2 deficiency. To study the oxygen dependence of VO2 in muscle fibers we developed a method to register and interpret O2 disappearance curves (ODC).

Method

We employed PQM using a probe distributed in the interstitium. Rapid pressure onset to the tissue extruded most of the RBCs from the muscle vessels making the ODC independent of hemoglobin. The contribution to the O2 consumption by PQM was evaluated and accounted for. The corrected ODC was converted into the PO2 dependence of VO2. Fitting equations obtained from a model of heterogeneous PO2 distribution was applied to recover the parameters of respiration in muscle fibers.

Results

In 6 muscles the rate of O2 consumption at rest was Vmax = 139 ± 6 nl O2/cm3•s, Km = 12 ± 2 torr and the correlation between them was 0.94. The PO2 drop across a fiber was Ä = 4.1 to 4.9 torr. Critical PO2 was estimated as 22 torr at VO2 = 90 nl O2/cm3•s.

Conclusion

The oxygen dependence of respiration can be measured in thin muscles under different experimental conditions. In resting muscle Km and critical PO2 are substantially lower than interstitial PO2 = 51 ± 3 torr, a result discordant with the hypothesis of metabolic regulation of oxygen supply to tissue. The PO2 gradient across a muscle fiber was found to be small.

Support: NIH grant R01HL18292
Effects of wall shear stress on NO simulation in arterioles
Authors: Kim Sangho , Cho Seungkwan , Namgung Bumseok
Published: June 2021

The shear stress acting on the endothelium is a dominant mechanical stimulus that modulates the production rate of NO in the endothelial cells. Thus, determination of the wall shear stress (WSS) is important in studying the flow‐induced vasodilation. Recently, we have reported a way to estimate WSS in small arterioles by measuring the cell‐free layer width and edge velocity of red cell core, and used the method in NO simulation studies. In using that method, we assume that the flow within the cell‐free layer is steady. However, it has been known that variations in flow velocity due to the arterial pulse would exist in arterioles. Thus, in this study, we have considered temporal changes of the edge velocity of red blood cell core resulting from the temporal cell‐free layer width variations in estimating the WSS in the vessels. Linear and non‐linear velocity profiles within the layer were used to estimate the WSS. The temporal variation results of the WSS were utilized to predict NO bioavailability in the arterioles. We found a slight but significant difference in estimated WSS by using different velocity profiles in the cell‐free layer. However, NO simulation showed that this slight difference of WSS did not lead to a significant change in NO bioavailability.

This work was supported by NUS URC Grant R‐397‐000‐091‐112.
Numerical simulation of time‐dependent NO/O2 transport in arterioles
Authors: Cho Seungkwan , Ong Peng Kai , Namgung Bumseok , Kim Sangho
Published: June 2021

We developed a new time‐dependent computational model for coupled NO/O2 transport in small arterioles that incorporates potential physiological responses (temporal changes in NO scavenging rate and O2 partial pressure in blood lumen and NO production rate in endothelium) to the temporal cell‐free layer width variations. Two relations between wall shear stress (WSS) and NO production rate based on the linear and sigmoidal functions were considered in this simulation study. The cell‐free layer data used for the simulation were acquired from arteriolar flows (D = 48.3 ± 1.9 μm) in the rat cremaster muscles under normal flow conditions (WSS = 3.4–5.6 Pa). For both cases of linear and sigmoidal relations, temporal layer width variations were found to be capable of significantly enhancing NO bioavailability and this effect was more pronounced in the latter (P < 0.0005) than the former (P < 0.005). In contrast, O2 bioavailability in the arteriolar wall was not considerably altered by the temporal layer width variations, irrespective of the relation. Prominent enhancement (P < 0.005) of soluble guanylyl cyclase (sGC) activation in the smooth muscle by the temporal layer width variations were predicted for both relations. The extent of sGC activation was generally lower (P < 0.01) in the case of the sigmoidal relation than that of the linear relation, suggesting a lesser tendency for arterioles to dilate with the former.
Specific Nox components mediate the age‐related impairment in collateral growth
Authors: Miller Steven J , Wenning Mary J , Bills Randall G , Chittajallu Nandita , Unthank Joseph L
Published: June 2021

Collateral growth (CG) is impaired by aging, but the impairment is reversed by antioxidant treatment. The aim of this study was to determine if NADPH oxidase (Nox) mediated the age‐related impairment of CG. CG (% luminal expansion) and Nox expression were assessed with an established model of ileal artery ligation in young (2–3 mo; Y) and retired breeder (10–12 mo; RB) Wistar Kyoto (WKY) rats, as well as RB treated with p22phox siRNA (RB+siRNA) or the Nox2 peptide inhibitor Nox2ds‐tat (RB+dstat). CG capacity at 7 days post ligation was restored in RB+siRNA (49.2±8.3%, p<0.001 vs. control) and in RB+ds‐tat (26.6±4.5%, p<0.003 vs. control), verifying the role of Nox in CG impairment. Measurement of Nox subunit basal mRNA expression with qPCR showed that Nox4 and p47phox, but not Nox2, were significantly (p<0.05) increased compared to Y WKY. Nox1 expression was too low for quantification. Changes in RB collateral artery Nox expression at 1, 3, and 7 days relative to same‐animal control arteries are shown in the table. The results suggest an important role for specific Nox isoforms/subunits in age‐related CG impairment, and the inhibitor data implicate the Nox2 isoform as a primary mediator of CG impairment. Support: HL092012
The mechanism of local blood acidification in the swimbladder by spatially organized monocarboxylate transporters
Authors: Kato Akira , Umezawa Takahiro , Ogoshi Maho , Ookata Kayoko , Munakata Keijiro , Doi Hiroyuki , Romero Michael F. , Hirose Shigehisa
Published: June 2021

The gas in the swimbladder of teleost fish is composed primarily of O2, and the swimbladder volume is regulated by O2 transfer between the luminal space of the swimbladder and the blood. In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the “Root effect.” To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 16 members of the H+/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute‐specific transcellular transport. Electrophysiological analyses demonstrated that MCT1b is a high‐affinity lactate transporter whereas MCT4b is a low‐affinity/high‐conductance lactate transporter. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.
Matrix Metalloproteinase‐9 in Homocysteine‐Induced Intestinal Microvascular Endothelial Paracellular and Transcellular Permeability
Authors: Munjal Charu , Tyagi Neetu , Lominadze David , Tyagi Suresh C
Published: June 2021

An elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with inflammatory bowel disease, the mechanism of Hcy action is unclear. Therefore we hypothesize that HHcy activates matrix metalloproteinase‐9 (MMP‐9), which in turn enhances permeability of human intestinal microvascular endothelial cell (HIMEC) by decreasing expression of junction proteins and increasing caveolae. HIMECs were grown in Transwells and treated with Hcy in the presence or absence of MMP‐9 activity inhibitor. Hcy induced permeability was assessed by measuring fluorescence intensity of solutes in the Transwells’ lower chambers. The cell‐cell interaction and cell barrier function was estimated by measuring Trans‐ endothelial electrical impedance. Confocal microscopy and flow cytometry were used to study cell junction protein levels. Hcy‐induced changes in transcellular transport of HIMECs were estimated by observing formation caveolae and paracellular permeability was associated with degradation of vascular endothelial cadherin and zona occludin‐1. Elevation of Hcy content increases permeability of HIMEC layer affecting both paracellular and transcellular transport pathways, and this increased permeability was alleviated by inhibition of MMP‐9 activity. These findings contribute to clarification of mechanisms of inflammatory bowel disease development.
Infection of human pericytes by HIV‐1 disrupts the integrity of the blood‐brain barrier
Authors: Toborek Michal , Nakagawa Shinsuke
Published: June 2021

HIV‐1 infection of the brain affects the cross‐talk between the individual cell types of the neurovascular unit, which then contributes to the disruption of the blood‐brain barrier (BBB) and the development of neurological dysfunctions. While the toxicity of HIV‐1 on neurons, astrocytes, and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV‐1 on pericytes. Therefore, the purpose of this study was to evaluate whether pericytes can be infected with HIV‐1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV‐1 receptor CD4 and coreceptors CXCR4 and CCR5. Importantly, we demonstrate for the first time that HIV‐1 can replicate, though at a low level, in human brain pericytes. Pericytes are susceptible to infection with both the X4‐tropic and R5‐tropic HIV‐1 strains. Moreover, HIV‐1 infection of pericytes results in compromised integrity of the in vitro model of the BBB. These findings suggest that infected pericytes may be involved in progression of HIV‐1‐induced CNS damage. Supported by MH63022, MH072567, DA027569, NS39254, and ES 07380.
Activation of the ERα and ERβ pathway downregulates voltage‐gated Ca2+ channels in coronary arteries
Authors: Gray William R. , Dalton Robin J. , Sketas Greg , Hill Brent J.F.
Published: June 2021

Our lab has previously demonstrated that a physiological concentration (1 nM) of estrogen (E2) can downregulate voltage‐gated, L‐type Ca2+ (VGCC) channels in coronary arteries. The distal ends of coronary arteries obtained from hearts of female pigs were sectioned into longitudinal strips and incubated for 24 hrs in 1 nM E2, EtOH, an estrogen receptor (ER) α/β antagonist (ICI 182,780), and a G protein‐coupled ER antagonist (GPER; G15). The arterial strips were homogenized for Western blot analysis using an antibody reactive to the VGCC alpha1C subunit and protein kinase Gα. Our preliminary results suggest that the E2 induced VGCC downregulation is mediated through ER α/β, and not GPER. Furthermore, ER activation by E2 in endothelium intact arteries have an increase in PKG expression (known to inhibit VGCCs), decrease in VGCC expression, and less coronary arterial reactivity. Overall, our results suggest that the classical ERs can be therapeutically targeted in women to reduce the density of VGCCs. Support: NCRR of the NIH, Grant #P20 RR‐16460 and the Arkansas SURF program.
Oxidation‐reduction state modifies vascular reactivity
Authors: Choi Hyehun , Webb R. Clinton , Lamb Fred S
Published: June 2021

Oxidation‐reduction (redox) reactions of cell surface thiol/disulfide couples regulate cellular processes and Cysteine/Cystine (Cys/CySS) is the predominant extracellular low‐molecular‐weight thiol/disulfide couple. We hypothesized that redox state regulates vascular reactivity independent of reactive oxygen species. Aortic rings from C57BL/6 mice were treated with varying concentrations of Cys/CySS, and vascular contraction and relaxation assessed. Acute exposure (40 min) to a reduced (−150 mV) state decreased relaxation to acetylcholine (Ach), not contraction to phenylephrine (PE), and an oxidized (0 mV) state had no effect on either Ach or PE compared to a neutral (−80 mV) state. After 4 hours, relaxation to Ach was impaired in the reduced, but enhanced in the oxidized state (control Emax = 88 ± 1.9 %, reduced Emax = 67 ± 2.3 %; control pD2 = 7.3 ± 0.07, oxidized pD2 = 7.7 ± 0.07, *p<0.05, n=10 to 12). Relaxation to sodium nitroprusside was unaltered. Contraction to PE was enhanced by reduced and attenuated by oxidized conditions (control Emax = 110 ± 3.4 %, reduced Emax = 143 ± 4.1 %, oxidized Emax = 94 ± 3.8 %, *p<0.05, n=10 to 12). Contractions to KCl and serotonin were unaffected. Thus redox state specifically modifies vascular reactivity in a receptor‐dependent fashion. It remains to be determined if these effects are physiologically significant in disease states associated with altered serum redox states.
Sub‐contractile sphingosylphosphorylcholine enhances vasoreactivity via reactive oxygen species
Authors: Ward Jeremy PT , Aaronson Philip I , Snetkov Vladimir A
Published: June 2021

Sphingosylphosphorylcholine (SPC), at concentrations that do not cause contraction (<1uM), causes potentiation of vasoconstriction to other agonists or depolarisation (Snetkov et al, Hypertension 2008;51:239–245). Our objective was to determine whether this was related to activation of NADPH oxidase and production of reactive oxygen species (ROS).

METHODS

Small pulmonary or mesenteric arteries were mounted on a myograph, challenged with depolarising solutions containing ~23 mM KCl to elicit a small contraction, and incubated with SPC (1uM) before being re‐challenged.

RESULTS

0.2 to 1uM SPC caused substantial potentiation of depolarisation‐induced force to between 400 and 800% of control in both rat and mouse arteries. Tempol (3mM) suppressed potentiation (e.g. PA: 526 +/− 220%; +tempol: 200+/−60%, n=5). SPC enhanced ROS production estimated by lucigenin and DCF. Potentiation was abolished in p47phox −/− mice (n=6). 0.2 uM SPC enhanced voltage activated calcium current in whole cell patch by ~30% (p<0.01), and this was mimicked by exogenous ROS. We conclude that low concentrations of SPC may cause hyperreactivity via NADPH oxidase and elevation of ROS, which enhances calcium entry via L‐type channels. Funded by the Wellcome Trust
Differential modulation of cardiac β1 and β2 adrenergic receptor contractile effects by adenosine A1 and A2A receptors
Authors: McIntosh Victoria , Zhan Enbo , Lasley Robert
Published: June 2021

The cardiac adenosine A1 receptor (A1AR) anti‐adrenergic effect and its modulation by the adenosine A2A receptor (A2AAR) has been described using the non‐selective β‐adrenergic receptor (βR) agonist Isoproterenol (ISO). The purpose of this study was to determine how the contractile effects of selective β1R vs. β2R activation are altered by A1AR and A2AAR. Dose response studies were performed in isolated perfused hearts from wild type (WT), A1KO, and A2AKO mice measuring LV systolic pressure (LVSP) and +dP/dt (n≥3/group). The responses to a single dose of β1R partial agonist xamoterol (Xam) and β2R agonist procaterol (Pro) are shown in Table 1. Similar responses were seen to Xam in WT, A1KO and A2AKO hearts, but the response to Pro was potentiated in A2AKO compared to WT and A1KO hearts (*p<0.05). This contrasts with previously reported effects of ISO in A1KO and A2AKO hearts. The A1AR agonist CCPA exerted significant antiadrenergic effects on both Xam and Pro responses (% increase in LVSP, +dP/dt with 100 nM Xam‐ 4 ± 2, 10 ± 5; with 10 μM Pro 17 ± 3, 25 ± 5). β1R and β2R positive inotropic effects in the intact heart are differentially modulated by A1AR and A2AARs, potentially due to heterodimerization between ARs and βRs. Support from NHLBI R01HL066132.
Restoration of serum euthyroidism is inadequate to reverse cardiac dysfunction in experimental hypothyroidism
Authors: Weltman Nathan Y , Savinova Olga V , Chen Yuefeng , Schlenker Evelyn H , Youmans Steven , Gerdes A. Martin
Published: June 2021

Clinical observations suggest a strong link between hypothyroidism (H) and poor outcomes in heart disease. We evaluated the efficacy of 2 weeks of Triiodothyronine (T3) in drinking water administered to hypothyroid rats with cardiac dysfunction to characterize the dosage of T3 that restores serum TH levels and cardiac function in propylthiouracil (PTU) induced H. PTU treatment reduced serum THs, small resistance vessels, and caused cardiac atrophy and LV dysfunction. PTU led to increased wall stress, primarily owing to a relative decrease in LV posterior wall thickness. Treatment with 3μg/kg/day restored serum TH levels while 5μg/kg/day reversed cardiac atrophy and wall thinning. A significant elevation in serum THs was necessary for complete LV functional restoration.

LV Function

HR
LV ESP
dP/dT
−dP/dT
LV FS

Control
366 (48)
142 (14)
10790 (2499)
−11910 (2299)
41 (5)

PTU
229 (38) *
105 (12) *
4209 (626) *
−4266 (891) *
35 (3) *

PTU + 3 T3
270 (27) * †
107 (13) *
5366 (718) *
−5623 (1120) *
36 (6)

PTU + 5 T3
313 (27) * †
113 (10) *
6223 (845) *
−6399 (999) *
38 (4)

PTU + 10 T3
363 (47) †
139 (21) †
9380 (1890) †
−11097 (3075) †
42 (4) †

p<.05 vs Control;

p<.05 vs PTU

In PTU induced H, restoration of serum THs was not associated with restoration of cardiac function suggesting that serum TH levels might not accurately reflect cardiac TH status. Short term T3 dosing to restore serum THs is inadequate to fully reverse cardiac dysfunction in PTU induced H.
Identifying the Molecular Mechanisms of Diastolic Dysfunction in Drosophila
Authors: Kaushik Gaurav , Nishimura Mayuko , Graham Suzanne , Fuhrmann Alexander , Bodmer Rolf , Cammarato Anthony , Engler Adam Jeffrey
Published: June 2021

Due to its short lifespan, Drosophila melanogaster is a tractable system in which to study the effects of senescence on cardiac function. Female yellow‐white flies experience a significant decrease in diastolic diameter between 1 and 5 weeks of age (>20%). An atomic force microscopy‐based micromechanical analysis on the intact heart tubes of juvenile and geriatric flies revealed that their cardiomyocytes stiffen with age proximal to the intercalated discs (1.8 ± 0.13 to 3.8 ± 0.30 kPa). Another strain, white 1118, is significantly more arrhythmic but experiences less diastolic dysfunction with age (<10% decrease in diastolic diameter). White flies were found to have no change in stiffness with age (p > 0.10). These first in situ mechanical analyses of living myocardium have resolved differences in passive cardiac mechanics due to age and suggest that phenotype and mechanism differ significantly between Drosophila strains. DNA‐microarrays have identified a host of mechanically‐sensitive proteins to have significantly altered expression with age. Mechanical analysis of flies with RNA‐interference targeted against promising candidates indicates several molecular mechanisms of age‐related myocardial stiffening and diastolic dysfunction. [NIH 1T32HL105373‐01 to GK; NIH 1D092OD006460 to AJE; AHA 10SDG4180089 to AC]